1Department of Physiology, Faculty of Veterinary Sciences, University of Khartoum, Sudan; and 2Department of Veterinary Physiology, Free University of Berlin, Berlin, Germany
Submitted 24 September 2004 ; accepted in final form 6 April 2005
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ABSTRACT |
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rumen; ammonia; sodium transport; sodium-hydrogen exchanger; potassium channel
Ammonia influences a variety of epithelial functions. In T84 human intestinal crypt epithelia, ammonia blocks a K+ channel required for maintaining the driving force for electrogenic apical chloride secretion (30). Acid secretion is reduced in bullfrog oxyntic cells by the possible involvement of an apical K channel (28). Na transport is impaired in rat colon by NH4+ competing with the Na-binding site of the Na+/H+ exchanger (NHE) (13). Cougnon et al. (17) have shown that NH4+ is transported by the colonic H+/K+-ATPase of the rat expressed in Xenopus oocytes.
Ammonia absorption takes place in the rumen (6), small intestine (41), and the colon (15) and occurs primarily by simple diffusion of the nonionized lipid-soluble NH3 form. However, ionic diffusion (NH4+) has also been demonstrated (5, 8, 12, 15).
In the kidney (31, 52), where NH4+ has to be transported against a considerable concentration gradient from the renal cortex (<0.1 mM) to the medulla and, ultimately, the urine, transport of NH4+ has to be coupled to other ions and replaces other cations in transport proteins such as the NHE3, Na-K-2Cl exchange, K/H exchangers, or NH4+/H+ exchangers belonging to the Rh gene family (31, 52). Conversely, in the rumen and the colon, where NH4+ is in abundant supply, transport via channels is energetically feasible and would explain the increase in short-circuit current observed both in rat colon and rumen epithelium after exposure to this ion (5, 14).
It is generally known that cellular uptake of ammonia affects intracellular pH (pHi) (7, 46). Predominant NH3 uptake tends to alkalinize the cytoplasm, whereas the predominant uptake of NH4+ acidifies it. The magnitude and direction of this change in pHi depends on the relative transport rates of NH3 and NH4+, which can be altered by the luminal pH according to the Henderson-Hasselbalch equation. The wide variations in ruminal ammonia concentrations (up to 70 mM) (11) and of pH (5.47.4) cause corresponding alterations of NH3 and NH4+ concentrations and flux rates across the rumen epithelium (25). Note that in rumen, NH4+ transport can also be influenced by changes in the potential difference (PD) across the apical membrane (PDa) (5), whereas exchangers are typically not affected by membrane potential (54).
Alteration of pHi is well known to affect cellular transport of Na+ via NHE in various systems (7, 46), including the rumen epithelium (42), and a modulation of Na transport is to be anticipated. In a recent study (1), it was demonstrated that interaction between ammonia and Na+ transport across the isolated rumen epithelium of sheep is modulated by the diet.
The mechanisms underlying this adaptation to an increase in the uptake of nitrogen (urea or protein) are poorly understood. However, it should be noted that luminal application of ammonia induced a significantly higher increase in short-circuit current (Isc) in tissues of concentrate-fed animals than in epithelia of hay-fed animals (1). Therefore, we suggested that the ammonia-induced change in Isc is caused by the uptake of NH4+ in its protonated form. Dissociation of NH4+ within the cytosol should increase the amount of H+ available for exchange with sodium.
Alkalinization of ruminal epithelial cells by uptake of ammonia in the form of NH3 was demonstrated by Müller et al. (42), whereas experiments by Bödeker and colleagues (5, 6) show uptake of NH4+. In these experiments, the ammonia-dependent increase of Isc was abolished by pretreatment with mucosal quinidine, and the authors suggested involvement of a potassium channel (5, 6). There are conflicting reports on the permeability of the apical membrane of rumen epithelium for potassium, depending on whether luminal potassium was high (20, 35, 50), or low (21, 35). This question obviously needs further clarification.
It was therefore the aim of the present study 1) to determine the flux of ammonia through the rumen epithelium, (2) to change the relative flux rates (NH4+ or NH3) by variation of luminal pH and PDa, 3) to look for effects of altered ammonia flux rates on Na transport, and 4) to characterize the NH4+ conductance.
The patch-clamp data in this study show that ruminal epithelial cells express quinidine-sensitive channels of 130 pS that conduct both potassium and NH4+ but not sodium. Relative permeability of the membrane for potassium rose with concentration. Anomalous mole-fraction effects (29) between K+ and NH4+ suggest competition for a common pathway that may limit apical potassium efflux from the rumen epithelium under in vivo conditions.
Ussing chamber data demonstrate that variations of luminal pH determine the form of ammonia transport as NH4+ or NH3 and consequently affect the absorption of Na+ depending on the transported form of ammonia. Predominant uptake of NH4+ through potassium channels stimulated and predominant diffusion of NH3 inhibited Na transport across the rumen epithelium of sheep. Under physiological conditions (pH 6.50 or lower), ammonia stimulates Na transport and may be a modulator of Na absorption via NHE, significantly contributing to the classic effect of short-chain fatty acids (24, 33).
