Na transport in sheep rumen is modulated by voltage-dependent
cation conductance in apical membrane
Ingo
Lang1 and
Holger
Martens2
2 Department of Veterinary
Physiology, Free University of Berlin, 14163 Berlin; and
1 Department of Zoophysiology
and Cell Biology, University of Potsdam, 14471 Potsdam, Germany
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ABSTRACT |
The effects of clamping the transepithelial
potential difference (PDt; mucosa
reference) have been studied in sheep rumen epithelium.
Pieces of ruminal epithelium were examined in Ussing chambers, in a
part of the experiments combined with conventional intracellular
recordings. After equilibration, the tissue conductance (Gt) was 2.50 ± 0.09 mS/cm2, the potential
difference of the apical membrane
(PDa) was
47 ± 2 mV,
and the fractional resistance of the apical membrane
(fRa) was 68 ± 2% under short-circuit conditions. Hyperpolarization of the
tissue (bloodside positive) depolarized
PDa, decreased fRa, and
increased Gt
significantly. Clamping PDt at
negative values caused converse effects on
PDa and
fRa. All changes
were completely reversible. The determination of individual
conductances revealed that the conductance of the apical membrane
increased almost linearly with depolarization of
PDa. The PD-dependent changes were
significantly reduced by total replacement of Na. These observations support the assumption of a PD-dependent conductance in the apical membrane that permits enhanced apical uptake of Na even at depolarized PDa. This mechanism appears to be
important for the regulation of osmotic pressure in forestomach fluid.
sheep rumen; sodium transport; potential difference-dependent
conductance
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INTRODUCTION |
THE RUMEN IS an important site of Na absorption in the
digestive system of sheep, and it has long been known that the
absorption of Na from the rumen is mediated by an active transport
mechanism (8). This conclusion has been supported by all the subsequent in vitro studies (7, 17, 28), which have further revealed that the flux
in net Na (JNanet) is
considerably higher than the (Na-dependent) short-circuit current
(Isc). The discrepancy between
Isc and
JNanet has led to the
assumption of two parallel transport mechanisms for Na, namely
electrogenic and electroneutral (7, 28). These mechanisms enable the
rumen epithelium to cope with the wide range of ruminal Na
concentrations between 21 mmol/l (31) and 145 mmol/l (2). At low Na
concentrations, Na is mainly transported via the electrogenic pathway,
whereas, at higher Na concentrations, the electroneutral Na/H exchange
mechanism is predominant (26). However, this extended knowledge of
ruminal Na transport does not explain a very old observation: an
increase of K intake and, consequently, of ruminal K concentration
enhances Na absorption from the rumen (38, 42); this causes a very
close and reciprocal relationship between ruminal Na and K
concentration, i.e., the concentration of Na is low at high K and vice
versa. Consequently, the sum of the Na and K concentrations in ruminal
fluid is kept almost constant (38). The physiological meaning of this
mechanism can readily be appreciated, because the K-dependent Na
absorption prevents an increase of osmotic pressure in the ruminal
fluid and hence a flow of water into the forestomachs when diets with a
high K content are consumed. The underlying mechanism of the
K-dependent Na transport is unknown. Stacy and Warner (42) have
suggested a stimulation of Na absorption by an increase of luminal
osmotic pressure. In a previous study, we tested the hypothesis that
the K-dependent Na transport is mediated by electroneutral Na-K-2Cl
cotransport, but mucosal addition of furosemide or bumetanide (1 mM),
which are potent inhibitors of Na-K-2Cl cotransport, does not change Na
fluxes in isolated epithelia of sheep rumen (28). Alternatively, a link
between the electrogenic Na transport and the ruminal K concentration appears to be contradictory and not in accord with the
electrophysiological consequences of high ruminal K concentrations. An
increase in ruminal K concentration depolarizes the apical membrane of
rumen epithelium (25) and hence reduces the driving force for apical Na
uptake. Furthermore, a positive correlation between the ruminal log of
the K concentration and the transepithelial potential difference (PDt, bloodside positive) has been
demonstrated (11); this would enhance passive (paracellular) backflow
of Na from the blood into the rumen. These effects would obviously
reduce net Na absorption, in contrast to the well-known experimental
observation of enhanced Na transport at high ruminal K.
A different explanation for these contradictions might be provided by
our unpublished observation that the rumen epithelium exhibits a
reversible increase in total tissue conductance
(Gt) at
increasing transmural PD (bloodside positive). If this change in
Gt is primarily
or solely located in the apical membrane, the following working
hypothesis could explain K-dependent Na transport: an increase in
ruminal K concentration depolarizes the apical membrane and increases
or induces a PD-dependent cation conductance, which enhances Na uptake
(despite a reduced electrical driving force) and finally increases
transepithelial Na transport via the basolateral Na-K-ATPase. The aim
of the present study was to test this hypothesis with the use of Ussing
chambers and microelectrode techniques.
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METHODS |
Animals.
The sheep used varied in breed, age, and sex. They had liberal access
to drinking water and hay. The animals were intended for human
consumption and killed at a local slaughterhouse.
Epithelia.
Immediately after slaughter, pieces of the ventral rumen sac were
excised. They were immersed in a transport buffer solution (maintained
at 38°C and gassed with carbogen) and stripped of the attached
muscle layers and the serosa.
Ussing chambers.
Pieces of epithelia were mounted between the halves of an Ussing
chamber with an exposed area of 0.95 cm2. Edge damage was minimized by
rings of silicon rubber between the chamber halves and the tissue.
Above each side of the tissue was a reservoir with 16 ml buffer
solution, maintained at 38°C and continuously stirred by a gas lift
system with oxygen.
For the experiments with microelectrodes, a modified Ussing chamber
with a tissue area of 0.79 cm2 was
used. The chamber had a volume of 0.6 ml on the mucosal side and 0.5 ml
on the serosal side. Both halves were perfused with oxygenated buffer
solution at a flow rate of 14 ml/min; the solution was heated to
38°C immediately before entering the chamber.
Microelectrodes.
