Ca-sensitive Na transport in sheep omasum

Gerhard Schultheiss1 and Holger Martens2

1 Department of Physiology, Faculty of Veterinary Medicine, University of Giessen, D-35392 Giessen; and 2 Department of Veterinary Physiology, Free University of Berlin, D-14163 Berlin, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-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.

calcium-sensitive sodium transport; nonselective cation channel; Ussing chamber; microelectrodes; apical conductance


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha ), 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of the Tissue

The preparation and incubation of forestomach epithelia have been described in detail by Leonhard-Marek and Martens (35). Briefly, sheep of different ages and sex were killed at a local slaughterhouse. The omasum was removed from the abdominal cavity within 4 min after sheep were stunned and exsanguinated. The omasum was opened by a cut along the omasal canal, and several leaves were removed. The epithelia were carefully rinsed in a buffer solution (gassed with 95% O2 and 5% CO2, 38°C). The two epithelial surfaces of each leaf were separated by blunt dissection and quickly taken to the laboratory in a buffer solution. The heterogeneous origin of sheep caused unavoidable variations of Isc and transepithelial conductance (Gt). Consequently, control and treatments were done with tissues from the same animals.

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 · h-1 · 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)
<IT>J</IT> = <IT>J</IT><SUB>d</SUB> ⋅ &xgr; + <IT>J</IT><SUB>m</SUB> (1)
where J represents the total unidirectional flux of an ion, Jd · xi  is the component of the flux that varies with the electrical driving force (xi ), which is defined by
&xgr; = (<IT>z</IT> ⋅ <IT>F</IT> ⋅ PD<SUB>t</SUB>/<IT>R</IT> ⋅ <IT>T</IT>)/[exp (<IT>z</IT> ⋅ <IT>F</IT> ⋅ PD<SUB>T</SUB>/<IT>R</IT> ⋅ <IT>T</IT>) − 1] (2)
and Jm represents the electroneutral (PDt independent) part of flux.

PDt is the potential difference across the epithelium, z is valence, T is absolute temperature, F is Faraday's constant, and R is the gas constant. (Note that with PDt near 0 mV xi  approaches "1." At PDt = 0 mV, xi  is not defined but is set to "1" because of the given approach.)

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 MOmega 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 MOmega , 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 alpha  = apical resistance/basolateral resistance (Ra/Rb) Delta Va/Delta 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 (Delta Vb) was calculated using the formula Delta Vb Delta VT - Delta 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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

General Properties

All mounted omasal tissues exhibited under control conditions a serosa-positive Isc (range of 1-4 µeq · h-1 · cm-2). The tissue conductance varied between 1.5 and 6 mS/cm2 and declined very rapidly during the first 20-30 min after the tissues were mounted. After these rapid changes occurred, Isc and Gt commenced along a very small and linear decay with time (some 8-12% per hour, see Fig. 2). In Na-free solutions (replacement by NMDG), the positive Isc was totally abolished or, in a small number of tissues, was negative (0-0.1 µeq · h-1 · cm-2). When ouabain (0.1 mM) was added to the serosal compartment, the Isc disappeared within 20-30 min (data not shown). In a few cases, a negative Isc was observed. These findings suggest that the Isc in sheep omasum is predominantly Na dependent. We examined this possible relation by varying Na concentrations in the buffer solution and found a saturation with a Michaelis-Menten constant for transport of Na (Na concentration vs. current) of 6.7 mM and maximal transport capacity of Na of 1.56 µeq · h-1 · cm-2 (Fig. 1A). All these observations are typically for tissues with the amiloride-sensitive Na channel in the apical membrane. As previously reported (41), mucosal amiloride (1 mM) did not change Isc (data not shown, see Mucosal Amiloride and Phenamil). Consequently, we considered whether the suggested electrogenic omasal Na transport exhibits characteristics of the recently described NSCC.


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Fig. 1.   A: increasing Na concentration and short-circuit current (Isc) in sheep omasum under control conditions. Inset: double reciprocal plot that reveals a Michaelis-Menten constant for transport of Na (Na concentration vs. current) = 6.7 mM and maximal transport capacity of Na = 1.56 µeq · h-1 · cm-2 (n = 8-10, N = 3). B: relationship between Na concentrations and Ca-sensitive Isc after removal of mucosal Ca. Ca-sensitive (peak) Isc linearly increased with increasing Na concentrations. C: Ca-sensitive Isc () exhibited a saturation behavior after downregulation of Isc (Isc at the end of the plateau phase; see Fig. 3). In the presence of mucosal Ca (open circle ), Isc is almost identical to data in A.