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MATERIALS AND METHODS |
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The mounted tissues were bathed on each side with 18 ml buffer solution by using a gas lift system and were gassed with 95% O2-5% CO2 or 100% O2 (HCO3-free buffer solutions) at 38oC. The standard electrolyte solution contained (in mM) 90 Na+, 5 K+, 1 Ca+2, 2 Mg+2, 25 HCO3, 59 Cl, 1 H2PO4, 2 HPO42, 25 acetate, 10 propionate, 5 butyrate, 10 glucose, and 30 D()-N-methylglucamine-hydrochloric acid (NMDGCl). In ammonia containing buffer solutions, NMDGCl was replaced by equimolar NH4Cl (5, 15, and 30 mM). In HCO3-free buffer solutions, HCO3 was replaced by gluconate, and the solutions were buffered with HEPES (8 mM). Amiloride was obtained from Sigma (Munich, Germany). Radioisotopes (22Na and 36Cl) were purchased from Amersham (Braunschweig Germany). All reagents were of analytical grade.
Electrical Measurements and Calculation of Na and Cl Flux Rates
For details, see Martens et al. (38). The transepithelial conductance (Gt) was determined by briefly applying a 100-µA current across the tissue in both directions and measuring the resulting change in transmural potential difference (PDt), from which Gt can be calculated using Ohm's law. The sum of all electrogenic ions moving across the tissue was determined by measuring the external current (equivalent to Isc) necessary for clamping PDt to zero.
Fluxes were measured in the short-circuit mode. 22Na and 36Cl (80 and 100 kBq, respectively) were added to the "hot" side of the epithelium, and three flux periods of 30 min were performed after an equilibration time of 30 min. Samples from the "hot" site were taken before the first and after the last flux period for the calculation of the specific radioactivity. The sample volume was replaced by the corresponding buffer. 22Na and 36Cl fluxes were determined in separate experiments. Total ammonia flux {mucosal-to-serosal flux [Jms (NH4+ + NH3)]} was calculated from aliquots taken from the serosal side at the beginning and the end of the flux period and determined directly by using an ion analyzer (gas-sensitive electrode; EA 940; Orion).
Radioactivity
22Na and 36Cl were assayed by using a well-type crystal counter (LKB Wallace-Perkin-Elmer, Überlingen, Germany) and a -counter (LKB Wallace-Perkin-Elmer), respectively.
Statistics
Statistical evaluations were carried out by using the SPSS program version 10.0 for Windows. Results are given as means ± SE. The comparison between the groups was carried out in the form of a repeated-measurement analysis of variance with a two-factorial model without interaction. P values <0.05 were considered significant. N refers to the number of experimental animals, and n refers to the number of tissues.
Patch-clamp experiments. Cells from rumen epithelium were cultured on glass coverslips according to established methods (36, 49). Briefly, the stratum corneum (26) was removed from ruminal papillae by fractional trypsinisation, and the last (fourth or fifth) fraction was plated out in cell culture dishes. The number of cornified cells in the culture increased rapidly after seeding. Coverslips were removed from the culture dishes for experiments 38 days after seeding or, alternately, 15 days after reseeding from primary culture. Previous studies in our lab suggest that cells at this stage have differentiated and express proteins that are found in the apical membrane of ruminal epithelium (36, 48).
Immediately before use, cells were gently cleansed with trypsin (0.02%, Biochrome, Berlin, Germany) for 1 min and introduced into a perfusion chamber on the stage of an inverted microscope where they were superfused by warmed Ringer solution (37.5°C; TC01 and PH01, Lohmann Research Equipment, Castrop-Rauxel, Germany). All patch-clamp experiments were performed essentially as in a previous study (36). Only cells in which washout occurred, demonstrating seal stability, were used for evaluation. Capacitance was measured regularly; cells in which swelling occurred were excluded. Note that usually the seals of such cells ruptured quickly, and stable measurements with washout could not be obtained.
Currents were recorded using an EPC 9 patch-clamp amplifier (HEKA Elektronic, Lambrecht, Germany). Pulse generation, data collection, and analysis was performed using TIDA for Windows software (HEKA Elektronic) and filtered with a 2.9-kHz Bessel filter. Records were corrected for capacitance. Positive ions flowing into the pipette correspond to a negative current and are depicted in figures as going downward. For inside-out patches, the pipette potential corresponds to the negative membrane potential.
Two types of pulse protocols were used. Either current response was recorded at 100 Hz using a protocol that generated steps of 200-ms duration to voltages between 120 and 100 mV in 20-mV steps, returning to a holding potential of 40 mV for 200 ms in between ["pulse protocol I" (36)]. This protocol was repeated continuously to allow the monitoring of current responses of the cells to changes in external solution. In addition, conventional voltage pulse protocols were used that recorded_data at a much higher sampling rate of 5 kHz ["pulse protocol II" (36)]. As before, holding potential was 40 mV, and voltages ranged from 120 to 100 mV, but the step size was 10 mV. For single-channel experiments, data were sampled at 10 kHz.