Microelectrodes were pulled from filamented borosilicate glass and
filled with 0.5 M KCl, yielding resistances of 15-25 M
. Rumen
epithelial cells were impaled across the apical membrane with a
motorized micromanipulator with a piezo element. The PD of the apical
membrane (PDa) was measured with
reference to the mucosal solution.
PDa and
PDt were observed on an
oscilloscope. Impalements were accepted if
1) there was an abrupt fall in
PDa during advancing of the
microelectrode, 2)
PDa remained stable for at least 1 min, and 3)
PDa returned to 0 ± 3 mV on
withdrawal of the electrode.
Electrical measurements.
The preparations in the conventional Ussing chambers were connected to
a computer-controlled voltage-clamp device (AC-microclamp, Aachen,
Germany). The PDt was measured
through KCl (3 M) agar bridges near the tissue and calomel electrodes.
External current could be passed through the epithelium via another
pair of agar bridges. The
Isc and the
technical current
(It) needed to
clamp PDt to defined voltages were
recorded. The Gt
was determined from the change in
PDt caused by unidirectional
current pulses superimposed on
It. The pulse
duration was 500 ms, with the start of the recording after 250 ms. In
the microelectrode studies, transepithelial voltage pulses had an
amplitude of 10 mV and a duration of 160 ms. The pulse frequency was
0.5 Hz. Pulses were generated and measurements were performed with a
microelectrode amplifier and voltage-clamp device (Biomedical
Instruments, Munich, Germany). The fractional resistance of the apical
membrane (fRa)
was calculated from the pulse-induced changes in
PDa relative to the changes in
PDt
(fRa =
PDa/
PDt).
PDt,
Gt,
It,
PDa,
fRa, and the
electrode resistance were permanently displayed on a chart recorder and
stored on a PC.
Calculations.
The method for the calculation of membrane resistances described by
Frömter and Gebler (12) was modified. The voltage divider ratio
= Ra/Rb
(where Ra is
resistance of the apical membrane and
Rb is resistance
of the basolateral membrane) was calculated as
fRa/(1
fRa). It was
assumed that the use of Ca- and Mg-free solution on the mucosal side
altered only the
Ra membrane and did not influence the parallel shunt pathway. The parameters measured with no divalent cations in the mucosal solution are marked with an
asterisk. Ra,
Rb, and
resistance of the paracellular pathway (Rp) were
calculated as
These resistances are converted to conductances in
RESULTS.
Solutions.
The buffer solution used for the transport of the epithelia from the
slaughterhouse to the laboratory contained (in mmol/l) 1.2 CaCl2, 1.2 MgCl2, 2.4 Na2HPO4,
0.4 NaH2PO4,
25.0 NaHCO3, 5.0 KCl, 115.0 NaCl,
and 5.0 glucose. It was gassed with carbogen. The other solutions were
gassed with oxygen and buffered to pH 7.4 with Tris. Osmolarity was
adjusted to 300 mosmol/l with mannitol. The control solution contained
(in mmol/l) 2.0 K2HPO4,
1.0 KH2PO4, 10.0 glucose, 8.0 MOPS, 120.0 NaCl, 1.0 CaCl2, and 1.0 MgCl2.
In the Na-free solution, Na was replaced by
N-methyl-D-glucamine
(NMDG). The nominally Ca- and Mg-free solutions contained 1.0 mmol/l
EDTA and no added CaCl2 or
MgCl2.
Statistics.
Results are given as means ± SE or as single values;
n is the number of tissues in the
Ussing chamber studies or the number of impalements in the
microelectrode studies. Statistical comparisons were made by a paired
Student's t-test;
P values of <0.05 were considered significant.
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RESULTS |
Capacitive currents.
During the passage of short pulses across a tissue as complex as the
rumen epithelium, capacitance effects occur. To check the duration of
the capacitive currents and to choose a pulse duration long enough to
avoid artifacts in the calculation of Gt, we clamped
the epithelium to +40 mV with a rectangular voltage pulse and observed
It at a high time
resolution. Figure 1 shows an example of
such a recording. In Fig. 1B it is
evident that after 160 ms, the earliest time we used for the
calculation of Gt, capacitive
currents have disappeared.

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Fig. 1.
A: typical example of effect of a
rectangular voltage pulse hyperpolarizing rumen epithelium to +40 mV on
technical current
(It). Pulse
duration was 10 s, and sampling frequency was 100 Hz. After capacitive
currents at onset of pulse, there was no further change in
It.
B: part of
A at a higher time resolution. In our
experiments, determination of tissue conductance
(Gt) with short
pulses was performed 250 or 160 ms after onset of pulses. Figure
clearly shows that, after this time, capacitive currents have
disappeared.
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Voltage dependence of
Gt.
The PDt of the rumen epithelium
was linearly correlated with increasing log of the K concentration in
the ruminal fluid with a range of 20-60 mV (bloodside positive;
Ref. 11). The applied PDt included
this physiological range and was extended to negative values, from
80 mV to +80 mV. The
PDt-Gt
relationship is shown in Fig.
2A. Only
small alterations of
Gt were seen when
PDt was changed from
80 to
0 mV. In contrast,
Gt curvelinearly
increased from 2.50 ± 0.09 mS/cm2 under short-circuit
conditions (0 mV) to 3.68 ± 0.13 mS/cm2 at 80 mV
(P < 0.05). It should be noted that
alterations of Gt were pronounced at physiological (in vivo)
PDt (20-60 mV). The PD-induced variations of
Gt were
completely reversible and independent of the sequence of the applied
PDt (stepwise from minus to plus or alternating polarity pulses). The
PDt-It
relationship from the same tissues is given in Fig.
2B. The
It intercept at 0 PDt represents the
Isc (12.4 ± 1.27 µA/cm2). Despite the
noticeably enhanced
Gt at positive
PDt, the
PDt-It relationship deviated only slightly from linearity. To analyze this
discrepancy, we calculated a current
(Icalc), with
the clamped PDt and the measured
Gt (Fig.
2B). The difference
between Icalc and
It when
PDt = 0 mV represented the
Isc. Moreover, at
any other PDt, this difference
represented the current caused by active rheogenic ion transport
(Iact; Fig.
