1 Department of Physiology, Na transport
across a preparation of sheep omasum was studied. All tissues exhibited
a serosa-positive short-circuit current (Isc), with a
range of 1-4
µeq · h
calcium-sensitive sodium transport; nonselective cation channel; Ussing chamber; microelectrodes; apical conductance
THE OMASUM, THE THIRD PART of the forestomach in
ruminants, is an important site of water and electrolyte absorption in
the proximal part of the digestive tract (10, 11). A positive and
linear relation has been observed between amounts of water and Na
flowing into the omasum and absorbed from it: 40-60% in calves
(10) and 10-20% in sheep and goats (11). Despite this well-established knowledge, very little is known about the possible mechanism(s) of epithelial Na transport in the omasum. Harrison et al.
(24) observed a net Na transport in the mucosal-to-serosal direction
and a positive short-circuit current
(Isc) in in
vitro studies with an isolated preparation of sheep omasum. Martens and
Gäbel (41) confirmed these results and concluded from ion exchange studies and the effects of various inhibitors of Na transport mechanisms that Na is predominantly transported by an electroneutral Na/H exchange. High mucosal concentrations of amiloride (1 mM) significantly reduced net Na flux without any effect on
Isc. Serosal addition of ouabain (0.1 mM) or replacement of Na abolished the Isc. This
probably indicates that the
Isc represents an
electrogenic Na transport that is working in parallel with the proposed
Na/H exchange (41).
Since the early publications of Koefoed-Johnsen and Ussing (30), an
overwhelming number of studies on electrogenic Na transport have been
published for a variety of tissues and species (20, 21, 51). There is
little doubt that the electrogenic Na transport might be one of the
best-studied transport systems in epithelia. Conducting properties
(44), regulation by hormones (63), molecular biology (6), and
inhibition by amiloride, which is the most common marker and potent
inhibitor of this transport mechanism (5), have all been studied.
However, there is growing evidence that electrogenic Na transport
mechanisms may exist that are only to some extent amiloride sensitive
[in human colon in vitro (47), leech skin (64), alveolar type II
cells (43), and nasal airway epithelium of rabbit (45)] or
totally insensitive against amiloride [in rabbit cecum (7, 25,
48, 49), colon of Xenopus laevis (32), and rumen epithelium of sheep (42)]. Furthermore, it has been shown that removal of divalent cations from the mucosal solution enhances Isc and
Na transport in various amphibian epithelia (32, 61), rumen epithelium
of sheep (36), coprodeum of chicken embryo (26), and rabbit cecum (48),
which probably represents a nonselective cation conductance or channel (NSCC).
At the present time, the available data are very limited regarding the
supposed electrogenic Na transport in sheep omasal epithelium and do
not allow any integration within the current concepts of electrogenic
Na transport mechanisms. Therefore, this study aimed to characterize
the assumed electrogenic Na transport in sheep omasum using Ussing
chamber and microelectrode techniques. Our results show that the
electrogenic Na transport is not sensitive to mucosal amiloride (1 mM)
and is not stimulated by serosal addition of aldosterone (1 µM).
Removal of mucosal divalent cations (Ca and Mg) considerably enhances
Isc and net Na
flux and decreases the voltage divider ratio ( Preparation of the Tissue
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 · cm
2.
A Michaelis-Menten-type kinetic was found between the Na concentration and the Isc
(Michaelis-Menten constant for transport of Na = 6.7 mM; maximal
transport capacity of Na = 4.16 µeq · h
1 · cm
2).
Mucosal amiloride (1 mM), phenamil (1 or 10 µ), or serosal aldosterone (1 µM for 6 h) did not change
Isc. Removal of
divalent cations (Ca and Mg) enhanced
Isc considerably
from 2.61 ± 0.24 to a peak value of 11.18 ± 1.1 µeq · h
1 · cm
2.
The peak Isc
(overshoot) immediately declined to a plateau
Isc of
~6-7
µeq · h
1 · cm
2.
