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Am J Physiol Gastrointest Liver Physiol 294: G429-G440, 2008. First published November 21, 2007; doi:10.1152/ajpgi.00251.2006
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MUCOSAL BIOLOGY

Ammonium transport in the colonic crypt cell line, T84: role for Rhesus glycoproteins and NKCC1

Roger T. Worrell,1,2 Lisa Merk,1 and Jeffrey B. Matthews3

Departments of 1Surgery and 2Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio; 3Department of Surgery, University of Chicago, Chicago, Illinois

Submitted 8 June 2006 ; accepted in final form 15 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although colonic lumen NH4+ levels are high, 15–44 mM normal range in humans, relatively few studies have addressed the transport mechanisms for NH4+. More extensive studies have elucidated the transport of NH4+ in the kidney collecting duct, which involves a number of transporter processes also present in the distal colon. Similar to NH4+ secretion in the renal collecting duct, we show that the distal colon secretory model, T84 cell line, has the capacity to secrete NH4+ and maintain an apical-to-basolateral NH4+ gradient. NH4+ transport in the secretory direction was supported by basolateral NH4+ loading on NKCC1, Na+-K+-ATPase, and the NH4+ transporter, RhBG. NH4+ was transported on NKCC1 in T84 cells nearly as well as K+ as determined by bumetanide-sensitive 86Rb-uptake. 86Rb-uptake and ouabain-sensitive current measurement indicated that NH4+ is transported by Na+-K+-ATPase in these cells to an equal extent as K+. T84 cells expressed mRNA for the basolateral NH4+ transporter RhBG and the apical NH4+ transporter RhCG. Net NH4+ transport in the secretory direction determined by 14C-methylammonium (MA) uptake and flux occurred in T84 cells suggesting functional RhG protein activity. The occurrence of NH4+ transport in the secretory direction within a colonic crypt cell model likely serves to minimize net absorption of NH4+ because of surface cell NH4+ absorption. These findings suggest that we rethink the present limited understanding of NH4+ handling by the distal colon as being due solely to passive absorption.

RhBG; RhCG; colon; hyperammonemia

THE OBSERVATION THAT PORTAL vein ammonium (NH3 + NH4+) concentration (~350 µM) is substantially higher than systemic levels (~45 µM) is prima facie evidence that NH4+ production and/or absorption occurs in the gastrointestinal tract (7, 41). In particular, the colon is an ammonium-rich organ because of bacterial fermentation of nonabsorbed dietary nutrients in the luminal compartment, leading to concentrations typically around 14–20 mM but as high as 100 mM (41). It is well accepted that the colonic mucosa absorbs ammonium, that is, that the direction of net colonic movement of NH3/NH4+ (Jnet, representing the difference between mucosal-to-serosal and serosal-to-mucosal fluxes, Jms – Jsm) is in the absorptive direction. However, the extent to which the colonic mucosa can generate a significant secretory flux of NH3/NH4+ (i.e., a nonzero Jsm) against the larger absorptive vector Jms is unknown. Moreover, the possibility that transepithelial secretion and absorption of NH3/NH4+ might occur in different cell types (as it does for Na+ and Cl–) has not been addressed previously. However, it is intuitively appealing to consider that a subset of colonic epithelial cells might actively extrude NH4+ so as to temper the otherwise unopposed interstitial accumulation of this toxic substance. Indeed, there are a number of examples in nature where epithelia faced with a high external NH4+ concentration are able to actively excrete NH4+ against an uphill gradient (38, 40, 43, 47).

Although there are some data that address absorptive NH3/NH4+ flux (Jms), the cell type and pathways remain incompletely defined. It is controversial whether absorption occurs by the primary movement of NH3 or NH4+. Early studies supported a NH3 diffusion model on the basis of the pH sensitivity of ammonium movement (3, 4, 55); however, this does not appear to be the case for ammonium movement across mammalian ileum (28, 29). Recent studies indicate that colonic crypts, gastric glands, and renal cells have a low apical NH3 permeability (2, 18, 42, 44). Entry of NH4+ in the absorptive direction has been postulated to occur via apical Ba2+-sensitive K+ channels in the surface epithelium (10, 21) or perhaps via aquaporin-1 (33).

In contrast, there is almost no information concerning NH4+ secretory flux (Jsm) in intestine. NH4+ secretion in other epithelia occurs via its transport on known K+ transporters owing to the similar hydrated radius of K+ and NH4+. In most models, the basolateral loading step for NH4+ uses the K+ site on Na+-K+-ATPase and/or the Na+-K+-2Cl– cotransporter NKCC1. Apical ammonium exit pathways vary from system to system. Passive diffusion of NH3+ across the apical membrane in combination with H+ secretion by H+-ATPase results in an "ion trapping" mechanism of secretion in fresh water fish gill (51) and possibly in mammalian inner medullar collecting duct (IMCD) (45). In contrast, in the case of salt water fish gill (51) and the desert locust, Schistocerca gregaria, rectum (43), apical secretion of NH4+ occurs by replacing H+ on an apical Na+-H+-exchanger (NHE). In the giant mudskipper, Periophalmodon schlosseri, gill, both ion trapping and NHE are used to maintain NH4+ gradients up to 100 mM (37, 38). In the green crab, Carcinus maenas, NH4+ transport across the apical membrane occurs by first vesicular ion trapping of NH4+ and then microtubule-dependent vesicular release (40, 47). An alternative model for mammalian IMCD involves NH4+ exit on the H+ site of colonic-type H+-K+-ATPase, with K+ recycling occurring on ROMK1 K+ channel (30). A common feature of the above systems is physiological exposure to a significant NH4+ concentration ([NH4+]) (20–100 mM). Human colonic epithelia exposure is within this elevated range (41). Apical NHEs and colonic-type H+-K+-ATPase are present in the apical membrane of colonic epithelia (22). Likewise, Na+-K+-ATPase and NKCC1 are present. Given a similar environment and the fact that many of the same basolateral and apical membrane transport pathways are present in colonic cells circumstantially, this suggests the colon may, indeed, actively secrete ammonia as do these other tissues.