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-1 · cm-2) within 8-10 min. This peak Isc was only transient, and an almost exponential decline of Isc followed until a new plateau (with Isc of 7.15 ± 0.71 µeq · h-1 · cm-2) ensued after 20-40 min (if not otherwise mentioned, all experiments were performed during the plateau phase). The alteration of Isc was accompanied by an increase in Gt (peak of 8.49 ± 0.83 and plateau of 7.47 ± 0.71 mS/cm2). The parallel changes of Isc and Gt were closely correlated (r = 0.96; see Estimation of ENa). A linear relation was found between the Isc under control conditions and the Ca-sensitive Isc (peak Isc minus Isc before mucosal change of buffer): y = 1.22 + 3.79x, in which y = Ca-sensitive Isc and x = Isc under control conditions (r = 0.88; n = 16, N = 6). This suggests that the Isc measured under control conditions is enhanced by mucosal removal of divalent cations; a high Isc under control conditions led to a high Ca-sensitive Isc and vice versa. Mucosal addition of Ca decreased the Isc and Gt to baseline (Fig. 2). The Ca-sensitive Isc is closely related to the Na concentrations. Thus Na concentrations were varied from 10 to 115 mM in the buffer solution, and the responses in Isc were determined after mucosal removal of divalent cations. Varying the Na concentration caused a concentration-dependent response of Isc after mucosal removal of divalent cations (Fig. 3). Isc significantly increased at all Na concentrations within 10-15 min (P < 0.05). A pronounced peak of Isc followed by a downregulation was only observed at high Na concentrations. A linear correlation was found between the Na concentration and the Ca-sensitive component of the Isc (Fig. 1B). This close correlation supports the assumption that removal of divalent cations activated a conductance in the luminal membrane through which Na enters the cells driven by its electrochemical gradient. It is worth noting that the downregulation of Isc limited the Na uptake during the plateau phase, eliciting a saturation behavior (Fig. 1C). Isc is clearly saturated in the presence of mucosal Ca (Fig. 1C), confirming the data of Fig. 1A.


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Fig. 2.   Response of Isc on removal of divalent cations (-Ca2+, up-arrow ) from the mucosal side. Addition of 2 mM Ca (down-arrow ) to the mucosal bath resulted in a reversible decrease in Isc (P < 0.001; n = 10, N = 4). t, Time.



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Fig. 3.   Na-dependent Isc in sheep omasum. At all Na concentrations, a Ca-sensitive Isc (overshoot and plateau) was activated after mucosal removal of divalent cations. A pronounced overshoot followed by downregulation can only be observed at high Na concentrations (n = 10, N = 3). Error bars (±SE) are in some cases smaller than symbols.

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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Fig. 4.   Effects of repeated removal of Ca (up-arrow ) from the mucosal solution or addition of 2 mM Ca (down-arrow ) to the mucosal solution on Isc. A: addition of Ca at peak Isc (before downregulation) (n = 14, N = 5). B: addition of Ca to the mucosal solution during the plateau phase (after finishing downregulation) demonstrates that the Ca-sensitive Isc can be repeatedly inhibited (or activated by removal of Ca) without significant decrement (n = 10, N = 50). Error bars (±SE) are in some cases smaller than symbols.

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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Fig. 5.   A: reduced serosal Na concentration caused a significantly lower Isc after removal (up-arrow ) of divalent cations (peak and plateau; P < 0.001, Wilcoxon's matched pairs test; n = 12, N = 3). open circle , 115 mM serosal Na. , 10 mM serosal Na. up-arrow *, Na was replaced by N-methyl-D-glucamine. B: effect of serosal Ca concentrations on Isc. Tissues were equilibrated with the normal buffer solution (1 mM Ca) for 15 min. Buffer solution was then removed from both sides of the epithelium (up-arrow *) and replaced by a solution containing 0.5 mM Ca (open circle ) or 4.0 mM Ca (). Changing the serosal Ca concentration from 1 to 0.5 or 4.0 mM, respectively, influenced peak as well as plateau Isc, when Ca (and Mg) was removed from the mucosal side (up-arrow ). However, this response in Isc was significantly lower with 4.0 mM Ca in the serosal solution (P < 0.005). Error bars (±SE) are in some cases smaller than symbols.