Na flux measurements showed a close correlation between changes in
Isc and Na
transport. Transepithelial studies demonstrated that K, Cs, Rb, and Li
are transported, indicating putative nonselective cation channels,
which are inhibited by divalent cations (including Ca, Mg, Sr, Ba) and
by (trivalent) La. Intracellular microelectrode recordings from the
luminal side clearly showed changes of voltage divider ratio when
mucosal divalent cations were removed. The obtained data support the
assumption of a distinct electrogenic Na transport mechanism in sheep omasum.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
), which supports the
assumption of a NSCC in the luminal membrane. The determinations of
apical and basolateral membrane and paracellular resistances reveal
that the omasal epithelium of sheep is moderately tight and resembles rabbit cecum.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Incubation
Pieces were mounted between two halves of Ussing chambers with an exposed area of 0.95 cm2. Edge damage was minimized by rings of silicon rubber on both sides of the tissue. The mounted epithelium was bathed on both sides of the epithelium with 16 ml of buffer solution using a gas lift system.For studies performed with microelectrodes, the tissue was horizontally mounted in a modified chamber with an opening of 0.79 cm2. For microelectrode studies, the chamber had a volume of 0.7 ml on the mucosal and of 0.4 ml on the serosal side. Both chamber halves were perfused with a solution from gassed reservoirs, and the solution was heated to 38°C immediately before it entered the chamber.
The time from killing the sheep to mounting the epithelium was
30-40 min, and a further period of 30 min was allowed for
equilibration. Pieces of epithelia were rejected if the
Isc was <1
µeq · h1 · cm
2
or the conductance was >8 mS/cm2
at the end of the equilibration period.
Electrical Measurements
Ussing chambers. Electrical measurements were continuously obtained with a computer-controlled voltage-clamp device (Mussler, Aachen, Germany): KCl-agar bridges were positioned near each surface of the tissue and connected to Ag-AgCl electrodes for the measurement of the transepithelial potential difference (PDt). The same type of bridges, with a larger diameter, were inserted ~2 cm from the surface for the application of current. The tissues were incubated under short-circuit conditions. The displacements in the potential difference caused by bipolar pulses of 100 µA and 1-s duration were measured; from the change in PDt and pulse amplitude, Gt was calculated. All parameters, i.e., Isc, PDt and Gt, were printed out in regular intervals and were corrected for fluid resistance and junction potentials.
The electromotive force (ENa) was calculated using the method described by Yonath and Civan (68).Variations in transmural potential difference. Unidirectional fluxes measured at different potential differences were divided into their potential-dependent and potential-independent components using a mathematical model, which was described by Frizzell and Schultz (16)
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Microelectrode measurements.
For microelectrode experiments, a similar computer-controlled
voltage-clamp system (Biomedical Instruments, Germering, Germany) was
employed. Microelectrodes were pulled from borosilicate glass (Science
Products, Frankfurt am Main, Germany) and filled with 0.5 M KCl. The
tip resistance was 10-30 M in standard buffer solution.
Impalements were performed under open-circuit conditions across the
apical membrane using an electric microdrive (Science Products).
Microelectrode potentials were referenced to the mucosal solution and
measured using high-impedance electrometers (Biomedical Instruments).
The total tissue resistance, the microelectrode resistance
(Rel), the
apical potential difference
(PDa), and
PDt were continuously recorded on
a chart recorder (BBC Metrowatt, Nuremberg, Germany).
PDt and
PDa were continuously displayed on an oscilloscope (Gould, Dietzenbach, Germany). Impalements were accepted if the change in PDa was
abrupt, PDa was stable for >1 min, Rel during
and after the withdrawal of the microelectrode was not below 10 M
,
Rel did not
change ±15% during the measurement and after the withdrawal of the
microelectrode, and the change in tip potential did not exceed ±3
mV. The individual membrane resistances were calculated using the
method of Frömter and Gebler (17) with the
= apical
resistance/basolateral resistance
(Ra/Rb) =
Va/
Vb.
[Note that the voltage deflection in the potential difference
across the epithelium
(VT) and the
apical membrane (Va) was
directly observed, whereas that for the change in the basolateral
membrane
(
Vb) was
calculated using the formula
Vb =
VT
Va.]