Perhaps one of the most significant recent discoveries involving NH3/NH4+ permeability is that certain mammalian nonerythroid cells express members of the SLC42 solute carrier family (14, 32, 48). The SLC42 family is comprised of the human Rhesus-associated glycoproteins (RhG), which are homologous to the yeast Mep family of proteins (15). These proteins transport both ammonia, as well as methylammonium (MA). It is notable that the Mep transporters can concentrate MA by ~1,000-fold (9, 39). Not surprisingly, RhGs are found predominately in tissues that are exposed to elevated NH4+ levels such as liver, kidney, and stomach (24, 27, 48, 49, 50). RhGs have been detected in the gut, including proximal colon (13). Interestingly, two of the family members show polarized expression; RhBG is expressed in the basolateral membrane, and RhCG is apically expressed (48). Thus each is poised to be a potential player in the vectorial transport of NH3/NH4+ (32). The actual mode of NH3/NH4+ transport by the SLC42 family is not clear. Transport has been shown to occur in an electrogenic manner (31, 34) as electroneutral transport by acting as an NH4+-H+ exchanger (11, 12, 25, 26) and as an NH3 "gas channel" (17, 20, 23). Part of the confusion may derive from the results showing that the affinity for NH4+ is affected by NH3 (1). Regardless, the functional significance of RhG proteins in gut has not yet been elucidated.

As a first step in understanding the possible contribution of an NH3/NH4+ secretory vector (Jsm) to ammonium handling by the colonic mucosa, we used the colonic cell line T84 as a model of crypt enterocytes to define the relative contributions of NKCC1 and RhGs in basolateral uptake and transepithelial movement of NH3/NH4+. Our data suggest that colonic transport of ammonium includes a secretory vector that is nonzero and may significantly limit net mucosal absorption and, by extension, systemic accumulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. T84 cells (8) obtained from Jim McRoberts (University of California, Los Angeles) were grown to confluence at pH 7.4 in 162-cm2 flasks with DMEM-Hams's: F-12, 1:1 mix supplemented with 6% fetal bovine serum, 15 mM HEPES, 14 mM NaHCO3, 170 µM penicillin G, and 69 µM streptomycin sulfate. Amphotericin B was not included in the media to avoid potential complications due to its ionophoretic activity. Cells were maintained in culture with weekly passage by trypsinization in Ca2+- and Mg2+-free phosphate-buffered saline at a split ratio of 1:2. Cells for experimentation were plated on uncoated 12- or 24-mm Costar Transwell (0.4-µm pore size) inserts at a seeding density of 4–5 x 105 cells/cm2 and cultured 8–14 days with feeding in the above media three times per week. Cell monolayers were determined to be of acceptable use when the transepithelial resistance reached >1,200 {Omega}·cm2 as measured by an electrovoltohmmeter (EVOM) (World Precision Instruments) (see below). All experiments were performed at 37°C.

Epithelial monolayer integrity. The quality of high resistance monolayer formation was monitored using an EVOM as described previously (52, 54). The T84 cell line used in these experiments typically had a transepithelial resistance of >2,000 {Omega}·cm2. Both transepithelial potential (mV) and transepithelial resistance (k{Omega}) were measured with this instrument. Transepithelial current was calculated by Ohm's law and expressed as µA/cm2. EVOM measurements proved consistent and reliable for detecting changes in transepithelial voltage, resistance, and current (52–54). Moreover, measurements in the open-circuit mode represent the normal condition (e.g., not short circuited) of native epithelia.

Real-time RT-PCR of RhBG and RhCG. Total RNA was extracted from T84 cells with TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription was performed by using 50 µg/ml oligo(dT) 20 primer by using SuperScript reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Amplification reactions were performed with 1x SybrGreen PCR master mix (Applied Biosystems) or with a gene-specific fluorescent-labeled probe, gene-specific primers, and 200-ng sample cDNA in a 50-µl final volume. Real-time RT-PCR was performed on a DNA Engine Opticon 2 detector (DYAD). Thermal conditions (50°C for 2 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min) were used for all primer sets. Emitted fluorescence was measured during the annealing/extension phase; amplification plots were generated by using the Opticon Monitor Analysis software. Standard curves were generated for each primer set (Table 1) by using a known concentration of plasmid DNA; the amount of mRNA was then extrapolated and normalized to GAPDH for comparison.


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  		Table 1. Primer sets used for PCR

 
Plasmids were made by obtaining a portion of the gene by performing RT-PCR on cDNA from T84 cells that were positive for the gene. The RT-PCR product was then cloned into a TA cloning vector. Real-time RT-PCR was performed to confirm that the plasmid contained the insert. A standard curve was then generated from known concentrations of plasmid DNA for each primer set. Well behaved standard curves for each primer set were obtained, r2 ≥ 0.993.

14C-MA uptake and flux. T84 cell monolayers were grown on 1-cm2 Transwell inserts. Uptake and flux measurements were carried out in the following base solution (in mM): 135 NaCl, 3 KCl, 0.5 CaCl2, 2 MgCl2, 10 Na-HEPES at a pH of 7.4. Apical media volume was 0.5 ml and basolateral media volume 1 ml. For apical uptake and apical-to-basolateral flux (Jab) MA studies, apical volume was reduced to 250 µl to conserve 14C-MA tracer. Cells were incubated in cold MA for 5–10 min before uptake/flux initiation. Use of tracers, which are transported as well as diffusible through the cell membrane (exist in both charge and uncharged form), present a unique situation to classical ion tracer experiments. It is noteworthy that the steady-state condition may not be obtained during this incubation time; thus uptake and flux measurements may represent an overestimate of true values. However, experiments shown are comparable to those published by Handlogten et al. (11, 12) and thus support the functional presence of RhGs in T84 cells. Long-term measurements were carried out in culture media with cells kept in the tissue culture incubator between time points. Under these conditions "steady state" does exist. Short- and long-term fluxes are consistent, thus supporting the physiological relevance of the methods used. For consistency with initial studies and those involving NKCC1-mediated NH4+ transport, study of ammonium transport by RhGs was done under conditions of active cAMP-dependent Cl– secretion (stimulated by basolateral 10 µM forskolin, 5 min). As subsequently demonstrated by data in Fig. 8, A and B, RhG function is not altered by forskolin. When bumetanide or ouabain was used, drugs were applied 5 min before initiation of uptake and flux.