2B). Because
Icalc was always higher than the measured
It, active
electrogenic ion movement must have contributed to the
PDt; this consequently reduced the measured
It
at a given PDt.

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Fig. 2.
A: transepithelial potential
difference
(PDt)-Gt
relationship. Hyperpolarization of tissue strongly enhances
Gt;
n = 30. B:
PDt-It
relationships of same tissues as in A
are represented. With positive
PDt, relationship deviates
slightly from linearity but less than expected from
PDt-dependent increase in
Gt
(A). We calculated a current from
PDt and
Gt, namely
Icalc. Difference
between these two currents represents active rheogenic ion transport
(Iact). Under
short-circuit conditions, this current is by definition identical to
short-circuit current
(Isc). At
positive PDt, it significantly
increases. This may be explained by increased rheogenic Na transport.
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Na and PD-dependent changes of
Gt and
It.
Iact
significantly increased with positive
PDt. When
PDt = 80 mV,
Iact was 76.2 ± 3.1 µA/cm2. At negative
PDt up to
60 mV, there was
no significant difference between
Isc and
Iact. A possible
explanation for the difference between
Iact and
Isc could be an
electrogenic Na transport in the mucosal-serosal direction that is
stimulated at positive PDt. This
would be in agreement with the well-known in vivo observations and our
working hypothesis. To examine whether Na represents or significantly
contributes to this current, Na was replaced by NMDG on both sides of
the tissues, and the responses of
Gt and It were measured
upon the alteration of PDt. The
PDt-Gt
relationship is given in Fig.
3A. Na
replacement significantly reduced
Gt, from 2.49 ± 0.14 to 1.98 ± 0.09 mS/cm2, under short-circuit
conditions. A small PD-dependent increase of
Gt was still
present under Na-free conditions; at 80 mV, a Gt of 2.43 ± 0.10 mS/cm2 was obtained. This is
an increase of 24 ± 1.7%, which differs significantly from the
increase in percentage in the controls from the same animals (Fig.
3A).

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Fig. 3.
A:
PDt-Gt
relationship. After control experiment, same tissue samples were
incubated with Na-free solution. Under each voltage clamp condition,
Gt is
significantly smaller than that in control.
PDt-induced changes in
Gt are also less
distinct; n = 15. B:
Iact was
calculated under both control and Na-free conditions. Under Na-free
conditions in which PDt = 0 mV,
Iact = Isc and is almost
zero. Under control conditions, active rheogenic ion transport is
significantly more stimulated by a hyperpolarization of the epithelium
than under Na-free conditions.
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Replacement of Na abolished the
Isc almost
completely, from 13.5 ± 2.32 µA/cm2 (control) to 1.1 ± 1.00 µA/cm2. The most important
information deduced from this experiment was the calculation of
Iact (Fig.
3B). Under Na-free conditions, Iact increased to
31.0 ± 2.3 µA/cm2 at
PDt = 80 mV . This is 29.9 ± 2.6 µA/cm2 larger than
Isc. In controls
from the same animals, this difference is 75.7 ± µA/cm2, which is significantly
higher. The difference between treatment and control may indicate that
Na contributes to the enhanced
Iact at positive
PDt.
PDt and PDa.
Because the concurrent changes of
PDa and/or the PD of the
basolateral membrane (PDb) when
altering PDt were not known, these PD were measured under voltage clamp conditions of
PDt from
80 to +80 mV by
mucosal impalement with microelectrodes. The relationship between
PDt and
PDa is almost linear:
PDa = 0.66 PDt
47.7 mV (r2 = 0.92),
n = 15.
Voltage dependence of
fRa.
The working hypothesis in this study is the assumption that the
depolarization of PDa by high
ruminal K concentrations increases or induces a PD-dependent (cation)
conductance in the apical membrane; this may explain the increase in
Gt upon
hyperpolarization of PDt
(bloodside positive). If this assumption is correct, the measurement of
the fractional resistance,
fRa = Ra/(Ra + Rb), should
be sensitive upon manipulation of
PDa. The determination of these
parameters has been possible by microelectrodes for many years (12).
Figure 4 shows a representative mucosal
impalement of rumen epithelium. PDt,
Gt,
PDa, and
fRa were recorded
simultaneously. Alterations of PDt
caused the known effects on
Gt. A
hyperpolarization was accompanied by a decrease in
PDa and
fRa. All
PD-induced alterations were completely reversible. Figure
5 summarizes the relationship between
PDt,
Gt, and
fRa and clearly
shows the PD-dependent reciprocal relationship of
Gt and
fRa. An increase
in Gt is
accompanied by a decline in
fRa and vice
versa. It is worthwhile mentioning that fRa linearly
decreased within the physiological range of
PDt (20-60 mV). A very close
and linear correlation was obtained between PDa and
fRa (Fig.
6).

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Fig. 4.
Original tracing of an impalement under various voltage-clamped
conditions. With a hyperpolarization of the epithelium
(PDt positive),
Gt increases,
potential difference of apical membrane
(PDa) depolarizes, and
fractional resistance of the apical membrane
(fRa)
decreases. For technical reasons, no calculation of
Gt or
fRa could be
performed with first voltage pulse after changing
PDt. This is indicated by dotted
lines in traces of
Gt and
fRa.
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Fig. 5.
Gt and
fRa were recorded
simultaneously under various PDt
(n = 15). Every increase in
Gt is accompanied
by a fall in fRa
and vice versa.
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Fig. 6.
fRa becomes
significantly smaller when apical membrane is depolarized. Each symbol
stands for an individual recording (n = 15). Linear regressions have been fitted to the plots (solid lines).
Means ± SE of the regression equations are:
fRa(%) = ( 0.2 ± 0.0) · PDa(mV) + (57 ± 2); r2 = 0.93 ± 0.01.
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Calculation of the membrane conductances.
The preceding experiments demonstrated that the rumen epithelium
exhibited PD-dependent alterations of
Gt and
fRa, supporting the assumption of changes in the conductance(s) in the apical membrane.