Solutions and Chemicals (Patch Clamp)
Pipette solution for whole cell experiments, designated as "K-gluconate" contained (in mM) 1 KH2PO4, 10 HEPES, 0.8 CaCl2, 0,9 MgSO4, 5 EGTA, 123 K-gluconate, and 10 NaCl. Extracellular NaCl solution contained the following ion concentrations (in mM): 130 NaCl, 1 NaH2PO4, 5 KCl, 10 HEPES, 1.7 CaCl2, and 0.9 MgCl2. With the use of this basic recipe, NaCl was substituted by either choline chloride or NH4Cl or KCl in the solutions designated by these ions. In solutions labeled "NH4" (65 mM), "K" (65 mM), or "NH4 + K", the chloride salts of the designated cations (each at 65 mM) were used to substitute for NaCl. Osmolarity was adjusted with choline chloride (65 mM). In experiments in which BaCl2 and TEACl were added to the solutions, osmolarity of the different solutions was adjusted by mannitol.
For inside-out single-channel experiments, standard 130 mM NH4Cl solution was used to fill the pipette (facing the external side of the membrane), whereas calcium was reduced in the KCl bath solution facing the cytosolic side and contained (in mM) 130 KCl, 3 NaCl, 5 EGTA, 0.9 MgSO4, 0.8 CaCl2, 10 HEPES, and 1 KH2PO4. The solutions designated as NH4Cl, NaCl, choline chloride, or K-gluconate were equal in composition but contained the respective ions instead of KCl. Liquid junction potentials were corrected according to established methods (3).
Analysis
To compare whole cell data from different cells with each other, initial inward and outward current in NaCl solution at 120 and 100 mV were assigned the value of 100%. All other currents were seen in relationship to these values. Significance testing was performed using the paired Student's t-test and standard software. The number n refers to the number cells used; care was taken to include cells from different animals.
Reversal potentials were estimated by linear regression between the zero-current values just above and just below the zero level for each cell and corrected for liquid junction potential. Relative permeability ratios (Pb/Pa) were calculated by switching the major ion in the external solution, measuring the corresponding reversal potential (Erev), and using Goldman-Hodgkin-Katz theory (29): Erev,b Erev,a = RT/zF x ln(Pb[B]o)/(Pa[A]o).
In single-channel experiments, current-voltage relationships were fitted using the Goldman-Hodgkin-Katz equation for two ions (29): I = E x F2/(R x T) x (PNH4[NH4]i + PK[K]i (PNH4[NH4]o + PK[K]o) x exp{[E x F/(R x T)]/[1 exp(E x F/(R x T)]} and standard software (SigmaPlot for Windows, 8.02).
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RESULTS |
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In a first series of experiments, the effect of adding different concentrations of ammonia to the luminal side of rumen epithelium was studied using the Ussing chamber technique. At the end of the equilibration period, the Isc and the total tissue Gt exhibited constant values of 0.680.80 µeq·cm2·h1 and 2.132.68 mS/cm2, respectively, reflecting electrogenic sodium transport (34, 36). This did not change significantly during the next 30 min in control tissues. As in the study by Bödeker and Kemkowski (5), luminal ammonia caused a significant increase of Isc up to 0.45 µeq·cm2·h1 (Fig. 1). A new steady state of Isc was obtained after 1015 min, and the rise of Isc (Isc) exhibited saturation kinetic properties (Fig. 1; pH 7.4).
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Determination of Ammonia Flux Rates
It is impossible to distinguish between transport of NH4+ through a potassium channel and of Na+ via the electrogenic pathway (34, 36) by measuring Isc alone. In addition, no information on the transport of ammonia as NH3 is obtained. Therefore, ammonia flux rates were measured directly.
Total ammonia was kept constant (30 mM), and concentration of NH3 varied by increasing the pH from 6.4 to 6.9 and 7.4. This should induce an increasing gradient in NH3 concentration at an almost constant concentration of NH4+. At pH 6.4, a steady-state ammonia flux [Jms (NH3 + NH4+)] of 0.82 µmol·cm2·h1 could be measured from the mucosal to the serosal side. An increase in pH on the luminal side of the membrane resulted in a significant (P < 0.05) stimulation of ammonia flux rates (Fig. 2).
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Effect of Ammonia on Na and Cl Transport at pH 7.4
Increasing luminal ammonia concentrations in a CO2/HCO3-free buffer at a luminal pH of 7.4 significantly (P < 0.05) decreased the mucosal-to-serosal flux of sodium [Jms (Na)]. As a consequence, the net flux [Jnet (Na)] (P < 0.05) decreased from 2.01 ± 0.11 (control) to 0.74 ± 0.12 µeq·cm 2·h1 at 30 mM NH4Cl (Table 1).
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Effect of Ammonia on Na and Cl Transport at pH 6.9 or 6.4
The above results support the preliminary conclusion that at pH 7.4, ammonia enters the cells predominantly via diffusion of the lipophilic form NH3. Lowering the luminal pH decreases the concentration and diffusion of NH3 and should diminish effects on pHi. Two series of experiments (luminal pH of 6.9 and 6.4) were performed to test this hypothesis. Ammonia did not influence Na fluxes at pH 6.9 (Table 3).