Figure 8
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  		Fig. 8. cAMP effect on MA and NH4+ transport. A: basolateral MA uptake and Jba flux in the absence or presence of 10 µM forskolin (FSK). B: apical MA uptake and Jab flux in the absence or presence of 10 µM forskolin. For A and B, symmetrical 1 mM MA and a 2-min sample time were used. C: Jba flux of NH4+ in the absence or presence of 10 µM forskolin. Initial condition was 0 mM apical and 10 mM basolateral NH4+. D: Jab flux of NH4+ in the absence or presence of 10 µM forskolin. Initial condition was 10 mM apical and 0 mM basolateral NH4+. For C and D, a 5-min sample time was used, and n = 6 for each bar. NS, no statistical significance and *P < 0.05 for control vs. forskolin-treated groups.

 
Jab and basolateral-to-apical flux (Jba) are used to indicate absorptive (Jms) and secretory (Jsm) vector (direction) of flux as would occur in intact colon. Jnet is used to indicate the combined effect of each vector, i.e., Jba – Jab (When plotted together, Jab is assigned a negative value.). 14C-MA (1 or 0.5 µCi/ml for the long-term uptake and flux) was used. Radioactivity in an aliquot of initial loading media was determined to establish specific activity for each experiment. At the end of the flux period (typically 2 min) the entire volume of transmedia was collected and radioactivity determined to calculate transepithelial flux. Uptake was ended by sequentially dunking inserts into three reservoirs of >500 ml ice-cold 0.1 M MgCl2, 10 mM Tris·HCl (pH 7.4). Inserts were then cut from the supports and placed in scintillation vials with scintillation cocktail to determine total cellular radioactivity. For uptake calculations, the radioactivity of the cellular fraction and transmedia fraction were combined. Previously, we determined that correcting for protein content by protein measurements on individual inserts did not lead to a significant decrease in variability and were thus omitted to simplify the assay (54); data is expressed per unit area of the epithelial monolayer.

Ammonium assay. Ammonium determination was accomplished using a spectrophotometric ammonium assay kit (Megazyme, Wicklow, Ireland) on the basis of the enzymatic conversion of NADPH to NADP in the presence of 2-oxoglutarate and glutamate dehydrogenase. The assay has a variability between duplicate measurements of 1–2 µM NH4+ (0.005–0.010 absorbance units) with a linear range of ~10 µM to 5.5 mM NH4+. Media from experiments was sampled (0.1 ml) and immediately placed on ice, and necessary samples were frozen at –20°C until the time of the assay. Unknown samples with anticipated [NH4+] >5 mM were diluted accordingly to achieve an assay [NH4+] approximating 2.5 mM. Data are presented either as actual NH4+ movement (nmol·min–1·cm–2) or as accumulated NH4+ (mM) for longer-term experiments. As for 14C-MA uptake and flux, drugs were applied 5 min before initiation of the flux (exposure to NH4+).

Reagents. Forskolin was obtained from Calbiochem, and FBS and PenStrep were from GIBCO BRL; all other chemicals were of the highest grade obtainable from Sigma, Invitrogen, or Stratagene.

Significance tests. Data are presented as means ± SE. Significance was estimated using the Student's t-test and two-way ANOVA when indicated, with a P value of less than 0.05 indicating a significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
T84 cells express ammonium transporters RhBG and RhCG. The presence of ammonium transporters, RhBG and RhCG, along the gastrointestinal tract have been shown by Handlogten et al. (13). Each isoform was determined to be present in mouse ileum and proximal colon. Although the authors concluded that expression was predominantly in the surface epithelium of proximal colon, their study did not include distal colon. Thus we sought to determine the presence of RhBG and RhCG mRNA in a human secretory cell line of colonic crypt origin (T84). By using specific human primers and real-time RT-PCR, mRNA for both RhBG and RhCG were readily detected in T84 cells, thus suggesting the expression of these transporters within secretory cells characteristic of colonic crypt cells (Fig. 1). Quantitative analysis of real-time RT-PCR showed approximately equal expression of RhBG and RhCG in T84 cells, ratio of RhBG/RhCG = 1.10 ± 0.15 (n = 8).


Figure 1
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  		Fig. 1. mRNA for the ammonium transporting Rh glycoproteins (RhGs) is present in T84 cells. The end products at RT-PCR cycle completion of representative real-time RT-PCR reactions (2 for each RhBG and RhCG) are shown by PAGE gel electrophoresis. Quantitative analysis of real-time RT-PCR reactions yielded a ratio of RhBG/RhCG of 1.10 ± 0.15 (n = 8). Products of the predicted size (~70 bp) are indicated by the arrow. Control RT-PCR reactions did not reveal any product (not shown).