Flux Studies
22Na and [14C]mannitol (Amersham Buchler, Braunschweig, Germany) were added to one side of the epithelium to yield a specific activity of 55 kBq/M and 50 kBq/M, respectively, in the buffer solution. After an equilibrium period of 30 min, aliquots were sampled at 30-min intervals and replaced by aliquots of unlabeled solution. [14C]mannitol was counted in a beta-scintillation counter (Packard) and 22Na in a well-type crystal gamma-counter (Berthold). Unidirectional fluxes were calculated from the rate of tracer appearance on the other side of the tissue. Paired determinations of fluxes were accepted if tissue conductances differed <20%.Solution, Drugs, and Chemicals
All chemicals were of analytic grade. The serosal and mucosal standard buffer solution contained (in mM) 115 Na, 119 Cl, 5 K, 1 HPO4, 2 H2PO4, 1 Ca, 1 Mg, 8 MOPS, 10 Tris, 10 glucose, and 47 mannitol, which was gassed with 100% O2. In ion exchange studies, all Na was replaced by Rb, Cs, Li, K, or N-methyl-D-glucamine (NMDG), respectively. For transport and preparation of the tissue, the buffer solution contained (in mM) 141 Na, 85 Cl, 5 K, 25 HCO3, 1 HPO4, 2 H2PO4, 1 Ca, 1 Mg, 13 acetate, 13 propionate, 13 butyrate, and 10 glucose, gassed with 95% O2 and 5% CO2. The osmolarity and the pH of all solutions were adjusted to 290 ± 10 mosM and 7.4 ± 0.1, respectively. Amiloride, aldosterone, theophylline, and calcimycin (A-23187, Ca ionophore) were purchased from Sigma Chemical (Munich, Germany). Radioisotopes (22Na and [14C]mannitol) were obtained from Amersham Buchler. The free Ca concentration was calculated by means of a computer program (46).Statistics
Results are given as means ± SE. N is the number of animals, and n is the number of tissues mounted. The results were tested for significance using the Wilcoxon-Mann-Whitney U test or, if called for, the Wilcoxon's matched-pairs test. Values of P < 0.05 were considered significantly different. ![]() |
RESULTS |
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General Properties
All mounted omasal tissues exhibited under control conditions a serosa-positive Isc (range of 1-4 µeq · h
|
Mucosal Replacement of Divalent Cations (Ca and Mg)
Figure 2 illustrates the time course of Isc under control conditions and after the mucosal solution (nominally Ca and Mg free) was changed. Removal of Ca and Mg caused an increase of Isc (from 2.61 ± 0.24 to 11.18 ± 1.1 µeq · h
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An evident reversibility and reproduction of changes in
Isc (and
Gt) were
observed when divalent ions were frequently removed from or added to
the mucosal solution during the plateau phase (Fig.
4B). In
contrast, the (first) addition of Ca to the mucosal compartment at the
peak Isc caused a
very different pattern of inhibition or activation of
Isc (Fig.
4A). The magnitude of the first peak
Isc was not
reached again, and a plateau
Isc was not obtained. Figure 4A shows that, due to
removal and administration of Ca, only the time course of the
downregulation was prolonged, without an influence on the decline in
Isc.
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Feedback Inhibition of Ca-Sensitive Current
The decline of Isc from the peak value clearly indicated that autoregulatory mechanisms exist to regulate Na uptake to the activity of the Na-K-ATPase in the basolateral membrane, in order to maintain intracellular ionic composition and cell volume. It is generally accepted that intracellular Ca modulates Na permeability of the apical membrane and that the Ca concentration in the cytosol is kept very low by a Na/Ca exchange (3Na/1Ca) in the basolateral membrane (65). This countertransport mechanism mediates the extrusion of Ca from the cell against a very steep electrochemical gradient. It is energized by the electrochemical gradient of Na (65), which can therefore be easily modulated by variation of Na concentrations in the serosal solution. A reduction of the serosal Na concentration, i.e., from 115 to 10 mM, leads to a minimal decrease of Isc (see Fig. 5A). The removal of divalent cations from the mucosal solution caused in both groups the known increase of Isc. However, the response in current (peak and plateau) was significantly (P < 0.001) lower in tissues with 10 mM Na in the serosal solution (Fig. 5A). The Ca-sensitive current was rapidly blocked by mucosal addition of Ca, and again no significant differences from baseline Isc were observed (Fig. 5A). The possible role of intracellular Ca was tested in a second approach in which the equilibrium position of the Na/Ca exchanger was altered by an increase of the extracellular Ca concentration. Tissues were incubated under control conditions, and the Ca concentrations were changed on both sides of the tissues after equilibration. An increase of Ca from 1 to 4 mM did not change Isc (Fig. 5B). Lowering Ca from 1 to 0.5 mM caused a slight (~10%) but insignificant increase in Isc (Fig. 5B). The activation of Ca-sensitive Isc by mucosal removal of divalent cations caused in both groups a significant increase in Isc (P < 0.005). However, the response in Isc (peak and plateau phase) was significantly lower in tissues incubated with 4 mM Ca on both sides of the tissue (Fig. 5B).