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 (Jmright-arrow s), net flux, and Isc are almost identical and not significantly different. This good agreement strongly supports the assumption that the enhanced Isc represents an electrogenic Na transport. The net transport of Na under control conditions significantly exceeded the Isc, which indicates an electroneutral Na component in parallel to the electrogenic transport (Table 1) and corroborates our previous conclusion regarding the assumption of a Na/H exchange in the luminal membrane of sheep omasum (41).

                              
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Table 1.   Effect of mucosal Ca removal on Na transport, Isc, and Gt

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. Jmright-arrow s and serosal-to-mucosal flux (Jsright-arrow m) were linearly correlated with the Eq. 1 under control and nominally divalent cation-free conditions, which indicates that the model of Frizzell and Schultz (16) that was used for studying the effects of PDt was satisfactory. The Jsright-arrow m fluxes included both potential-dependent and potential-independent (intercept) components and did not significantly differ between treatments. Provided that the potential-dependent Na flux in the serosal-to-mucosal direction represents the passive flow through the paracellular shunt, then Ca- and Mg-free solutions did not alter the permeability of this pathway for Na. The intercepts, i.e., potential-independent components of Na flux in the mucosal-to-serosal direction, were also not significantly different between treatments and probably represent the electroneutral Na transport via Na/H exchange. However, the slopes differed significantly (P < 0.05). Jmright-arrow s of Na was much more influenced by the transmural potential difference under divalent cation-free conditions, which is in accordance with the assumption of an electrodiffusible flow of Na through a putative conductance in the luminal membrane.


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Fig. 6.   Effects of variation of the transepithelial potential difference (PDt; -25, 0, and +25 mV) on the Na fluxes with (solid symbols) or without (open symbols) mucosal Ca. Slope of mucosal-to-serosal (mright-arrows) flux was significantly increased by mucosal Ca omission. PDt of +25 mV (xi  = 0.60; serosal side positive) decreases mucosal-to-serosal flux of Na; PDt of -25 mM (xi  = 1.54, serosal side negative) increases mucosal-to-serosal flux of Na. Effects PDt on serosal-to-mucosal (sright-arrowm) flux of Na are reversed. P < 0.05; n = 12, N = 4. See MATERIALS AND METHODS for definition of xi .

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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Table 2.   Effect of mucosal Ca on mannitol transport, Isc, and Gt

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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Fig. 7.   A: theophylline, a phosphodiesterase inhibitor (10 mM, mucosal and serosal side, down-arrow *, ) significantly reduced the Isc under control conditions (P < 0.01). Response in Isc clearly exhibited significant differences after removal of divalent cations (-Ca2+). Pretreatment with theophylline caused a significantly lower increase in Isc (peak and plateau; P < 0.01, Wilcoxon's matched pairs test; n = 17, N = 4). B: administration of aldosterone (1 µM, serosal side, 60 min, ) did not influence the Isc [with Ca in the mucosal solution or after removal (up-arrow ) of divalent cations; n = 8, N = 4]. Error bars (±SE) are in some case smaller than symbols.

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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Table 3.   Ca-sensitive Isc of various monovalent cations in the mucosal solution

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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Fig. 8.   A: original tracing of Isc after removal or addition of different polyvalent cations (2 mM, mucosal side, down-arrow ). Ca-sensitive Isc can only be partly blocked by the cations Mg, Ba, La, or Sr; 1 mM Ca (down-arrow **) caused in all cases a further decrease in Isc. B: dose-response curve of increasing Mg (open circle ) or Sr () concentrations (mucosal side only) on decrease of Ca-sensitive Isc. Both divalent cations did not totally block the Ca-sensitive Isc, even at a concentration of 3.6 mM. Addition of 1 mM Ca (in addition to 3.6 mM Mg or Sr, dotted line) further reduced the Isc in both groups (with Mg: P < 0.001; with Sr: P < 0.01; n = 7, N = 4).