 
RhBG (typically basolateral) and RhCG (typically apical) (48) are known to transport MA; thus they can be functionally assayed using 14C-MA uptake and flux. Fig. 2 supports the functional presence of each transporter in T84 cells. Apical side 14C-MA uptake was significantly less than that of basolateral side uptake, 139 ± 5 vs. 390 ± 16 pmol·min–1·cm–2 MA, respectively (Fig. 2A). NH4+ competitively reduces the magnitude of RhG protein-mediated MA transport (11, 12). Indeed, 14C-MA uptake was found to be reduced in the presence of 10 mM NH4+. Inclusion of 10 mM NH4+ reduced apical 14C-MA uptake ~36% to 25 ± 1 pmol·min–1·cm–2 MA. Basolateral 14C-MA uptake was reduced ~44% to 217 ± 16 pmol·min–1·cm–2 MA by 10 mM NH4+. These data together indicate the functional presence of RhG proteins at both the apical and basolateral membrane of polarized T84 monolayers. In addition, the data suggest that basolateral loading of NH4+ exceeds apical loading (presumably by RhBG and RhCG, respectively, although this has not been directly determined in T84 cells). Transepithelial flux with symmetrical 1 mM cold MA reveals a net secretory direction of flux in T84 cells (Fig. 2B). The apical-to-basolateral flux (Jab) of MA was –134 ± 7 pmol·min–1·cm–2 MA, whereas the basolateral-to-apical flux (Jba) was 241 ± 7 pmol·min–1·cm–2 MA, resulting in a calculated net flux (Jnet) of 106 ± 14 pmol·min–1·cm–2 MA. Inclusion of 10 mM NH4+ reduced Jab by ~22% to –104 ± 6 pmol·min–1·cm–2 MA, Jba by 31% to 167 ± 6 pmol·min–1·cm–2 MA, and Jnet by ~41% to 63 ± 12 pmol·min–1·cm–2 MA.


Figure 2
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  		Fig. 2. Functional determination of RhG activity in T84 cells. In addition to NH4+, methylammonium (MA) is transported by the RhGs; thus their activity can be assessed by [14C]-MA uptake and flux. A: uptake of [14C]-MA when applied to the apical or basolateral side of T84 cell monolayers. The magnitude of uptake is significantly greater when applied basolaterally. MA uptake from either side is attenuated by the addition of NH4+ characteristic of RhG activity. B: transepithelial flux of MA in T84 cells. Tracer [14C]-MA was added to either the apical or basolateral side to determine unidirectional flux in either direction. The combined results demonstrate net secretion of MA. Transepithelial MA flux was attenuated by the addition of NH4+ characteristic of RhG activity. In A and B, symmetrical 1 mM cold MA was used with 1 µCi/ml 14C-MA applied to either the apical or the basolateral side. Uptake and flux period was 2 min. C: long-term MA uptake over a 60-min period obtained in cells bathed in culture media and kept in tissue culture incubator. D: long-term MA flux over a 60-min period for cells bathed in culture media and kept in tissue culture incubator. The combined results are qualitatively similar to those in A and B. In C and D, symmetrical 1 mM cold MA was used with 0.5 µCi/ml 14C-MA applied to either the apical or the basolateral side; n = 6 for all groups. Jab, apical-to-basolateral flux; Jba, basolateral-to-apical flux; Jnet, combined effect of each vector.

 
To determine if net secretion occurs over a longer time period under culture conditions, long-term flux experiments were carried out with T84 cell monolayers over a 60-min period in complete culture media. Results are qualitatively similar to short term (2-min flux period). Calculated rates (Fig. 2C) were 257 ± 11 for basolateral and 107 ± 5 pmol·min–1·cm–2 MA for apical uptake. Jba was 113 ± 8, and Jab –44 ± 3 pmol·min–1·cm–2 MA, resulting in a secretory direction flux (Jnet) of 69 ± 7 pmol·min–1·cm–2 (Fig. 2D). The lower values for long-term flux rates may be due to the different bathing conditions (culture media vs. minimal media) or possibly could represent the effect of closer to steady-state intracellular MA concentration ([MA]) in the long-term experiments. Nonetheless, qualitatively the data, in whole, support an appreciable secretory direction flux due to RhG activity.

Specificity of 14C-MA as a tracer for RhG-mediated uptake and flux. 14C-MA is known to be transported by the RhGs. However, it is uncertain the degree to which MA may also be transported by NKCC1 (as defined by bumetanide-sensitivity). We therefore examined the effect of bumetanide on 14C-MA uptake and flux. Figure 3A shows the dose response relation for basolateral MA uptake (5 min) in T84 cell monolayers in the absence and presence of 100 µM bumetanide. Bumetanide produced a small decrease in MA uptake, which was apparent at greater concentrations of cold MA. We also observed [consistent with results of Handlogten et al. (12)] that, as [MA] increases, there develops in parallel a nonsaturable component of MA uptake, representing linear diffusive entry, particularly at concentrations above ~5 mM MA. To determine the time course of MA uptake and flux, as well as the sampling rate at which RhG activity could be more accurately accessed, 3 mM [MA] was used. This concentration was chosen to provide an extra safety margin in sampling times. The time course of MA uptake (Fig. 3B) and transcellular flux (Fig. 3C) indicate little effect of bumetanide on MA uptake or flux at time points less than ~3 min. However, at longer time points (>5 min), a more significant bumetanide-sensitive component for MA uptake is revealed. Since these data were obtained at 3 mM MA, use of 1 mM MA in combination with a 2-min sample period is more than adequate to access specific RhG function. Transepithelial flux of MA is less sensitive to bumetanide treatment, even at extended flux durations. There was no discernable K+ competition (measured by Dixon plot with 1 and 3 mM MA and 0–10 mM K+) for the total (Fig. 3D), as well as bumetanide-sensitive (Fig. 3E) component of MA uptake, as would be expected if MA were carried on the K+ site of NKCC1 or the pump, suggesting that this small effect of bumetanide on MA uptake is indirect. The greater impact of bumetanide at longer time periods and a slightly greater effect on uptake vs. transepithelial flux are supportive of an indirect effect as well.