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Isc and Na Fluxes
The parallel changes of Isc and Gt, after mucosal removal of divalent ions, suggest an activation of a Ca-sensitive electrogenic ion transport. Transport rates of Na were compared under nominally Ca- and Mg-free conditions (mucosal) during the plateau phase and after mucosal addition of Ca in the same tissue (sequential design). The removal of divalent cations caused the known alteration of Isc and Gt. The Na flux rates during the plateau phase are summarized in Table 1. It is important to note that the changes of mucosal-to-serosal flux (Jm
|
Na Fluxes and Variation of Transmural Potential Difference
A putative model of electrogenic Na transport includes the luminal uptake through a cation conductance and the basolateral extrusion via the Na-K-ATPase. The uptake depends on the chemical and electrical gradients across the luminal membrane. Alterations in PDt result in a variation of the electrical gradient across the luminal membrane, therefore changing electrogenic Na fluxes. Figure 6 illustrates the effects of variations of PDt on fluxes of Na under control and divalent cation-deprived conditions. Jm
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Gt and Mannitol Fluxes
The tissue conductance measured with the Ussing chamber technique represents the sum of conductances of epithelial cells (Gc) and of the shunt pathway (Gs). The removal of divalent cations caused a significant increase of Gt. This was closely correlated with the altered Isc, which suggests that primarily Gc has been changed. Because divalent cations, such as Ca, have been reported to modulate tight junctions (22), a possible effect on Gs cannot be excluded. To distinguish between these two possibilities, measurements of unidirectional fluxes of mannitol were performed. Mannitol is poorly absorbed by cells, and transport across the epithelium takes place between the cells. The known alteration of Isc and Gt after removal of divalent cations from the mucosal solution did not influence mannitol transport, indicating that Gs did not change (Table 2).
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Inhibition by Theophylline
Theophylline (10 mM on both sides of the epithelium) significantly reduced the Isc within 10-20 min by ~30% (Fig. 7A, P < 0.01, Wilcoxon's matched-pairs test) and Gt by 10-15%. When the Isc exhibited a new steady state (30 min), Ca and Mg were removed from the mucosal solution. Figure 7A shows that the peak Isc was significantly (P < 0.01) lower in tissues treated with theophylline and that the downregulation of current was much more pronounced. Addition of Ca to the mucosal side caused the known decrease of current. Again, significant differences (P < 0.01) were observed between control results and results from theophylline-treated tissues.
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Serosal Aldosterone
Aldosterone is the major mineralocorticosteroid regulating Na homeostasis in vertebrates and elevates transepithelial Na transport (63). Because possible modulations of Na transport across forestomach epithelia by this corticosteroid have not been reported, the effect of serosal addition of aldosterone (1 µM) was tested. The tissues were mounted in Ussing chambers, and, after equilibration, the Ca-sensitive Isc was activated by mucosal removal of divalent cations. Ca was added to the mucosal side at peak Isc (see Fig. 7B), and aldosterone (1 µM) was added to the serosal side when the Isc had declined to baseline values (Fig. 7B). Aldosterone influenced neither the Isc under control conditions nor the Ca-sensitive current after 6 h of incubation. It is interesting to note that the Isc commenced along a time-dependent decay after 3 h of incubation, with a pronounced decrease on the magnitude of the Ca-sensitive current after 6 h (Fig. 7B).Ion Substitution
It has been shown in previous studies (61) that divalent cation-free mucosal solutions activate NSCCs. We totally replaced Na on the mucosal side and examined Li, K, Rb, and Cs with normal buffer solution in the serosal compartment. All tested monovalent cations demonstrated a Ca-sensitive current (Table 3), which clearly indicated that a nonselective conductance was activated.
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Effects of Divalent Cations
The normal buffer solution contained 1 mM Ca and 1 mM Mg. Activation of the Ca-sensitive Isc was achieved by mucosal replacement of this buffer with a nominally Ca- and Mg-free solution and addition of 1 mM EGTA for chelating traces of divalent cations. The activated current was reversibly and completely inhibited by repeated mucosal supplementation of 2 mM Ca (see Fig. 4). Higher Ca concentrations did not cause a further decrease in Isc. Other polyvalent cations have been shown to block Ca-sensitive currents as well as Ca (48, 61). We tested the effects of 2 mM Ba, Mg, Sr, and La during the plateau phase in a random manner. All cations significantly (P < 0.05) reduced the Ca-sensitive current, and a new steady-state Isc was observed within 10-20 min. However, the decrease of current varied in dependence on the divalent cation applied. None of them blocked all the Ca-sensitive current. Mucosal addition of Ca (1 mM) during the new steady-state Isc (after mucosal addition of 2 mM Ba, Mg, Sr, or La) further reduced the current. A typical time course of an experiment with different divalent cations is shown in Fig. 8A. The fraction of Ca-sensitive Isc inhibited by other divalent cations (2 mM) increased from 48% for Mg to 59% for Sr and La and to 76% for Ba. Consequently, higher concentrations of divalent cations were used. Because La and Ba appear to precipitate at higher concentrations, only Mg and Sr were applied. The inhibition of Ca-sensitive Isc by Mg and Sr was concentration dependent, but, even at 3.6 mM Mg, <50% of the current was blocked (Fig. 8B). Sr was more effective at this high concentration (>95% blocked). Further administration of 1 mM Ca to Mg and Sr decreased the Isc significantly (Fig. 8B; with Mg, P < 0.001; with Sr, P < 0.01).