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Effect of PDt
Because NH4+ is charged, uptake across the luminal membrane through a K channel should be modulated by the PDa. PDa was altered by variation of PDt (34), and the effect on the transport of Na both in the presence and the absence of luminal ammonia (30 mM) was studied. All solutions were titrated to a pH of 6.4, so that ammonia should be present almost exclusively in the ionized form.
Imposing a PDt of + 25 mV (serosal side positive) significantly depolarizes PDa by 15 mV from some 50 to 35 mV (34). This PDt is too low to induce significant stimulation of electrogenic sodium transport (34) against a high background of electroneutral sodium transport (stimulated by presence of short-chain fatty acids in the current study).
In line with this, a PDt of +25 mV only led to a small, nonsignificant reduction of Jms (Na) and Jnet (Na) (Table 5). Likewise, serosal-to-mucosal flux (Jsm) (Na) was not significantly changed either by changing PDt or by the addition of ammonia. All changes probably represent effects on paracellular, passive flow.
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However, in the presence of ammonia (30 mM), Jms (Na) and Jnet (Na) could be reduced significantly by increasing PDt from 0 to +25 mV (P < 0.05). The potential-induced decrease in Jnet(Na) (Jnet at PDt 0 mV Jnet at PDt +25 mV) was significantly greater in the presence of ammonia (1.59 ± 0.27 µeq·cm2·h1) than in its absence (0.77 ± 0.28 µeq·cm2·h1). This suggests that depolarization of the apical membrane reduced the influx of NH4+ needed to stimulate the NHE in the presence of ammonia.
Effect of Amiloride
The assumption that the stimulatory effect of ammonia (30 mM) on Na transport at pH 6.4 is caused by enhanced NHE activity was tested by blocking this exchanger by 1 mM mucosal amiloride (38), completely blocking the effects of ammonia on net Na transport (Table 6). Note that amiloride had no effect on Isc.
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To test for mutual inhibition of NH4+ and K+ in a common pathway, we studied the modulation of Na transport by K. Luminal solutions contained 15 mM ammonia at pH 6.4. Increasing luminal K concentration from 5 (control) to 45 mM caused a significant reduction of Jms (Na) from 8.70 ± 0.67 (control) to 6.01 ± 0.65 µeq·cm2·h1, respectively, with concurrent significant reduction of Jnet(Na). Jsm(Na) remained unchanged (Table 7).
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Patch-clamp experiments in the whole cell configuration.
K-GLUCONATE PIPETTE SOLUTION: EFFECT OF NH4CL.
In a first series of experiments, rumen epithelial cells were brought into the whole cell configuration using the K-gluconate solution described in the MATERIALS AND METHODS (Fig. 4).
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When NaCl was replaced by NH4Cl (130 mM) in the external solution, inward current rose to 189 ± 17% of the original value (100%) measured in NaCl solution at a pipette potential of 120 mV (P < 0.01, n = 17). Outward current at +100 mV rose to a mean level of 132 ± 10% (P = 0.007, n = 17; Fig. 4A). After washout of NH4Cl with NaCl, inward and outward currents recovered to 112 ± 12 and 102 ± 15%, respectively, of the original values in NaCl solution (P = 0.3 and P = 0.9, no significant difference to the value before application of ammonia). Reversal potential rose in all cells studied from a mean value of 33 ± 4 (NaCl) to 6 ± 4 mV (NH4Cl; n = 17, P < 0.001) and back to 25 ± 3 mV (NaCl; P < 0.01).
Conversely, replacement of NaCl in the bath solution with choline chloride did not induce significant changes in either inward current (85 ± 12%, n = 7, P = 0.2), outward current (88 ± 9%, P = 0.9), or reversal potential level (25 ± 7 mV, P = 0.7), in line with a previous study (36).
Exposure to BaCl2 (5 mM), known to block potassium channels, resulted in a significant decrease in NH4Cl-induced inward current to 70 ± 9% (n = 7, P < 0.05, 120 mV) of the level in NH4Cl solution without BaCl2 (100%), whereas outward current dropped to 77 ± 7% (n = 7, P < 0.01, +100 mV) of the original outward current level. Conversely, TEACl (10 mM) had no significant effect on the inward current induced by NH4Cl (102 ± 20%, n = 4, P = 0.9), whereas outward current dropped slightly to 80 ± 7% (n = 4, P < 0.05). Reversal potential was not significantly altered by either the addition of BaCl2 (3 ± 8 mV, n = 7, P = 0.1) or TEA (10.3 ± 15 mV, n = 4, P = 0.05) to NH4Cl bath solution.
K-GLUCONATE PIPETTE SOLUTION: EFFECT OF KCL.
Similar effects were observed when NaCl bath solution was replaced with KCl bath solution (Fig. 4B). This induced a rise of both inward current and outward current to 344 ± 66% (n = 8, P = 0.007, 120 mV) and 218 ± 80% (n = 8, P = 0.003, +100 mV), respectively, with reversal potential rising to 10 ± 7 mV (n = 8, P = 0.01).