Figure 3
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  		Fig. 3. MA uptake and flux in T84 cells. A: dose response for MA uptake in the absence and presence of basolateral 100 µM bumetanide. Total (bullet), bumetanide-insensitive ({blacktriangledown}), and bumetanide-sensitive ({blacksquare}) uptakes are shown. Uptake period was 2 min. Uptake represents combined uptake due to RhBG transporter activity and liner diffusive uptake (>5 mM MA). B: time course of MA uptake with 3 mM symmetrical cold MA. Total (bullet), bumetanide-insensitive ({blacktriangledown}), and bumetanide-sensitive ({blacksquare}) uptakes are shown. A significant bumetanide-sensitive component of MA uptake is observed only at uptake times exceeding 3–4 min. C: time course of completed transepithelial MA flux in the secretory direction. Total (bullet), bumetanide-insensitive ({blacktriangledown}), and bumetanide-sensitive ({blacksquare}) fluxes are shown. 3 mM symmetrical MA was used. A slight bumetanide-sensitive component of MA flux is observed only at time points exceeding 5 min, and n = 4 for each data point. *P < 0.05 D: Dixon plot of total basolateral MA uptake at varying K+ concentration [K+] with 1 mM (bullet) or 3 mM ({blacktriangledown}) MA demonstrating lack of competitive inhibition. E: Dixon plot of bumetanide-sensitive basolateral MA uptake at varying [K+] with 1 mM (bullet) or 3 mM ({blacktriangledown}) MA demonstrating no clear relation and lack of competitive inhibition. *P < 0.05.

 
Increasing extracellular pH has been shown to increase MA uptake in mIMCD-3 cells (12). Figure 4A demonstrates a similar increase in MA uptake with increased basolateral extracellular pH and a decrease with basolateral extracellular acidification. Total MA uptake increased by approximately threefold from 1,349 ± 49 to 4,379 ± 242 pmol·min–1·cm–2 on increasing outside (extracellular) pH (pHo) to 9.0, whereas MA uptake decreased by ~77% to 315 ± 16 pmol·min–1·cm–2 at pHo 5. Transcellular flux of MA (Fig. 4B) in the secretory direction also increased with an increase in basolateral pHo. A pHo increase from 7.4 to 9.0 increased secretory direction MA flux ~3.6-fold from 148 ± 3 to 534 ± 24 pmol·min–1·cm–2. Acidifying pHo to 5.5 produced an ~54% decrease in MA uptake (68 ± 11 pmol·min–1·cm–2). Change in basolateral extracellular pH did not produce any significant increase in a bumetanide-sensitive component of MA uptake, which further supports lack of MA transport on NKCC1 and Na+-K+-ATPase.


Figure 4
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  		Fig. 4. Basolateral MA uptake and secretory direction flux at various basolateral pH levels. A: MA uptake. Total (bullet), bumetanide-insensitive ({blacktriangledown}), and bumetanide-sensitive ({blacksquare}) uptakes are shown. Although basolateral pH has a significant effect on uptake, no bumetanide-sensitive component is observed. B: secretory direction MA flux. Total (bullet), bumetanide-insensitive ({blacktriangledown}), and bumetanide-sensitive ({blacksquare}) fluxes are shown. For A and B, apical media pH was 5.5. C: apical MA uptake (bullet) and absorptive direction MA flux ({blacktriangledown}) at varying apical pH. Basolateral pH was 7.4. Uptake and flux time was 2 min with 1 mM symmetrical MA, and n = 4 for each data point.

 
Change in apical extracellular pH had a similar effect on apical MA uptake and absorptive direction flux (Fig. 4C). Total apical MA uptake increased by approximately fourfold from 211 ± 9 to 890 ± 26 pmol·min–1·cm–2 on increasing apical pHo to 9.0, whereas MA uptake decreased by ~23% to 162 ± 6 pmol·min–1·cm–2 at pHo 5.5. Transcellular flux of MA in the absorptive direction also increased with an increase in apical pHo. An apical pHo increase from 7.4 to 9.0 increased MA Jab approximately eightfold from 54 ± 3 to 437 ± 15 pmol·min–1·cm–2. Acidifying pHo to 5.5 produced an ~54% decrease in MA uptake (25 ± 2 pmol·min–1·cm–2). These data combined demonstrate that apical media (luminal) alkalization has a more significant effect on MA absorption than does basolateral alkalization on MA secretion (approximately eightfold vs. ~3.6-fold increase).

It has been established that NH4+ will compete with MA uptake on RhG proteins (11, 12). Thus we used a transepithelial NH4+ gradient to ascertain the degree to which basolateral MA (and thus NH4+) loading and secretory direction NH4+ transport could occur. The rationale is that apical solution NH4+ might retard the exit of MA across the apical membrane (presumably RhCG), or, if apical NH4+ were to enter and remain in the cell at significant levels, a trans effect on basolateral transport (presumably RhBG) might be revealed. In this case 14C-MA is being used as a tracer for NH4+ transport to determine whether NH4+ secretion can occur against a substantial gradient. A caveat is that MA will only sample a subset of the available secretory mechanisms since it is not transported by NKCC1 or, to our knowledge, by other known K+/NH4+ transporters. Initial experiments with 1 mM MA and 2-min uptake showed an ~50% decrease in basolateral MA uptake from 1,514 ± 41 to 773 ± 29 pmol·min–1·cm–2 with the inclusion of 20 mM basolateral NH4+ (thus confirming effective NH4+ competition), whereas inclusion of 20 mM NH4+ on the apical side produced no significant decrease in basolateral MA uptake (1,578 ± 33 pmol·min–1·cm–2). Figure 5 shows that apical NH4+ concentrations as high as 100 mM have no effect on either basolateral MA loading or on the secretory direction flux of MA. These data indirectly indicate that NH4+ transport in the secretory direction driven by basolateral RhG (presumably RhBG) can occur against a significant NH4+ gradient, one within the physiological range of luminal [NH4+].


Figure 5
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  		Fig. 5. Basolateral MA uptake (bullet) and secretory direction flux ({blacktriangledown}) in the presence of apical NH4+. Apical NH4+ at concentrations up to 100 mM does not inhibit basolateral uptake of MA or secretory direction flux of MA, suggesting that NH4+ uptake and transport via RhBG can occur against a physiologically relevant NH4+ gradient. Uptake and flux time was 2 min with 1 mM symmetrical MA. Apical media pH was 5.5 and basolateral pH 7.4. Initial condition was 0 mM basolateral NH4+, and n = 4 for each data point; *significant difference from the zero time point.