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Mucosal Ca and Isc
Previous studies in epithelia have clearly demonstrated that Ca inhibited ion transport through NSCC (48, 61). Increasing Ca concentrations in the mucosal solution decreased the Ca-sensitive current in a dose-dependent manner (Fig. 9) with a Michaelis-Menten-type kinetic. The best approximation was obtained by using a double Michaelis-Menten model (Michaelis-Menten constant values of 92 ± 24 µM and 0.4 ± 0.05 µM Ca for the low- and high-affinity binding sites, respectively; Fig. 9, r2 = 0.997), indicating two different binding positions for Ca.
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Mucosal Amiloride and Phenamil
Amiloride is a potent blocker of the classic Na channel, and this diuretic drug is considered a marker for the electrogenic Na transport (5). We determined previously (41) and in the present study that mucosal amiloride (1 mM) did not change Isc under control conditions. Because a possible influence of amiloride on Ca-sensitive current was not known, we tested mucosal amiloride during the plateau phase. The data are summarized in Table 4. Increasing concentrations of amiloride from 1 to 100 µM did not change the Ca-sensitive Isc. Phenamil (1 and 10 µM) in the mucosal solution did not change Isc or Gt with or without divalent cations in the mucosal solution (data not shown). A 100 µM concentration of phenamil slightly decreased Isc from 1.97 ± 0.24 to 1.74 ± 0.22 µeq · cm
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Studies with Microelectrodes
Mucosal Ca- and Mg-free solutions increased the Gt. The studies with mannitol and the potential-dependent Js
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The reversible perturbation of the apical resistance encouraged us to
attempt the calculation of individual membrane resistances using the
method of Frömter and Gebler (17). The mean values are given in
Table 5. It is worth noting that Ca- and Mg-free solutions reduced
Ra from 2,024 ± 43 to 665 ± 23 · cm2, which
accounts for the change in transepithelial resistance. Consequently,
decreased from 10.1 ± 0.3 to 3.3 ± 0.1 due to the removal
of divalent cations.
Estimation of ENa
Yonath and Civan (68) described a method for estimating ENa in toad bladder. The underlying assumptions of this model include a decrease of resistance to Na entry into the transporting cell of the epithelium and, consequently, proportional changes in Isc and Gt. The ratio of these changes, i.e., in Isc and Gt, allows the calculation of ENa. The conditions of this model were obviously given by our results. The proposed plot of Yonath and Civan (68) between changes in Isc and Gt after mucosal removal of divalent cations exhibited a linear and close relationship (r = 0.99), with y = 1.9 ± 0.016x. The slope of this linear regression was 1/ENa, i.e., 0.016 or 62 ± 1 mV. ![]() |
DISCUSSION |
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The results of this study include findings that both resemble the classical electrogenic Na transport and exhibit similarities to NSCC. Consequently, the discussion will first deal with the classical Na transport and then focus on NSCC.
Electrogenic Na Transport
The omasal epithelium of sheep exhibits a positive Isc, which was closely correlated with the presence of Na in the buffer solution or with the undisturbed activity of the Na-K-ATPase in the basolateral membrane (24, 41). Furthermore, the Isc increased with increasing Na concentrations and followed Michaelis-Menten-type kinetics under control conditions. This saturation of Na transport very likely arises from a decline in luminal membrane permeability with increasing Na concentrations, as has been reported in studies on other Na-transporting epithelia (Refs. 7, 18 and see review in Ref. 51). The origin of the reduced luminal membrane permeability with increasing Na concentrations remains controversial: "self-inhibition" from the mucosal side (18), saturation of the single-channel conductance (31), or "feedback inhibition" by cytosolic factors (15, 39) may be involved. With the methods used in this study, we are not able to distinguish between the different alternatives of saturation.A further observation is in agreement with the classical Na transport
(40, 52). A sudden increase of Na transport in these tissues caused an
overshoot of Isc,
followed by an exponential decline and new steady state. The overshoot
Isc in omasal
tissue, which is several times higher than that in frog skin or rabbit colon, is downregulated to an almost constant
Isc of 5-7
µeq · h1 · cm
2,
which is in the range of the maximal transport rate in rabbit colon
(4-6
µeq · h
1 · cm
2)
(52). The enhanced inflow of Na into the epithelial cells of sheep
omasum is obviously essential for the induction of downregulation. Repeated addition of Ca to the mucosal compartment at the peak Isc interrupted
the possible responses in the cytosol and prevented a new steady-state
Isc (Fig.