Because fRa only
represents a quotient, information regarding the absolute changes is
still lacking. An analysis of epithelial resistances is possible when
only one resistance can be altered reversibly. In tissues with classic
electrogenic Na transport, this manipulation can easily be performed by
mucosal addition of amiloride, which is a potent and reversible
inhibitor of the Na channel (12). The electrogenic Na transport of the rumen epithelium is amiloride insensitive (30), but removal of the
divalent cations Ca and Mg from the mucosal side significantly enhances
Gt (and
Isc) of sheep
rumen epithelium (24). This reversible change of
Gt was therefore
used for the determination of resistances. Hence, we repeated the
microelectrode studies with or without divalent cations in the mucosal
solution at PDt of 0, 20, 40, 60, and 80 mV (negative PDt were
omitted because they caused a large and irreversible increase of
Gt upon removal
of Ca and Mg), and we recorded
Gt,
PDa, and
fRa from the same
impalement. The data thus obtained are summarized in Table
1. Removal of Ca and Mg from
the mucosal solution caused a significant increase in Gt and
Isc, depolarized
PDa, and reduced
fRa significantly
under short-circuit conditions. Furthermore, the known PD-dependent alterations of Gt
and fRa were also
observed in the absence of mucosal Ca and Mg. The calculation of the
individual resistances clearly showed that conductance of the apical
membrane (Ga)
was significantly enlarged from 1.34 ± 0.14 mS/cm2 under short-circuit
conditions to 2.60 ± 0.22 mS/cm2 at 80 mV
PDt (Fig.
7A).
Because conductance of the basolateral membrane
(Gb) and
conductance of the paracellular pathway
(Gp) (despite
some scatter of the data at 80 mV) remained unchanged (Fig. 7,
B and
C), the
PDt-dependent changes of
Gt must have been located in the apical membrane.

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Fig. 7.
A: conductance of the apical membrane
(Ga) was
calculated from data with Ca- and Mg-free solution on mucosal side of
epithelium and with control conditions
(n = 12) according to the method
described by Frömter and Gebler (12). There is a significant
potential dependence of
Ga; high
transepithelial PD leads to an increase in
Ga. Data from one
impalement are represented by the same symbol in
A, B,
and C.
B: no significant change in
conductance of the basolateral membrane
(Gb) resulting
from PDt changes can be detected.
C: conductance of the paracellular
pathway (Gp) is
unaffected by changes in PDt. At
higher PDt, data are not normally
distributed. Therefore, a Wilcoxon signed-rank test was performed. No
significant changes could be detected.
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DISCUSSION |
Previous studies have shown that an increase of apical K concentrations
elevates PDt (9, 11), depolarizes
PDa (25), and enhances Na
absorption from the rumen of sheep (8, 38). Because K-dependent
electroneutral Na transport has not been demonstrated in sheep rumen
(28), it has been hypothesised that the depolarization of
PDa activates a PD-dependent
cation conductance in the apical membrane; this mediates enhanced
apical Na uptake and finally Na transport across the rumen epithelium.
The results of the present study support this hypothesis.
PDt and
Gt.
The rumen epithelium clearly exhibits
PDt-dependent changes of
Gt; these are
completely reversible and independent of the sequence of applied
PDt (stepwise from minus to plus
or alternating polarity pulses). However,
PDt-dependent changes of
Gt are
complicated by possible capacitance effects when short pulses are used.
Sehested et al. (40) have determined, in studies with bovine rumen
epithelium, time-dependent changes of
PDt in response to a current. They
have found a monoexponential buildup of
PDt and a new steady state after
10 ms. This is in agreement with our own measurements (Fig. 1);
It reaches a new
steady state after some 50 ms. Because we use a time delay of 160 ms
(microelectrode) or 250 ms (Ussing chamber) for the calculation of
Gt, possible
capacitive effects are highly unlikely.
A further problem of the applied method could be the keratinized
multilayered structure of the rumen epithelium. The determination of
cellular electrophysiological parameters relies on the assumption that
the cells of the various layers are coupled and represent one
intracellular compartment. This assumption is probably true. Henrikson
(20) has described "complex intercellular channels" within the
rumen epithelium; these permit the diffusion of Na through the
different layers. Furthermore, we have not observed a change of
PDa after successful mucosal
impalement when the microelectrode is moved stepwise into deeper layers
of the epithelium.
Gt is the sum of
cellular conductances
(Gc) and of the
paracellular or shunt conductance
(Gp; Ref.
35), and consequently any change in
Gt could be
caused by alterations of
Gc and/or
Gp. The usual
Ussing chamber method does not discriminate between these alternatives.
Impalements of the epithelial cells with microelectrodes help to
localize alterations of
Gt to the apical
or the basolateral membrane and, if the resistance of one membrane can
reversibly be changed, aid the calculation of apical, basolateral, and
paracellular resistance. The
fRa decreases
significantly with increasing PDt and the simultaneous depolarization of
PDa. The decrease in
fRa is in keeping
with the determination of
Ga,
Gb, and
Gp by the use of
the reversible Ca-sensitive alteration of
Gt (and
Isc). The
response on removal of mucosal Ca and Mg is restricted to Ga, which is
enhanced from 1.40 mS/cm2 when
PDt = 20 mV to 2.53 mS/cm2 at 80 mV; this compensates
the diminution of electrochemical PD for Na
(
µNa) within this range of
PDt and permits enhanced Na uptake
across the apical membrane and transepithelial Na transport. Removal of
divalent cations from the mucosal solution causes increases in
Isc and
Gt in sheep rumen
epithelium and JNanet (
Isc =
JNanet; Ref. 25) and does
not change mannitol fluxes, indicating that the permeability of the
shunt pathway is not influenced. We have used this experimental design (± divalent cations) because the electrogenic Na transport in rumen
epithelium is not amiloride sensitive (30).
PDt and
Gt in other epithelia.
PDt-dependent changes of
Gt have been
observed in many epithelia (15, 18, 34, 43). In tissues with the
classic amiloride-sensitive Na transport, hyperpolarization (bloodside
positive) causes a decrease of apical conductance and cellular current
(33, 34) and an increase of transmural resistance (13). Consequently, Na transport is diminished with increasing hyperpolarization of the
tissue (5). Since single-channel recordings of amiloride-sensitive Na
channels of A6 cells exhibit near linearity in their current-voltage (I-V)
relationship over a range of ±80 mV (16), the decrease of Na
transport may be explained by the reduced driving force. This generally
accepted
I-V
relationship of amiloride-sensitive Na transport is in contrast to the
observation in the present study and suggests that the electrogenic Na
transport in sheep rumen, which is amiloride-insensitive (30) and
modulated by apical divalent cations (24), is a distinct Na pathway.