 
Transepithelial transport of NH4+ in T84 cells. Although 14C-MA uptake and flux is a useful tool to investigate the contribution of the RhG proteins to secretory direction movement of NH4+, it does not allow one to determine the contribution of Na+-K+-ATPase or NKCC1 to NH4+ secretion. Unfortunately, there is no convenient radioisotope to directly measure NH4+ transport. We have previously shown that NH4+ competes with K+ on Na+-K+-ATPase as well as NKCC1 in T84 cells and that both transporters will transport (load) NH4+ into T84 cells (54) and thus may contribute to NH4+ secretion.

Initial studies were designed to determine the relative magnitude of NH4+ flux in T84 cell monolayers under asymmetric conditions and were performed in the presence of forskolin to maximize NKCC1 activity. Unidirectional flux of NH4+ was determined by the inclusion of 10 mM NH4+ on one side of the cell monolayer and sampling the trans-side media. NH4+ flux in the secretory direction (Jba) exceeded flux in the absorptive direction (Jab) (Fig. 6), resulting in a calculated secretory direction for the net movement of NH4+. Flux rates were –6.7 ± 1.9 for Jab, 38.3 ± 8.1 for Jba, and 30.8 ± 7.8 nmol·min–1·cm–2NH4+ for the calculated net flux. These data add further support to the MA uptake and flux experiments indicating that T84 cells are capable of net NH4+ secretion.


Figure 6
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  		Fig. 6. Net NH4+ flux in the secretory direction occurs in T84 cells. Unidirectional NH4+ fluxes in forskolin-stimulated T84 cells under asymmetric NH4+ conditions. 10 mM NH4+ with 0 mM K+ was added to one side of the epithelium, and the other side was sampled at 5 min for NH4+ determination. The combined results demonstrate a calculated net secretion (Jnet) of NH4+; n = 4 for each group.

 
To determine whether transepithelial potential (apical negative because of Cl– secretion with forskolin stimulation) affected NH4+ secretion, flux studies were performed in an Ussing chamber system (4-ml symmetrical volume) either under open-circuit or short-circuit (transepithelial voltage = 0 mV) conditions. Asymmetric 0:10 mM NH4+ gradients were established and 0.1 ml transside bathing media sampled at 10-min intervals for 60 min. Calculated secretory rate for the initial 10-min period was not significantly different between open-circuit and short-circuit mode (29 ± 6 and 24 ± 2 nmol·min–1·cm–2, respectively). Similarly secretory rate did not differ over the full 60-min period between open-circuit and short-circuit modes (30 ± 3 and 26 ± 2 nmol·min–1·cm–2, respectively). These data indicate that transepithelial NH4+ movement is electroneutral (supportive of electroneutral movement on RhGs as well as NKCC1). With present techniques it is difficult to distinguish between paracellular NH4+ movement and cellular membrane NH3 movement, although the short-circuit current experiments suggest little contribution of paracellular NH4+ movement to the overall transepithelial flux.

To determine the degree to which NKCC1 and Na+-K+-ATPase contribute to the secretory direction flux, cells were treated with basolateral bumetanide or ouabain for 5 min before the flux period. Both bumetanide and ouabain decreased Jba NH4+ flux (Fig. 7A). Bumetanide decreased the unidirectional secretory direction NH4+ flux by ~44% from 73 ± 10 to 40 ± 5 nmol·min–1·cm–2, whereas pump inhibition reduced NH4+ transport by ~33% to 48 ± 6 nmol·min–1·cm–2, suggesting that NKCC1 and, to some extent, pump activity contribute to the secretory flux of NH4+. In a separate experiment, the effect of basolateral bumetanide on the unidirectional absorptive flux of NH4+ was assessed. Whereas inhibition of NKCC1 caused a decrease in secretory direction NH4+ flux, absorptive NH4+ flux was enhanced by NKCC1 inhibition. Jab for NH4+ was increased approximately twofold from –1.6 ± 0.2 in control to –3.2 ± 0.5 nmol·min–1·cm–2 in bumetanide-treated cells. These data suggest that NKCC1 may play a key role in scavenging NH4+ traversing the cell from the lumen, thus limiting NH4+ absorption. To further lend support for NKCC1 limiting NH4+ absorption, an additional experiment was designed under gradient conditions with NH4+ initially present on both sides of the monolayer (Fig. 7B). Depicted is the change in basolateral media [NH4+] in cells exposed to 10 mM apical NH4+ and 3 mM basolateral NH4+ in the absence and presence of basolateral bumetanide. Under control conditions, there was no significant increase in basolateral NH4+ (which would represent net absorption), or rather the monolayer is capable of preventing diffusive NH3 entry at the apical membrane from being delivered to the basolateral medium. However, in the presence of basolateral bumetanide, basolateral NH4+ levels increase by 0.42 ± 0.02 mM (14%) in 10 min and by 0.55 ± 0.04 mM (18%) by 20 min. The diminished increase in basolateral NH4+ with time suggests the possibility that other mechanisms of limiting absorptive NH4+ flux are acting in a compensatory manner.


Figure 7
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  		Fig. 7. Effect of basolateral bumetanide on NH4+ flux. A: secretory direction flux (Jba) in the presence of 100 µM basolateral bumetanide or 100 µM basolateral ouabain. A 5-min sample period was used. B: change in basolateral NH4+ concentration [NH4+] observed with an applied apical-to-basolateral NH4+ gradient of 10 mM apical to 3 mM basolateral. Data is shown for control (bullet) and with 100 µM basolateral bumetanide ({blacktriangledown}). Inhibition of NKCC1 leads to a significant increase in basolateral [NH4+], suggesting NKCC1 plays a key role in limiting an absorptive direction flux of NH4+. Cells were pretreated with basolateral drug for 5 min before initiating the NH4+ flux, and n = 4 for each group. *P < 0.05 vs. control.