In KCl solution, TEACl (10 mM) blocked KCl-induced inward and outward current to values of 58 ± 20 (n = 4, P < 0.05, 120 mV) and 66 ± 18% (n = 4, P < 0.01, +100 mV), respectively. Effects of TEACl on cells in NaCl solution did not reach significance level (n = 3). The effects of BaCl2 (5 mM) were less pronounced, with inward current dropping to 72 ± 17% (n = 4, P < 0.05) of the original level in the presence of KCl, whereas changes in outward current did not reach significance level [92 ± 7% (n = 4, P < 0.05)]. The currents were also blocked by quinidine (100 µM) with inward current sinking to 82 ± 3% (n = 3, P < 0.05), outward current to 47 ± 23% (n = 3, P < 0.05), whereas reversal potential remained the same as in KCl solution.
K-GLUCONATE PIPETTE SOLUTION: INTERACTION OF KCL AND NH4CL AT PH 6.4.
Ussing chamber experiments suggest that influx of NH4+ into ruminal epithelial cells is inhibited by application of K+. To test for the hypothesis that K+ and NH4+ use a common pathway, cells were exposed to solutions containing either 65 mM NH4Cl, 65 mM KCl, or a mix of both ions (osmolarity adjusted with choline chloride). All solutions were titrated to a pH of 6.4, at which the concentration of NH3 should be minimal (Fig. 4C).
Exchanging pH of external NaCl solution from 7.4 to 6.4 did not significantly alter either inward current, outward current, or reversal potential (33 ± 2 mV, n = 8, P = 0.1). Application of NH4Cl (65 mM) increased inward current (120 mV) from 100 to 167 ± 19% (n = 6, P = 0.01), whereas changes in outward current did not reach significance (116 ± 8%, P = 0.1). Reversal potential rose from 33 ± 2 to 24 ± 2 mV (P = 0.02), significantly less than that obtained with 130 mM of NH4Cl [P (unpaired) = 0.009]. Conversely, currents at 120 mV were not significantly different from those observed in solutions containing 130 mM NH4Cl (Fig. 4C), suggesting effects of saturation.
The application of KCl (65 mM) increased mean inward current to 180 ± 21% [n = 4, P (paired) = 0.03], whereas outward current and reversal potential only rose slightly to 137 ± 16% (P = 0.2) and 26 ± 4 mV (P = 0.1), respectively. These values are not significantly different from those obtained with NH4Cl (65 mM) solution.
If the independence principle holds (29), application of a mix of NH4Cl (65 mM) and KCl (65 mM) should result in additive depolarization. Indeed, after application of a mix of both salts, cells depolarized to an end value of 18 ± 2 mV (P = 0.004 vs. NaCl) in KCl + NH4Cl solution or by a difference of 15 ± 9 mV. This value corresponds roughly to the numeric sum of the depolarizations obtained with NH4Cl (65 mM) and KCl (65 mM; 12 ± 5 mV, P = 0.3) as calculated for the individual cells. However, the current responses to NH4Cl + KCl solution were much lower than those predicted by the independence principle. Inward current rose relative to NaCl, reaching 175 ± 27% (n = 7, P = 0.03). Relative to NH4Cl (65 mM), inward current dropped in three cells in KCl + NH4Cl solution (Fig. 4C), rose slightly in two others, and remained unaltered in one cell, with no significant net effect on the mean value. Independence theory would have predicted a rise in all_cells by 80%. A rise of this magnitude was not observed in any of the cells.
NH4Cl + KCl solution did not induce significant changes in outward current level vs. NaCl solution (112 ± 11%, P = 0.3). However, a decrease versus the level in NH4Cl (65 mM) solution could be observed, with outward current dropping visibly in five of six cells (to 85 ± 5% of the level in NH4Cl (100%; n = 5, P = 0.04; Fig. 4C).
Further reduction of NH4Cl concentration to 32.5 mM (choline replacement) resulted in a smaller, but still clearly visible, induction of inward current (n = 2).
Choline chloride pipette solution. To minimize interference between K+ and NH4+, cells were filled with choline chloride pipette solution (Fig. 5, A and B).
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NH4Cl pipette solution. Cells were filled with an NH4Cl pipette solution. In NaCl bath solution, reversal potential was 14 ± 4 mV (n = 12), significantly lower than in choline chloride-filled cells (P = 0.007), reflecting efflux of NH4+.
When cells were superfused with NH4Cl solution (pH 5.8, reduction of NH3), cells depolarized to 4.1 ± 3 mV (n = 8, P = 0.01) with inward current level at 404 ± 139% of the value in NaCl solution, (n = 8, P < 0.05, 120 mV) and outward current at 213 ± 66% (n = 8, P < 0.01, +100 mV; Fig. 5, C and D). Addition of quinidine (100 µM) to NH4Cl solution lowered inward current to 42 ± 17% of the value in NH4Cl solution (n = 3, P < 0.05) and outward current to 43 ± 3% (n = 3, P < 0.01). Reversal potential remained the same (5 ± 4 mV, P = 0.2). Values recovered to 90 ± 20% (n = 3, P = 0.6, inward) and 85 ± 4% (n = 3, P = 0.06, outward) of the level at the beginning of the experiment after washout with NaCl buffer. Superfusion with KCl raised both inward and outward current, both of which were blocked by quinidine (n = 1). Reversal potential changed from 6 (NaCl) to 26 mV (KCl) and to 6 mV (KCl + quinidine).