However, it should be mentioned that a voltage-sensitive (the open
probability increases with depolarization) amiloride-blockable Na
channel has been described in cultured human sweat duct cells (21) and in A6 cells (16). Recently, Marunaka et al. (32) have reported an
amiloride-sensitive Na-permeable nonselective cation channel in the
apical membrane of fetal alveolar epithelium with an increased open
probability when the apical membrane is depolarized.
Depolarization of the apical membrane potential results in a marked
decrease of fRa
in gallbladder epithelium (14, 15, 43). Indeed, depolarization of
PDa has disclosed the presence of
a voltage-dependent apical K conductance in these studies. Because the
rumen epithelium exhibits a K conductance in the apical membrane (25),
a possible contribution of this conductance to the PD-dependent change
of fRa cannot be
excluded. An increased open probability of the putative K channel on
depolarization would enhance K exit from the cell and transepithelial K
transport in the serosal-mucosal direction and would consequently
decrease or abolish
Iact, in contrast
to the obtained data. As mentioned above, the depolarization of
PDa increases cation absorption
and not secretion. However, uncertainties still remain. The K current of sheep rumen epithelium is very small (25) and might be outnumbered or probably overwhelmed by electrogenic Na transport. Garcia-Diaz et
al. (14), Stoddard and Reuss (43), and Gunter-Smith (15) have been able
to abolish the PD-dependent changes of
fRa in
gallbladder epithelium by mucosal addition of the K channel blocker
tetraethylammonium (TEA) or Ba. These inhibitors do not block the
apical K conductance of sheep rumen epithelium (TEA; Martens,
unpublished observations) or do so only to a small extent (Ba; Ref.
25). Present knowledge of ion transport in rumen epithelium supports
the assumption of a K (25) and a Na (7) conductance in the apical
membrane. The PD-dependent alteration of
fRa could hence
include the Na and/or the K conductance, or, alternatively, it could be
a conductance that is silent at normal
PDa and activated by
depolarization. These alternatives can only be distinguished by
single-channel studies.
Ruminal K, PDt, and
PDa.
Because PDt of the rumen
epithelium exhibits in vivo variations from 20 to 60 mV (bloodside
positive), the discussion here is restricted to this range of
PDt (11, 39). It is well known that the PDt of sheep rumen is
positively correlated with the rumen log of the K concentration (11,
39, 41). This correlation has been studied in more detail as it has
become evident that the absorption of Mg from the rumen, which is
essential for maintaining Mg homeostasis in ruminants (45), is
disturbed with increasing PDt (6,
27). Leonhard-Marek and Martens (25) have found that
PDa is depolarized and
PDt hyperpolarized with increasing apical K concentration. In their study,
PDa accounts for 0.6
PDt, which is in excellent
agreement with the present results and shows that the K-dependent
alterations of PDt,
PDa, and
Gt can easily be
simulated with a voltage clamp. The depolarization of
PDa reduces the driving force for
cation uptake and might be the predominant reason for decreased Mg
absorption upon high ruminal K (25). In contrast, Na transport is not
disturbed, despite the reduced
µNa, and can even be enhanced
under these conditions, because the reduced
µNa is compensated by an
increase of conductance of the Na pathway.
Another observation in keeping with a PD-dependent electrogenic Na
absorption is the difference between the current calculated from
PDt and
Gt,
Icalc, and the
current used by the voltage clamp at a given
PDt. At positive
PDt,
Icalc is
consistently higher than
It. This
difference between measured
It and calculated
current, Iact,
increases with increasing hyperpolarization of the tissue. This may be
caused by an increased electrogenic Na absorption. Because replacement
of Na significantly reduces the increase in Iact, in
agreement with the underlying working hypothesis, electrogenic Na
transport might represent the additional ion transport. Of course, we
cannot exclude possible contributions from other ions, as indicated by
the observation that even the total replacement of Na does not abolish
the increase in
Iact.
Ruminal Na transport.
It is well established that Na is absorbed from the rumen by active
transport (8). All in vitro studies have confirmed this conclusion and
have shown that
Isc is always
less than JNanet (7, 10, 28);
this has led to the assumption of two parallel Na transport mechanisms:
an electrogenic and an electroneutral Na transport (7). The
electroneutral transport is probably mediated by apical Na/H exchange,
since high mucosal concentrations of amiloride (1 mM) inhibit
JNanet significantly (28) and
other inhibitors of electroneutral Na transport (chlorothiazide, furosemide, or bumetanide) do not influence
JNanet (28). The lack of
effect of the loop diuretics furosemide and bumetanide make it very
unlikely that the K-dependent Na transport is represented by an
electroneutral Na transport.
The assumption of electrogenic Na transport in addition to
electroneutral transport relies on three observations.
Isc is abolished by Na replacement,
Isc is also
abolished by the serosal addition of ouabain (9), and
Isc equals
JNanet when Cl
and
HCO
3 are replaced by
SO2
4 and tricine (7). These
observations closely resemble results from studies with Na-transporting
tissues, such as amphibian skin (19) or rabbit distal colon (46).
However, significant differences are observed. Low mucosal
concentrations of amiloride, which effectively block electrogenic Na
transport in the tissues mentioned above, are without an effect on
Isc in rumen
epithelium (30). Mucosal removal of divalent cations enhances
Isc and
JNanet considerably (24),
indicating that the electrogenic Na transport in forestomach epithelia
exhibits properties distinct from the classic Na-absorbing tissues,
which is further substantiated by the data of the present study.