 
cAMP-stimulates ammonium but not MA transport. Because cAMP activates transepithelial Cl– secretion and NKCC1 in T84 cells, we examined the effect of forskolin on MA uptake and flux. Our previous studies demonstrated that basolateral ammonium dampens cAMP-mediated Cl– secretion in T84 cells (16, 35, 54). Since little is known regarding the regulation of RhG ammonium transport, uptake and flux measurements were determined in unstimulated T84 cells. As shown in Fig. 8, A and B, forskolin stimulation did not significantly alter MA transport. Basolateral MA uptake was 3,394 ± 255 pmol·min–1·cm–2 under control conditions and 3,024 ± 214 pmol·min–1·cm–2 in the presence of forskolin. Basolateral-to-apical flux was 312 ± 89 pmol·min–1·cm–2 for control and 260 ± 21 pmol·min–1·cm–2 in the presence of forskolin. Apical MA uptake was 1,535 ± 109 pmol·min–1·cm–2 under control conditions and 1,503 ± 125 pmol·min–1·cm–2 with forskolin. Under control conditions apical-to-basolateral MA flux was 157 ± 40 and 187 ± 8 pmol·min–1·cm–2 with forskolin. In contrast to the lack of effect of cAMP stimulation on MA transport, secretory direction NH4+ flux was increased ~24% in the presence of forskolin (Fig. 8C). Basolateral-to-apical NH4+ flux was 17 ± 1 nmol·min–1·cm–2 under control conditions and increased to 21 ± 2 nmol·min–1·cm–2 with forskolin. cAMP stimulation of NH4+ movement in the secretory direction is consistent with our previous data (54) and supportive of NKCC1 participation in basolateral NH4+ loading (see below). Consistent with NKCC1 activity limiting the transepithelial absorptive vector, forskolin stimulation results in decreased NH4+ Jab flux (Fig. 8D). Jab for NH4+ was 5.8 ± 0.5 nmol·min–1·cm–2 under control condition and decreased ~44% to 3.3 ± 0.8 nmol·min–1·cm–2 with forskolin stimulation. As with other classic secretory or absorptive processes the magnitude variability in NH4+ flux across experiments may, in part, relate to variability in the magnitude of forskolin response in T84 cells between cell platings and passages. However, despite the magnitude variability, the relative effect is consistent across different groups.

T84 monolayers maintain an applied ammonium gradient. The ability of T84 cells to maintain basolateral [NH4+] when exposed to a high apical-to-basolateral NH4+ gradient is shown in Fig. 9. Cell monolayers were exposed to various initial apical [NH4+] ranging from 0 to 30 mM, whereas basolateral [NH4+] was initially 3.11 ± 0.08 mM. Over a period of 30 min, cells in the control group (no added NH4+) did not produce significant amounts of NH4+. During the initial 10 min, basolateral media [NH4+] decreased in all groups. However, at apical NH4+ concentrations in excess of 10 mM, basolateral NH4+ increased by the 20-min time point. Basolateral [NH4+] continued to decrease when the initial apical [NH4+] was at or below that of basolateral NH4+ and was relatively unchanged with an initial apical (10 mM)-to-basolateral (3 mM) gradient. Thus despite increased apical membrane permeability to NH3, T84 cells can maintain at least a 10:3 NH4+ gradient, indicating an ability to transport NH4+ in the secretory direction to minimize back diffusion of NH3.


Figure 9
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  		Fig. 9. Basolateral [NH4+] over time in the presence of varying apical-to-basolateral gradients of NH4+. Initial gradients were (apical:basolateral): bullet, 0:0; {blacktriangledown}, 0:3; {blacksquare}, 3:3; {blacklozenge}, 10:3; {blacktriangleup}, 20:3; and hexagons, 30:3 mM NH4+. Apical and basolateral pH was 7.4; n = 4 for each data point.

 
The passive diffusion of NH3 from the apical media into and across T84 cells is supported by data obtained at differing apical pHo. Figure 10 shows the basolateral media [NH4+] in cells exposed to 10 mM apical NH4+ at apical media pH of 5.5, 7.4, and 9.0. Initial basolateral media [NH4+] was 2.88 ± 0.02 mM. At an apical media pH of 5.5 and 7.4, basolateral [NH4+] decreased to 2.69 ± 0.03 and 2.78 ± 0.01 mM in the first 10 min and then rose to levels not significantly different than the starting values. However, with an apical media pH of 9.0, at which more NH3 is present, basolateral [NH4+] increased to 3.00 ± 0.05 mM in 10 min and to 3.45 ± 0.09 by 20 min.


Figure 10
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  		Fig. 10. Effect of apical pH on basolateral [NH4+] with an initial apical-to-basolateral 10:3 mM NH4+ gradient. T84 cells can maintain a 10:3 NH4+ gradient at neutral (bullet) and acidic ({blacktriangledown}) apical pH but cannot overcome passive NH3 back flux at alkaline ({blacksquare}) apical pH. Basolateral pH was 7.4; n = 4 for each data point. *Significant difference from the respective zero time point.

 
The ability of T84 cells to actively minimize net NH4+ absorption when exposed to an apical-to-basolateral NH4+ gradient for an extended period was further tested with cells in culture medium. Cell monolayers were maintained in complete culture medium on the basolateral side with a measured value of 4.06 ± 0.03 mM NH4+ and serum-free culture media containing 20 mM NH4+ at pH 6.0 on the apical side. Apical pH 6.0 was used instead of pH 5.5 due to the buffering capacity of culture media. As shown in Fig. 11, basolateral [NH4+] decreased to 2.51 ± 0.03 mM by 10 min with only a slight increase (2.66 ± 0.04 mM) at 60 min.