4A). The suggested cytosolic factors of feedback inhibition are not known; however, it is very likely that
this effect is Ca mediated. In two experiments, the equilibrium position of the Na/Ca exchanger was altered. The assumed increase in
intracellular Ca decreased the
Isc (peak and
plateau; see Fig. 5B). Studies with
cortical collecting tubule (14) or A6 (39) cells have shown that Ca
indirectly mediated Na channel activity via activation of protein
kinase C (39) or
Ca2+/calmodulin-dependent protein
kinase II (14). Ismailov et al. (29) demonstrated a direct modulation
of bovine renal Na channels by Ca. The suggestion of a Ca-mediated
regulation of Na uptake in sheep omasum is speculative and equivocal
without the determination of intracellular Ca and without exclusion of
other Ca-regulating mechanisms. This tentative discussion of possible
explanations should be considered as a suggestion for further studies
with sheep omasum, but this was beyond the scope of the present study.
Saturation and overshoot results agree with studies of the classical Na
transport mechanism (20, 21, 30, 51). However, a growing number of
observations support the assumption of distinct Na transport pathways.
One important difference is the missing effect of amiloride and of the
amiloride analog phenamil (1 and 10 µM) on
Isc. A
concentration of 100 µM phenamil significantly reduced
Isc. However,
this effect appeared to be atypical for a block of a putative cation
channel because
Gt showed a
tendency to increase. Similar inconsistent observations have been made by Sellin and Dubinsky (48) and Sellin et al. (49). Amiloride is a
potent blocker of Na channels
(5)1.
The inefficacy of amiloride (with or without divalent cations) may
indicate that the putative Na conductance in sheep omasum is not
sensitive against this drug or, alternatively, has lost binding
properties. This suggestion has been discussed by Sellin and Dubinsky
(48) in conjunction with findings of Lewis and Clausen (37). These
authors found that luminal proteases caused a loss of
amiloride-sensitive Na current. Microbial fermentation in the
forestomachs includes activity of proteases (4). However, forestomach
epithelia from fetal sheep (at 120-140 days of gestation) exhibited a positive, amiloride-insensitive
Isc [mean
of 1.24 µeq · h1 · cm
2
and range of 0.90-1.45
µeq · h
1 · cm
2
(N = 3, n = 4); Martens, unpublished
observations (see acknowledgment)]. Serosal addition of
aldosterone (1 µM) influenced neither
Isc under control
conditions nor the Ca-sensitive
Isc.
Omasal Na Transport and NSCC
Extracellular divalent cations are potent modulators of channel functions in excitable (3) and nonexcitable tissues (61). In epithelia, the Isc is reversibly enhanced very similarly after mucosal removal of Ca and Mg (61). A further common property of the activated channels is the high selectivity for cations over anions and the poor discrimination between monovalent cations (50). Because the functions and structures of NSCC are incomplete and a conclusive classification is not possible (50), the following discussion will primarily consider studies with epithelia and a preliminary approach will be proposed for grouping the tissues under control conditions (Table 6).
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Group I
Group I tissues work in parallel with the amiloride-blockable channel and are not active in the presence of mucosal divalent cations; activation occurs by mucosal removal of divalent cations. Curran and Gill (8) published, to our knowledge, the first study in which Ca-free buffer solutions at the mucosal site reversibly enhanced Isc in frog skin. These authors suggested an increased Na permeability of the outer membrane. This early observation was more expanded by Van Driessche and Zeiske (62) who clearly demonstrated an inward Na movement through channels in the apical membrane of frog skin in the presence of mucosal amiloride. Ion replacement studies showed that these channels are permeable to other monovalent cations and that divalent cations in micromolar concentrations completely blocked this pathway (62). Because these observations exhibited similarities with Ca channels in frog muscle membrane (2) and heart (27), Van Driessche and Zeiske (62) attempted to measure Ca currents. Neither Ca currents nor effects of Ca channel blockers were detected (62). However, pretreatment of toad urinary bladder with Ag (40 nM) elicited a large inward current with Ca, Mg, or Ba as the principal mucosal cation (55). The results of studies of Das and Palmer (9) do not support the hypothesis of a Ca channel. They studied the single-channel properties of a cation pathway in toad urinary bladder and proposed an apical, outwardly rectifying K conductance, which is blockable by mucosal Ca and may play a physiological role in K transport. This suggestion has been recently confirmed (12, 57, 60).Two observations from studies with forestomach epithelia appear to be important. Höller et al. (28) reported a net Ca transport of sheep omasal epithelium in the mucosal-to-serosal direction. Furthermore, forestomach epithelia exhibited a K conductance in the luminal membrane (35). Unfortunately, our present knowledge on Ca or K transport in sheep omasum is very limited and a possible role of a putative Ca or K channel as NSCC cannot be excluded. However, some observations support the idea that the basal, amiloride-insensitive Na transport is primarily modulated by external Ca.