The proposed model of electrogenic Na transport, including a
PD-dependent conductance in the apical membrane, is complicated by the
supposition that an inflow of Na should depolarize
PDa with a consequential increase
in the PD-dependent conductance that would further enhance Na inflow
and hence the depolarization of
PDa. This positive feedback (and
vicious circle) of Na transport does not take place, as increasing Na
concentrations lead to the saturation of
Isc
[Michaelis-Menten constant
(Km) = 31.9 mM,
maximal transport capacity = 1.18 µeq · cm
2 · h
1;
Ref. 26]. It has been shown in several tissues that
PDa varies only within narrow
limits when the Na transport is altered (see review in Ref. 36). This
regulation of PDa seems to result
from a parallel change of Na transport rate and basolateral K
conductance (36). We have unsuccessfully attempted to alter
PDa of rumen epithelium by a rapid
change of mucosal Na concentrations during mucosal impalement with
microelectrodes. The changes in
PDa are very small or even absent,
a finding that may be explained by Ohm's law: the Na current of tens
of µA/cm2 passes through a
Ra of several
hundred
· cm2,
causing a voltage deflection of some millivolts. Moreover, the apical K
conductance could explain this observation: a possible depolarization
of PDa should enhance K efflux and
hence repolarize PDa. The apical K
conductance appears to accomplish two important functions that are
essential for the understanding of ruminal Na transport and that are
closely associated with PDa: at
low ruminal K concentrations, PDa
is stabilized, thus maintaining normal Na transport, whereas high
ruminal K concentrations depolarize PDa and activate the PD-dependent
conductance, which permits enhanced Na absorption.
The findings of the present study are in contrast to the early
observations of Stacy and Warner (42), who have suggested a stimulation
of Na transport by an increase of apical osmotic pressure, because
intraruminal application of KCl or a mixture of mannitol and urea
raises the osmotic pressure of ruminal fluid to ~400 mosmol/l and
enhances Na absorption from the rumen. The effect of KCl is much more
plausibly explained by the results of the present study in which we
assume a PD-dependent cation conductance. It is well known that urea is
very rapidly hydrolyzed by microbial urease to
NH+4, which causes K-like alteration of
PDt and
Isc (4).
Bödeker et al. (4) suggested that NH+4
passes through the K conductance in the apical membrane, which might
have similar effects on PDa and PD-dependent cation conductance. These conclusions are (indirectly) substantiated by recent studies in our laboratory showing that an
increase of apical osmotic pressure by mannitol decreases Na transport
(23).
Physiological implication of K-dependent Na transport.
One of the characteristics of digestive physiology in ruminants is the
high secretion rate of isotonic saliva: 10-20 l/day in sheep (22)
and 98-190 l/day in cows (1). The Na concentration in mixed saliva
is up to 168 mM and determines the Na concentration in the ruminal
fluid, which is some 18 mM lower than in mixed saliva (2).
Consequently, Na as the major cation in saliva and rumen content
predominantly influences the osmotic pressure of ruminal fluid, which
varies around isotonicity, being hypotonic (243-272 mosmol/l)
before and hypertonic (~400 mosmol/l) after a meal (48). K is the
most abundant mineral in plants and is rapidly dissolved in the
forestomach fluid (38). Indeed, a close linear correlation has been
observed between K intake and ruminal K concentrations (38). Because
the ruminal K exceeds 100 mosmol/l at high K intake, the sum of Na and
K should add up to 200-250 mosmol/l, resulting in high luminal
osmotic pressure and inflow of water into the rumen (49). This water
movement may impair the homeostasis of extracellular fluid and plasma
volume, because the secretion of saliva per se causes the withdrawal of
large amounts of electrolytes and water from the extracellular fluid volume, which is reduced after a meal (44) and which is accompanied by
a reduction of the plasma volume (3). These possible complications are
effectively prevented by the K-dependent Na absorption from the rumen
that keeps the sum of Na and K relatively low (some 140 mosmol/l) and
almost constant (38). Surprisingly, this important interaction between
K concentration and Na transport has rarely been considered in Na
transport studies. The results of the present study offer, for the
first time, a tentative model for this K-dependent Na absorption.
I-V
relationship in epithelia.
The epithelial and cellular effects of clamping the
PDt Na transport have been
investigated for many years (37), and it has been established that
epithelia exhibit electrical rectification and rarely behave as Ohm
resistors (13, 18). Since the perturbation of
PDt induces ion movement and,
consequently, changes of the driving forces for ions, two extreme
experimental conditions are usually used for the study of the
I-V
relationship: "instantaneous" and "steady state." (For a
careful discussion of these problems, see Ref. 37.) High ruminal K
concentrations cause the sustained (steady state) alteration of
cellular and transepithelial potential differences. We have simulated
this condition by choosing a pulse duration of 30 s in the Ussing
chamber studies. The obtained ratio of
PDa/
PDt
(0.6) is in excellent agreement with data from Leonhard-Marek and
Martens (25), who have induced electrophysiological alteration by
increasing apical K. Furthermore, the K-induced depolarization of
PDa and hyperpolarization of
PDt with the well-known effect on
ruminal Mg transport can be simulated by simple electrical manipulation
of the tissue (29). These observations support the conclusion that the
experimental conditions in the present study represent the K-dependent
electrophysiological alteration of ruminal epithelium.
The analysis of our data is based on the calculation of
Gt and
fRa from the
pulse-induced changes in PDt and
PDa. As we have shown,
Gt and
Ga are potential
dependent, a finding that implies a methodical problem: each pulse that
is generated by the voltage-clamp device changes
Gt, and hence all
data are subjected to an inevitable systematic error. However, these
deviations are much smaller than the observed effects caused by
depolarization or hyperpolarization. The same problems are present with
the calculation of
fRa as discussed by van Driesche and De Wolf (47).
In conclusion, it has been known for decades that an increase in
ruminal K concentration is accompanied by an enhanced Na absorption
from the rumen; this causes a decrease in Na concentration and
maintains the sum of Na and K concentrations in the ruminal fluid
almost constant (38). The data obtained in the present study support
the assumption of a tentative model of K-dependent Na transport in the
rumen epithelium. Depolarization of
PDa by high ruminal K
concentration increases a PD-dependent conductance, which permits an
enhanced apical Na uptake, despite the reduced driving force across
this membrane. Because the reverse observations have been made in
studies with classic amiloride-sensitive Na transport in frog skin (5,
33), the electrogenic Ca-sensitive amiloride-insensitive Na transport
in sheep rumen appears to be a distinct and novel mechanism of
epithelial Na movement, which, to our knowledge, has not been described
in other epithelial tissues.
 |
ACKNOWLEDGEMENTS |
We thank Dr. W. Nagel for critically reading the manuscript and for
methodical help and Dr. R. T. Jones for linguistic corrections.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: H. Martens, Institut f. Vet.-Physiologie,
FU-Berlin, Oertzenweg 19b, 14163 Berlin, Germany (E-mail:
martens{at}vetmed.fu-berlin.de).