At a concentration of 65 mM NH4Cl, inward current of the NH4Cl-filled cells rose to 197 ± 54% (n = 4, pH = 6.4), reversal potential to 2.5 ± 6. Inward current did not change significantly when KCl was given in addition to NH4Cl (each at 65 mM; 205 ± 52%, n = 4), whereas reversal potential rose to 19 ± 4 mV [n = 4, P < 0.01 vs. NH4Cl (65 mM) alone]. KCl (65 mM) hyperpolarized two of three tested cells, suggesting that in these cells, stimulation of NH4+ efflux (at 130 mM) exceeded the additional influx of K+ (65 mM).
Relative permeability ratios. In a previous study (36), we were able to show that K-gluconate-filled cells were depolarized by removal of external chloride, so that in NaCl solution, PK/PNa must be <1.8 ± 0.5. From these measurements, it appears that the larger part of the outward current and the reversal potential in NaCl solution are due to chloride influx and not to K efflux. A similar estimate of <1.6 can be obtained (indirectly) by comparing cells filled with Cs-methanesulfonate, CsCl, choline chloride, and K-gluconate (36).
On the other hand, superfusion of the cells with KCl solution also resulted in a depolarization of similar magnitude as that observed after removal of Cl. If PK/PNa is calculated from the reversal potentials (as described in MATERIALS AND METHODS) in NaCl solution and in KCl solution (both 130 mM), a much higher value of PK/PNa = 5 ± 2 is obtained. This discrepancy can be resolved if one assumes that permeability for potassium rises with the concentration of this ion in the bath solution. This assumption is confirmed by observing the increase in (TEACl sensitive) outward current after superfusion with KCl solution.
Other permeability ratios at 130 mM were: PNa/Pcholine = 1.0 ± 0.1, PNH4/PNa = 2.8 ± 0.4, and PNH4/PK = 0.7 ± 0.4.
A relative permeability ratio for NH4+ efflux in NaCl solution can be calculated from NH4Cl-filled cells superfused with NaCl solution and choline-filled cells superfused with NaCl solution, yielding PNH4/Pcholine = 2.6 (130 mM), which is in good agreement with the value obtained for PNH4/PNa (130 mM) from K-gluconate-filled cells. This suggests that the permeability of ruminal cells for NH4+ is not as dependent on concentration as that of K+.
Patch-clamp experiments in the single-channel configuration. Experiments in the inside-out configuration of the patch-clamp technique with symmetrical 130 mM NH4Cl solution in pipette and bath showed a channel with a conductance level of 135 ± 12 pS (n = 5; Fig. 6, A and D).
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DISCUSSION |
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In diarrhea, pH values of stool water >8.00 have been measured (44) with a concurrent rise in NH3 concentration. This should elevate intracellular pH and reduce the number of protons available for extrusion by the NHE. This mechanism could help to explain the loss of sodium and water in these conditions.
In sheep and cattle, animals not given sufficient time to adapt to a diet rich in energy and protein (as in fattening) are prone to a condition known as ruminal acidosis (22, 32). Large shifts in the NH4+/NH3 ratio occur, and uptake of protons not only due to the uptake of fatty acids as previously described (24) but also as NH4+, should be discussed. Thus a better understanding of the uptake routes for ammonia in the rumen and the interaction between ammonia and the NHE appears necessary.
The current study demonstrates that ammonia crosses the ruminal epithelium not only in the form of lipophilic NH3 but also in its ionized form as NH4+. The relative transport rates of the two forms depend on ruminal pH, with an impact on the absorption rate of Na+ by the rumen epithelium via NHE.
The suggestion of NH4+ uptake at pH 6.40 through a putative, quinidine-sensitive K channel by Bödeker and colleagues (5, 6) is supported by the present study. In patch-clamp experiments, we were able to show that ruminal epithelial cells express quinidine-sensitive channels of 130 pS that conduct potassium and NH4+, but not sodium, with interference when K+ and NH4+ are applied simultaneously. Competition between NH4+ and K+ for a common, electrogenic uptake pathway could also be demonstrated in Ussing chamber experiments.
The pH-dependent absorption of ammonia from the rumen has long been known (25). The increase of ammonia toxicity at a luminal pH greater than 7.30 (16) supports the assumption of an enhanced absorption of NH3 with increasing pH. This suggestion is supported by the determination of ammonia flux rates in this study. The wide physiological variation of ruminal pH (5.47.4) and ammonia concentrations (up to 70 mM) only has a minimal effect of 1.8% on the concentration of NH4+ but great impact on free NH3, which increases from 0.07 to 0.68 mM (at 30 mM luminal ammonia), resulting in an almost linear increase in NH3 flux through the epithelial cells (Fig. 2). Conversely, at the pH values below 6.9, which predominate in the rumen, uptake of NH4+ as a source of protons must be considered. It is a well-known fact that both NH3 and NH4+ affect pHi in opposite directions (7, 42, 46) and should thus have an impact on the amount of Na+ transported by the NHE.