Groups II and III
Tissues of groups 2 and 3 are characterized by an amiloride-insensitive Isc, which is remarkably enhanced by mucosal removal of divalent cations. In some of the tissues (group II), an amiloride-sensitive Na transport worked in parallel to this pathway (26, 64). This is not the case for rabbit cecum (7, 25), Xenopus colon (32), sheep rumen (42), and omasum (group III). The type of the (amiloride-insensitive) putative, Na-conducting ion channel in the apical membrane is not known (group III), and two alternatives should be considered concerning the Ca sensitivity [for details, see Krattenmacher et al. (33) and Heinz et al. (26)]. If there is only one Na channel in the apical membrane, the effect of divalent cations is probably related to changes of activity and selectivity of this channel. Alternatively, removal of divalent cations could activate a (second) cation channel that is totally blocked in the presence of divalent cations. Because single-channel studies have only been made with human keratinocytes (19), a clear differentiation is not possible. Two observations encouraged us to favor and propose the first possibility (one channel), which is the modulation of the amiloride-insensitive Na pathway. First, we observed a linear and close correlation (r = 0.88) between the Isc under control condition and the Ca-sensitive Isc. To our knowledge, such a correlation has never been reported in epithelia with Ca-sensitive Isc and suggests that the activity of the putative Na conductance, under control conditions, determines the Isc response when divalent cations are removed. A possible explanation for this relationship could be the speculative assumption that the open-channel probability of the putative Na conductance increases after removal of divalent cations. This has been observed in patch-clamp studies of a cation conductance in cultured human keratinocytes (19). Second, theophylline reduced the Isc under control conditions and the Ca-sensitive Isc, which again indicated the close relationship. This suggestion is supported by the correlation of Na concentrations and Isc (see Fig. 1A), by the missing effect of total replacement of Cl on Isc (41), and by the missing effect of theophylline on K and Cl fluxes in sheep rumen (67). Second messenger-induced changes of channel activity have been observed in tissues with NSCC and used for classification. The apical, outwardly rectifying K conductance of toad urinary bladder is stimulated by oxytocin (13), and a pronounced enhancement of channel activity was observed in the absence of mucosal Ca (12, 56). Erlij et al. (12) suggested that the K pathways enhanced by oxytocin are akin to those of the previously described Ca-blockable cation channel.Heterogeneity of NSCC
Selectivity. Van Driessche and Zeiske (62) reported that the apical membrane of frog skin becomes permeable to K, Rb, Cs, Tl, and NH4 when Ca is removed. This unspecific permeability to monovalent cations (33, 48, 62) and the sequence of permeability exhibited large variations (1, 26, 48, 62) and are different from the selectivity of sheep omasum.
Divalent cations. The Ca-sensitive current is blocked by micromolar concentrations of Ca in the mucosal solution (61). When other divalent (Mg, Ba, Sr) or trivalent (La) cations were tested, a variable inhibition of Isc was observed (61). Das and Palmer (9) roughly divided the divalent cations into two groups: Ca, Co, Cd, Mn, and Cu with a high affinity and Mg, Ba, and Sr with weaker effects, which resemble the results of the present study. Even at a Mg concentration of 3.6 mM, the Ca-sensitive current was only reduced by ~50%. In human cultured keratinocytes, 1 mM Mg did not decrease the open-channel probability of a Ca-sensitive cation conductance (19).
The possible interactions of divalent cations with NSCC have been discussed by Das and Palmer (9). A very simple explanation could be a fast block of the conducting pathway by divalent cations that bind to sites within the channel pore. This type of inhibition should be PD dependent, which was not shown in the single-channel studies of Das and Palmer (9) and Galietta et al. (19). Alternatively, Das and Palmer (9) considered changes of surface potential near the channel mouth or an allosteric effect on channel conformation. The blocking effect of micromolar concentrations of Ca in the present study is in general agreement with observations in tissues with NSCC (61). The approximation obtained by use of a double Michaelis-Menten plot supports the assumption of two binding sites but does not allow a discrimination between the different modes of modulation.Saturation. Aelvoet et al. (1) have observed a linear relationship between the Na concentration and Ca-sensitive Isc, which has been confirmed several times (9, 32, 58). This linear correlation was also obtained in sheep omasum by using (peak) Ca-sensitive Isc (Fig. 1B). The Isc of the plateau appears to exhibit a saturation (Fig. 1C), which has also been shown in rabbit cecum (48).