Received 20 October 1998; accepted in final form 15 June 1999.
 |
REFERENCES |
1.
Bailey, C. B.
Saliva secretion and its relation to feeding in cattle. 3. The rate of secretion of mixed saliva in the cow during eating, with an estimate of the magnitude of the total daily secretion of mixed saliva.
Br. J. Nutr.
15:
443-451,
1961.
2.
Bailey, C. B.
Saliva secretion and its relation to feeding in cattle. 4. The relationship between the concentrations of sodium, potassium, chloride and inorganic phosphate in mixed saliva and rumen fluid.
Br. J. Nutr.
15:
489-498,
1961.
3.
Blair-West, J. R.,
and
A. H. Brook.
Circulatory changes and renin secretion in sheep in response to feeding.
J. Physiol. (Lond.)
204:
15-30,
1969[Medline].
4.
Bödeker, D.,
and
J. Kemkowski.
Participation of NH+4 in total ammonia absorption across the rumen epithelium of sheep (Ovis aries).
Comp. Biochem. Physiol. A Physiol.
114A:
305-310,
1996[Medline].
5.
Canessa, M.,
P. Labarca,
D. R. Dibona,
and
A. Leaf.
Energetics of sodium transport in toad urinary bladder.
Proc. Natl. Acad. Sci. USA
75:
4591-4595,
1978[Abstract].
6.
Care, A. D.,
R. C. Brown,
A. R. Farrar,
and
D. W. Pickard.
Magnesium absorption from the digestive tract of sheep.
Q. J. Exp. Physiol.
69:
577-587,
1984[Medline].
7.
Chien, W.-J.,
and
C. E. Stevens.
Coupled active transport of Na and Cl across forestomach epithelium.
Am. J. Physiol.
223:
997-1003,
1972[Medline].
8.
Dobson, A.
Active transport through the epithelium of the reticulo-rumen sac.
J. Physiol. (Lond.)
146:
235-251,
1959.
9.
Ferreira, H. G.,
F. A. Harrison,
and
R. D. Keynes.
The potential and the short circuit current across isolated rumen epithelium of the sheep.
J. Physiol. (Lond.)
187:
631-644,
1966.
10.
Ferreira, H. G.,
F. A. Harrison,
and
R. D. Keynes.
Studies with isolated rumen epithelium of sheep.
J. Physiol. (Lond.)
175:
28P,
1964.
11.
Ferreira, H. G.,
F. A. Harrison,
R. D. Keynes,
and
A. H. Nauss.
Observations on the potential across the rumen epithelium of the sheep.
J. Physiol. (Lond.)
187:
615-630,
1966.
12.
Frömter, E.,
and
B. Gebler.
Electrical properties of amphibian urinary epithelia. III. The cell membrane resistances and the effect of amiloride.
Pflügers Arch.
371:
99-108,
1977[Medline].
13.
Frömter, E.,
J. T. Higgins,
and
B. Gebler.
Electrical properties of amphibian urinary bladder epithelia. IV. The current-voltage relationship of the sodium channels in the apical cell membrane.
In: Ion Transport by Epithelia, edited by S. G. Schulz. New York: Raven, 1981, p. 31-45.
14.
Garcia-Diaz, J. F.,
W. Nagel,
and
A. Essig.
Voltage-dependent K conductance at the apical membrane of necturus gallbladder.
Biophys. J.
43:
269-278,
1983[Abstract].
15.
Gunter-Smith, P. J.
Apical membrane potassium conductance in guinea pig gallbladder epithelial cells.
Am. J. Physiol.
255 (Cell Physiol. 24):
C808-C815,
1988[Abstract/Free Full Text].
16.
Hamilton, K. L.,
and
D. C. Eaton.
Single-channel recordings from amiloride-sensitive epihelial sodium channel.
Am. J. Physiol.
249 (Cell Physiol. 18):
C200-C207,
1985[Abstract].
17.
Harrison, F. A.,
R. D. Keynes,
J. C. Rankin,
and
L. Zurich.
The effect of ouabain on ion transport across isolated sheep rumen epithelium.
J. Physiol. (Lond.)
249:
669-677,
1975[Abstract].
18.
Helman, S. I.
Electrical rectification of the sodium flux across the apical barrier of frog skin epithelium.
In: Ion Transport by Epithelia, edited by S. G. Schulz. New York: Raven, 1981, p. 15-28.
19.
Helman, S. I.,
W. Nagel,
and
R. S. Fisher.
Ouabain on active transepithelial sodium transport in frog skin.
J. Gen. Physiol.
74:
105-127,
1979[Abstract].
20.
Henrikson, R. C.
Ultrastructure of ovine ruminal epithelium and localization of sodium in the tissue.
J. Ultrastruct. Res.
30:
385-401,
1970[Medline].
21.
Joris, L.,
M. E. Krouse,
G. Hagiwara,
C. L. Bell,
and
J. J. Wine.
Patch-clamp study of cultured human sweat duct cells: amiloride-blockade Na+ channel.
Pflügers Arch.
414:
369-372,
1989[Medline].
22.
Kay, R. N. B.
The influence of saliva on digestion in ruminants.
World Rev. Nutr. Diet.
6:
292-325,
1966[Medline].
23.
Kurkowski, S.,
and
H. Martens.
Auswirkungen eines erhhten osmotischen Druckes auf die Gewebeleitfhigkeit und den Na-Transport des isolierten Pansenepithels von Schafen (Abstract).
Proc. Soc. Nutr. Physiol.
7:
67,
1998.
24.