Recent in vitro studies with isolated rumen epithelial cells (REC) have demonstrated that recovery from acidification of pHi is reduced by inhibitors of NHE and that exposure of REC to 20 mM NH4Cl induces an increase in pHi to 7.8 (42), which inhibits NHE (2). In line with this observation, our findings indicate that in the intact rumen epithelium, electroneutral Na transport is abolished at pH 7.4. The remaining Jnet (Na) at 30 mM ammonia represents electrogenic Na transport and accounts for Isc. Jnet (Na) and Isc have the same magnitude if the measured Isc (1.02 µeq·cm2·h1) is corrected for the NH4+-dependent component (some 0.4 µeq·cm2·h1) of Isc [1.020.40 = 0.62 µeq·cm2·h1 vs. a Jnet (Na) of 0.74 µeq·cm2·h1; see Table 1].
Thus it emerges that at a ruminal pH of 7.4 and at physiological ammonia concentrations, electroneutral Na transport is inhibited (Fig. 7A). Lowering the luminal pH from 7.4 to 6.9 does not alter Na fluxes. A further decrease of luminal pH to 6.4 causes a concentration-dependent increase in Na transport (Fig. 7B). The involvement of the NHE in ammonia induced enhanced Na transport is supported by the observation that mucosal amiloride (1 mM) prevents the stimulating effect of ammonia at a luminal pH of 6.40.
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Despite the high value of PNH3/PNH4+, which can lead to the incorrect assumption that transport of NH4+ is "negligible," the amount of ammonia absorbed in the ionic form is considerable due to the low concentration of NH3 in relationship to NH4+ at physiological levels of pH. At neutral pH, and a total concentration of 30 mM ammonia, only 0.27 mM is in the form of NH3 so that even if the permeability of NH4+ is 175 times lower than that of NH3, the concentration is higher by a factor of 110. This means that for every millimole per liter crossing the membrane as NH3, 110/175 = 0.63 mmol or 40% of the total amount of ammonia will be transported as NH4+. At a slightly acidic pH of 6.4 ([NH3] = 0.07 mM), conversely, >70% is absorbed as NH4+, whereas at a pH of 7.4 ([NH3] = 0.68 mM), 80% of ammonia is absorbed as NH3. This is in good agreement with our observations concerning the stimulation of sodium transport via the NHE at a pH of 6.4 and its inhibition at a pH of 7.4.
The patch-clamp data in this study show that ruminal epithelial cells express quinidine-sensitive potassium channels, as suggested previously (5). Whereas additional uptake of ammonia through specific transporters cannot be ruled out (31, 53, 54), these transporters cannot explain the large NH4+-induced currents observed in both the Ussing chamber and patch-clamp data in this study. Note that transporters from the Rh gene family are typically saturated by a few millimoles per liter of NH4+, and that transport is not affected by a change in membrane potential (54).
Conductance of NH4+ through nonselective cation channels has been reported for a number of other tissues (10). In rumen, this appears unlikely. The internal solution for patch-clamp experiments was chosen to minimize contributions of the nonselective cation channel (36), and no appreciable increase in conductance of sodium compared with choline could be observed. Note that under physiological conditions with divalent containing solution, the sodium conductance of rumen epithelium is saturated at 30 mM Na+ (20, 47). However, exposure to NH4+ at pH 6.4 induced an additive increase in the transport rates of NH4+ and Na+, with a corresponding increase in Isc. Thus there was no sign of competition between Na+ and NH4+ for a common pathway. In contrast, K+ and NH4+ interfered with each other both in the patch-clamp and Ussing chamber experiments.
The question may arise if the currents observed in the patch-clamp measurements were induced by cell swelling or changes in pHi due to influx of NH3. However, capacitance remained stable, and both induction of current and depolarization could be observed at values of pH (5.8, 6.4), at which the concentration of NH3 was negligible. Note also that stimulation of current was observed in all cases. Stimulation of potassium and/or chloride currents should induce hyperpolarization in K-gluconate-filled cells, and not the depolarization observed. Effects of ammonia persisted after the removal of either intracellular chloride (as in the K-gluconate experiments) or potassium (as when NH4 or choline was used to replace K in the pipette solution). The experiments also show that reversal potential increases when NH4Cl is used to replace choline chloride externally and decreases when the same is done internally, in line with our assumption.
The NH4Cl-induced inward current was blocked by BaCl2 or quinidine but not by iberiotoxin. Single-cell experiments showed a conductance of 130 pS in symmetrical NH4Cl solution that was permeable to both K+ and NH4+ with a PNH4/PK of 0.5 ± 0.5. This permeability ratio, derived from the reversal potential (29), corresponds roughly to the slightly higher value of PNH4/PK = 0.7 ± 0.4 found in whole cell experiments at a 130 mM concentration of both ions.
However, note that under conditions in which the pore is saturated, the permeability ratio as derived from the reversal potential deviates from the ratio of the absolute permeabilities, as derived by dividing the absolute current values (29). This effect can be seen clearly in Fig. 4C, where current is not doubled by doubling the concentration of NH4+, as independence theory would demand.