Physiological Role of NSCC
The blockade of NSCC by very low mucosal Ca concentrations (µM) raises the question of whether the Ca-sensitive Na-transport is of physiological importance under normal in vivo conditions (32, 62). Continued studies with toad urinary bladder during the last decade have revealed that the Ca-sensitive Isc may play an important role in K transport and cell volume regulation (9, 12) because the Ca concentration in toad urine is often low enough (Isc and Gt
Mucosal removal of divalent cations caused a reversible change in the Isc and corresponding alterations in Gt, which presumably reflected variation of the conductance of the apical membrane. The (paracellular) flux rates of mannitol were not influenced (Table 2), and the PD-dependent JsStudies With Microelectrodes
The mucosal impalement of sheep omasum clearly showed that the Ca-sensitive pathway is located in the luminal membrane. Mucosal removal of divalent cations significantly reduced theEstimation of ENa
The concomitant variation of Isc and Gt, which were linearly correlated, was used for the calculation of ENa (68). The obtained ENa of 62 ± 1 mV is very similar to that shown by Turnheim et al. (54) in rabbit colon (65.4 mV). Insertion of ENa into the Nernst equation resulted in a calculated intracellular Na concentration of 8.2 ± 0.3 mM, which is within the range of other Na-absorbing epithelia (53).Channel Blockers
The early suggestion of Van Driessche and Zeiske (62) that the Ca-sensitive channels of amphibian epithelia resemble Ca channels gave rise to testing of Ca channel blockers. Nicardipine and diltiazem were without significant effects in frog skin (62), and verapamil inhibited rabbit cecum Isc in the presence and absence of mucosal Ca (48). There is some evidence for a potassium conductance in the luminal membrane of rumen epithelium in sheep that is blocked by verapamil (35). This possible complication impedes a satisfactory explanation of effects of Ca channel blockers and hindered us from using them.In conclusion, sheep omasal epithelium exhibits an electrogenic Na transport with typical properties of the classical Na transport: saturation and overshoot. These characteristics appear to be a general capability of epithelia to adjust luminal Na uptake with basolateral extrusion, independently of the type of luminal Na conductance. Mucosal amiloride (1 mM), the amiloride analog phenamil (1 and 10 µM), or serosal aldosterone (1 µM) did not change Isc, which clearly distinguishes sheep omasum from tissues with classical Na transport. Furthermore, removal of divalent cations from the mucosal solution considerably increased electrogenic Na transport. The physiological role of this modulation by divalent cations is not clear at the present time, but the sensitivity of this conductance against divalent cations appears to be a common property and could be used in the future as a test in tissues for amiloride-insensitive Na transport. The existence of amiloride-insensitive Na transport mechanisms in tissue of the gastrointestinal tract raises the question of whether these mechanisms may have more or other physiological functions than the classical Na transport. Recently, Lang and Martens (34) have described a PD-dependent cation conductance in the luminal membrane of sheep rumin epithelium. Similarly, Sellin and Dubinsky (48) demonstrated in planar bilayer studies with vesicles from rabbit cecum a cation conductance with voltage dependence. A possible connection between NSCC and the PD-dependent cation conductance is very speculative at the moment, but it could be a hint for functions that are not known in the classical Na transport.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the excellent technical assistance of K. Wolf and M. Ganz and thank Prof. A. D. Care (Aberystwyth, Wales) for providing us with the opportunity to study fetal forestomach tissue of sheep.
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FOOTNOTES |
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The study was supported by a grant of the Deutsche Forschungsgemeinschaft (Ma 699/11). G. Schultheiss was a recipient of a Scholarship from the Deutsche Studienstifung.
1 An inhibition by low concentrations of amiloride does not mean that only one type of Na channel exists. Significant differences of single-channel conductances or cation selectivity have been reported. For details, see Garty and Palmer (21).
Address for reprint requests and other correspondence: H. Martens, Dept. of Vet.-Physiology, Free University of Berlin, Oertzenweg 19b, 14163 Berlin, Germany (E-mail: martens{at}vetmed.fu-berlin.de).
Received 30 October 1997; accepted in final form 22 February 1999.
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