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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Fig. 9.   Decrease of Ca-sensitive Isc as a function of mucosal Ca concentrations. Fit was obtained using double Michaelis-Menten kinetics (Michaelis-Menten constant values = 92 ± 24 and 0.4 ± 0.05 µM for the low- and high-affinity binding sites, respectively; n = 20, N = 5).

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-2 · h-1 within 5 min (P > 0.05) in the presence of divalent cations without a change in Gt (3.32 ± 0.29 vs. 3.26 ± 0.31 mS/cm2; N = 4, n = 9). The effect of 100 µM phenamil during the plateau phase was a little bit more pronounced. Isc significantly decreased from 5.04 ± 0.48 to 4.12 ± 0.42 µeq · cm-2 · h-1, but Gt was not significantly enhanced (from 7.73 ± 0.81 to 8.23 ± 0.87 mS/cm2).

                              
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Table 4.   Mucosal amiloride does not change Isc in the absence of mucosal divalent cations

Studies with Microelectrodes

Mucosal Ca- and Mg-free solutions increased the Gt. The studies with mannitol and the potential-dependent Jsright-arrow m of Na suggest that a change in the paracellular conductance is not very likely or negligible and small compared with the observed change in Gt. Furthermore, the closely related alterations in Gt and Isc support the assumption of a change in the cellular conductance. Determination of the electrical resistance profile across frog skin with and without divalent cations in the mucosal solution has indicated that these ions act primarily on the apical membrane (59). Consequently, microelectrode techniques were used to localize the Ca-sensitive pathway. To determine whether the luminal membrane demonstrates a (monovalent) cation conductance, experiments were performed in which the divalent cations were removed during a microelectrode impalement. A representative experiment is given in Fig. 10. Without Ca in the mucosal solution, the omasal epithelial cells exhibit a PDa of -39 mV. Perfusion with Ca and Mg solutions at the luminal side caused an increase of PDa to -56 mV and a decay of PDt from 20 to 6 mV. These changes are consistent with an activation of an apical cation conductance after mucosal removal of divalent cations. The distribution of electrical potential differences across the luminal membrane of sheep omasum is illustrated in Fig. 11. PDa ranges from -51 to -74 mV under control conditions and from -31 to -54 in divalent-deprived buffer solutions. Table 5 summarizes the voltage profile across the epithelium, which exhibited a well-like potential profile.


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Fig. 10.   Original tracing of a mucosal impalement of sheep omasum. Changes of apical PD (PDa) and PDt are caused by mucosal addition of Ca (2 mM) to the Ca-deprived mucosal solution. Note the alterations in voltage deflection, indicating an increase in apical and transcellular resistance. Resistance of the microelectrode (Rel) exhibited only minor alterations during the impalement.



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Fig. 11.   Frequency distribution of PDa of all impalements without (open columns) and with (hatched columns) mucosal Ca. Range of PDa was -31 to -54 mV (mean of -42 ± 1 mV) or -51 to -74 mV (mean of -63 ± 1 mV), respectively [P < 0.001; number of impalements = 46 (without Ca) or 36 (with Ca), N = 6, n = 7]. In several tissues, more than one impalement was performed.


                              
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Table 5.   Electrophysiological parameters of sheep omasum with or without Ca in the mucosal solution

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 Omega  · cm2, which accounts for the change in transepithelial resistance. Consequently, alpha  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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · h-1 · 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 · h-1 · 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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Table 6.   A preliminary approach for grouping of NSCC in the luminal membrane of epithelia modulated by divalent cations in the mucosal solution

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 (<< 1 mM; Ref. 12). In contrast, the total concentrations of Ca and Mg in forestomach fluid of sheep are in the millimolar range (23), a range which very likely inhibits the Ca-sensitive Isc. This is probably true for all NSCC in the gastrointestinal tract (26, 33, 48). However, epithelia in the gastrointestinal tract (with NSCC) exhibited in all cases an amiloride- and Ca-insensitive Na transport (Table 6), which ensures Na absorption in the presence of physiological concentrations of mucosal divalent cations.

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 Jsright-arrow m of Na (Fig. 6), which are very likely passive and paracellular, were almost identical.

Studies 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 the alpha  (see Table 5), which indicated a decreased resistance of the apical membrane. Consequently, PDa depolarized because of the enhanced inflow of Na into the cells. The troughlike potential profile of sheep omasum remarkably resembles the electrical properties of rabbit cecum (7) or colon (66).

Estimation 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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