Permeation properties of K+ channels are generally similar in that they must reconcile two apparently contradictory properties: a high rate of ion conductance and a high selectivity (4, 29). A single-file, multi-ion pore is the preferred model and predicts the effects that we observed in ruminal epithelial cells, such as the fact that currents induced by a mix of K+ and NH4+ were lower than the sum of the currents induced by each ion alone (Fig. 4, C and D); in other words, that the two ions interfered with each other when flowing through the lumen of the pore(s). Typically, effects on reversal potential were not pronounced: at the reversal potential, the flux of ions through the pore comes to a halt.
Another seemingly paradox observation is that in the current study, we observed stimulation not only of inward, but also of outward current when cells were exposed to either K+ or NH4+ from the external side.
Again, the multi-ion channel model predicts such effects. Electrostatic repulsion speeds ion flow when such channels become multiply occupied. In many cases, channel pores have to be fully occupied (with a distinct number of ions) before a current begins to flow. Another property of multi-ion channels leading to concentration-dependent permeability is that permeant ions entering the pore from one side may nudge out ions blocking the pore from the inside. Thus the channel can discriminate between currents into the cell and out of it; in other words: the channel is (inwardly or outwardly) rectifying (29).
Alternately, occupancy of an ion-selective site in the pore by a permeant ion from either the outside or the inside via the "foot-in-the-door" mechanism is known to prevent entry into the inactivated state in potassium channels with C-type inactivation (29, 56).
These basic properties of potassium channels may explain an old paradox. It is undisputed that elevation of potassium depolarizes the membrane of ruminal epithelium (20, 35, 40) and that potassium is absorbed across the ruminal wall (50), stimulating Isc (5). However, various studies have shown that in NaCl Ringer, apical secretion of potassium is minimal (21, 35) despite the activity of the basolateral Na/K-ATPase.
We suggest that permeability for potassium is, indeed, low in apical NaCl solution and may be further reduced by the presence of NH4+ but is stimulated by the elevation of mucosal potassium. The physiological importance of this mechanism is evident in that it prevents a drain of potassium from the serosal side into the ruminal lumen under low-potassium conditions. Note that in multi-ion channel theory, NH4+ ions should be able to "sweep" individual potassium ions caught in the single file of NH4+ streaming through the pore into the cell, even against an electrochemical gradient for potassium (29).
Beyond this, we are unable to precisely identify the potassium channel or, more likely, group of channels that conducts ammonia in ruminal epithelium. Unfortunately, data on the large family of potassium channels are incomplete (27) and depend greatly on the tissue in which the channel is expressed. Conductance of NH4+ by various potassium channels is well documented (27), as is the presence of potassium channels in the apical membrane of intestinal tissues (18), which may represent a pathway for NH4+ efflux (8) from the lumen. Large-conductance calcium-activated potassium channels have been reported to conduct NH4+, but these channels display only minimal open probability at hyperpolarized voltages, ruling out these channels as a possible route for NH4+ influx in our experiments, as does the lack of an effect of iberiotoxin.
It is difficult to resist speculation concerning the role of NH4+ for the pathophysiology of hypomagnesaemia in ruminants. Hypomagnesaemia (grass tetany) is linked to use of artificial fertilization techniques with high concentrations of potassium and ammonia. Inhibition of magnesium absorption not only by K+ (11, 35, 36, 48) but also by ammonia has been known for many years (11, 23) as has the reduction of magnesium uptake via depolarization of the apical membrane (35, 48). Thus we suggest that uptake of NH4+ inhibits magnesium absorption via depolarization of the apical membrane, as previously described for K+ (11, 35, 48).
Care et al. (11) also noted that inhibitory effects of NH4+ and K+ on Mg2+ uptake were additive, which is in line with additive effects of NH4+ and K+ on membrane potential as suggested by this study. Inhibition of Na transport by ammonia at a pH of 7 was also reported, which can be attributed to the fact that at this pH, transport of NH3 is larger than transport of NH4+. However, the finding that addition of K+ results in a further decrease in Na absorption is in contrast to the effects of K+ alone (34, 36, 50). We suggest that in Care's experiments, K+ inhibits flux of NH4+ into the cells. Thus the stimulatory effect of NH4+ on NHE is terminated, and the inhibitory effect of NH3 (alone) leads to a net inhibition of sodium transport by elevation of K.
In conclusion, this study presents evidence that in the rumen, electroneutral Na transport is inhibited by ammonia entering the cytosol in the form of NH3 and is stimulated by entry of NH4+ through potassium channels. The luminal pH determines the predominant form of luminal ammonia uptake, NH3 or NH4+ and, hence, the effect of ammonia on Na transport. Because the physiological pH of the ruminal fluid is <6.9, ammonia enhances Na absorption. This modulation of NHE activity by ammonia appears to be as important as the "classic" effect of short-chain fatty acids (24, 33). Furthermore, it is interesting to note that electrogenic uptake of ammonium increases with adaptation to a high-protein diet (1).
The basic physiological mechanism outlined in this study should be valid not only for the rumen, but also for the human colon due to the similarities between the two tissues, namely electroneutral Na transport via NHE (19, 37, 43), abundance of K channels (33, 51), and a wide variation of ammonia concentrations and pH in the ingesta (9).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* K. Abdoun and F. Stumpff contributed equally to this work.
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