Leonhard, S.,
E. Smith,
H. Martens,
G. Gäbel,
and
E. Ganzoni.
Transport of magnesium across an isolated preparation of sheep rumen: a comparison of MgCl2, Mg aspartate, Mg pidolate, and Mg-EDTA.
Magnesium Trace Elem.
9:
265-271,
1990[Medline].
25.
Leonhard-Marek, S.,
and
H. Martens.
Effects of potassium on magnesium transport across rumen epithelium.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G1034-G1038,
1996[Abstract/Free Full Text].
26.
Martens, H.
Saturation kinetics of electrogenic and electroneutral Na transport of sheep rumen epithelium.
Proc. Soc. Nutr. Physiol.
3:
87,
1994.
27.
Martens, H.,
and
I. Blume.
Effect of intraruminal sodium and potassium concentrations and of the transmural potential difference on magnesium absorption from the temporarily isolated rumen of sheep.
Q. J. Exp. Physiol.
71:
409-415,
1986[Medline].
28.
Martens, H.,
G. Gäbel,
and
B. Strozyk.
Mechanism of electrically silent Na and Cl transport across the rumen epithelium of sheep.
Exp. Physiol.
76:
103-114,
1991[Abstract].
29.
Martens, H.,
G. Gäbel,
and
H. Strozyk.
The effect of potassium and the transmural potential difference on magnesium transport across an isolated preparation of sheep rumen epithelium.
Q. J. Exp. Physiol.
72:
181-188,
1987[Medline].
30.
Martens, H.,
M. Henseleit,
and
G. Gäbel.
Electrogenic, amiloride-insensitive Na-transport by sheep rumen epithelium is inhibited by serosal theophylline (Abstract).
Pflügers Arch.
431:
R4,
1989.
31.
Martens, H.,
O. W. Kubel,
G. Gäbel,
and
H. Honig.
Effect of low sodium intake on magnesium metabolism of sheep.
J. Agric. Sci.
108:
237-243,
1987.
32.
Marunaka, Y.,
N. Niisato,
H. O'Brodovich,
and
D. C. Eaton.
Regulation of an amiloride-sensitive Na+-permeable channel by a
2-adrenergic agonist, cytosolic Ca2+ and Cl
in fetal rat alveolar epithelium.
J. Physiol. (Lond.)
515:
669-683,
1999[Abstract/Free Full Text].
33.
Nagel, W.,
and
A. Essig.
Relationship of transepithelial electrical potential to membrane potentials and conductance ratios in frog skin.
J. Membr. Biol.
69:
125-136,
1982[Medline].
34.
Nagel, W.,
J. F. Garcia-Diaz,
and
A. Essig.
Voltage dependence of cellular current and conductances in frog skin.
J. Membr. Biol.
106:
13-28,
1988[Medline].
35.
Powell, D. W.
Barrier function of epithelia.
Am. J. Physiol.
241 (Gastrointest. Liver Physiol. 4):
G275-G288,
1981[Abstract/Free Full Text].
36.
Schultz, S. G.
Homocellular regulatory mechansims in sodium-transporting epithelia: avoidance of extinction by "flush-through."
Am. J. Physiol.
241 (Renal Fluid Electrolyte Physiol. 10):
F579-F590,
1981[Abstract/Free Full Text].
37.
Schultz, S. G.,
S. M. Thompson,
and
Y. Suzuki.
Equivalent electrical circuit models and the study of Na transport across epithelia: nonsteady-state current-voltage relations.
Federation Proc.
40:
2443-2449,
1981[Medline].
38.
Scott, D.
The effect of potassium supplements upon the absorption of potassium and sodium from the sheep rumen.
Q. J. Exp. Physiol.
51:
60-69,
1967.
39.
Scott, D.
The effects of sodium depletion and potassium supplements upon electrical potentials in the rumen of sheep.
Q. J. Exp. Physiol.
51:
60-69,
1966.
40.
Sehested, J.,
L. Diernaes,
G. Laverty,
P. D. Møller,
and
E. Skadhauge.
Methodological and functional aspects of the isolated bovine rumen epithelium in Ussing chamber flux studies.
Acta Agric. Scand. Sect. A Anim. Sci.
46:
76-86,
1996.
41.
Sellers, A. F.,
and
A. Dobson.
Studies on reticulo-rumen sodium and potassium concentration and electrical potentials in sheep.
Res. Vet. Sci.
1:
95-102,
1960.
42.
Stacy, B. D.,
and
A. C. I. Warner.
Balances of water and sodium in the rumen during feeding: osmotic stimulation of sodium absorption in the sheep.
Q. J. Exp. Physiol.
51:
79-93,
1966.
43.
Stoddard, J. S.,
and
L. Reuss.
Voltage-and time dependence of apical membrane conductance during current clamp in Necturus gallbladder epithelium.
J. Membr. Biol.
103:
191-204,
1988[Medline].
44.
Ternouth, J. H.
Changes in the thiosulphate space and some constituents of the blood of sheep after feeding.
Res. Vet. Sci.
9:
345-349,
1968[Medline].
45.
Tomas, F. M.,
and
B. J. Potter.
The site of magnesium absorption from the ruminant stomach.
Br. J. Nutr.
36:
37-45,
1976[Medline].
46.
Turnheim, K.,
R. A. Frizzell,
and
S. G. Schultz.
Interaction between cell sodium and the amiloride-sensitive sodium entry step in rabbit colon.
J. Membr. Biol.
39:
233-256,
1978[Medline].
47.
Van Driesche, W.,
and
I. De Wolf.
Microelectrode study of apical K channels in the skin of Rana temporaria.
Pflügers Arch.
418:
400-407,
1991[Medline].
48.
Warner, A. C. I.,
and
B. D. Stacy.
Solutes in the rumen of sheep.
Q. J. Exp. Physiol.
50:
169-184,
1965.
49.
Zhao, G. Y.,
M. Duric,
N. A. McLeod,
E. R. Ørskov,
F. D. Hovell,
and
Y. L. Feng.
The use of intragastric nutrition to study saliva secretion and the relationship between rumen osmotic pressure and water transport.
Br. J. Nutr.
73:
155-161,
1995[Medline].
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