Figure 11
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  		Fig. 11. T84 cells are able to maintain an apical-to-basolateral NH4+ gradient of 20:~2.5 mM for at least 60 min. Cells were kept in complete culture media at pH 7.2 on the basolateral side and serum-free culture media at pH 6.0 on the apical side (conditions that are more representative of in vivo conditions). Error bars are within the symbols; n = 4 for each point.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Excess systemic NH4+ levels can lead to hyperammonemia-associated encephalopathy, which can be life threatening. Although the kidney is responsible for carrying out the bulk of body NH4+ homeostasis, the possibility exists that some level of body NH4+ control can be accomplished via changes in colonic function. Our data demonstrate the following: 1) T84 cells express mRNA for two known NH4+ transporters RhBG and RhCG and display transport characteristics consistent with functional RhG proteins; 2) transepithelial MA flux in T84 cells is greater in the secretory than absorptive direction; 3) transepithelial transport of NH4+ in T84 cells is greater in the secretory than absorptive direction and is sensitive to cAMP stimulation and to inhibitors of NKCC1 and Na+-K+-ATPase; and 4) T84 cells are able to maintain an apical-to-basolateral NH4+ gradient despite having a greater apical membrane NH3 permeability than native colonic crypts (2, 16, 18, 42, 44). Taken together, these data provide evidence that model colonic crypt epithelia secrete NH4+ by a mechanism that involves both K+ transport and novel RhG protein-mediated pathways. The long-standing premise, based on greater [NH4+] in portal vein vs. systemic circulation, has been that NH4+ absorption occurs along the length of the intestine. No consideration has been given for an opposing NH4+ secretory vector, which is surprising considering that secretory epithelia in the intestine share similar transport mechanisms with renal and gill epithelium, which do effect NH4+ secretion. If confirmed in natural tissue, these findings indicate that active secretion by colonic crypts could oppose the larger absorptive NH4+ vector to limit net NH4+ delivery to the portal circulation.

RhBG and RhCG isoforms of the Rhesus glycoprotein family have been identified along the GI tract in mouse (13). Although limited RhBG and RhCG expression was observed in the upper portion of proximal colon crypts compared with surface cells in mouse tissue, both isoforms are present in T84 cells (human), which otherwise functionally resemble cells of crypt origin. Whether expression patterns of RhBG and RhCG differ along the length of the colon or along the entire crypt surface axis in mouse or human tissue remains to be determined as does distribution among the various cell types present within the colonic crypt. MA uptake in to T84 cells was observed from the basolateral side, thus suggesting the functional presence of RhBG. MA uptake was partially inhibited by the addition of basolateral NH4+, suggestive of RhBG function similar to reported results for RhBG in renal mIMCD-3 cells (12). In mIMCD-3 cells basolateral MA transport exhibits both diffusive and carrier-mediated components with the diffusive component becoming predominant at [MA] exceeding 10 mM. The dose response relation for MA uptake observed in T84 cells shows similar properties with an almost linear relation between 5 and 15 mM MA. Handlogten et al. (12) reported that MA uptake via RhBG in mIMCD-3 cells is not affected by the NKCC1 inhibitor bumetanide. In their study, excess NH4+ was used to distinguish between transporter-mediated and diffusive entry of MA. Since T84 cells have abundant NKCC1 activity (which, as discussed below, is capable of NH4+ transport), such a maneuver becomes somewhat problematic; therefore, we operationally defined transporter-uptake by performing assays at <5 mM MA and <3-min uptake periods. Consistent with RhBG in mIMCD-3 cells, transporter mediated uptake of MA (e.g., at <5 mM MA and <3-min uptake periods) is insensitive to bumetanide. With increased concentration of MA as well as with increased uptake times (at a greater fraction of diffusive MA entry), total MA uptake showed some sensitivity to bumetanide. This could be due to an indirect effect of bumetanide itself on diffusive MA entry or, alternatively, an indirect effect of NKCC1 inhibition on diffusive MA entry. The later possibility is perhaps more likely, given the abundant capacity for NKCC1 activity in T84 cells. It should be noted, however, that bumetanide had little impact on the transepithelial flux of MA, perhaps due to the apical exit of MA being rate limiting (suggested by less apical MA uptake vs. basolateral MA uptake).

Uptake of MA by either RhBG or RhCG expressed in Xenopus oocytes has been shown to be stimulated by extracellular alkalosis (26), similarly RhCG activity in mIMCD-3 cells is enhanced by extracellular alkalinization (11). Both RhBG and RhCG have been identified in mouse intestine, showing basolateral and apical immunostaining, respectively (48). In mIMCD-3 cells, functional distinction between RhBG and RhCG is primarily based on basal activity, pH sensitivity, and relative affinity (11). In T84 cells, both basolateral MA uptake (RhBG) and basolateral-to-apical flux of MA is enhanced by elevated basolateral pH and inhibited by basolateral solution acidification.

MA uptake into T84 cells was also observed from the apical side, thus supporting the functional presence of an RhG protein, likely RhCG. Uptake was partially inhibited by the addition of apical NH4+. Although conditions were not optimized for maximal competitive inhibition, since apical NH4+ will change intracellular pH (pHi) (16, 54), the data are suggestive of RhG activity as reported results for RhCG in renal mIMCD-3 cells (11). Apical MA uptake in T84 cells with changes in apical solution pH are consistent with published data for RhCG (11, 26), thus supporting the functional presence of apical RhCG in T84 cells.

The notion that NH4+ secretion can be driven by NKCC and Na+-K+-ATPase with an opposing apical exit pathway is supported by work in renal cells (19, 46). In fact, Kinne et al. estimated that it is energetically possible for NKCC and Na+-K+-ATPase under physiological conditions to generate an intercellular-to-extracellular [NH4+] ratio of ~2,000! As previously reported, NH4+ can be effectively transported on the K+ site of Na+-K+-ATPase and NKCC1 in T84 cells (54). Since similar loading pathways are used by various NH4+ secretory epithelia, it was logical to examine whether T84 monolayers demonstrate transepithelial ammonium transport. Indeed, we demonstrated a role for NKCC1 and perhaps Na+-K+-ATPase in transepithelial secretory flux of NH4+. Basolateral bumetanide as well as ouabain significantly decreased the movement of basolateral NH4+ to the apical compartment. Although it is difficult to assess the degree to which ouabain might indirectly affect NKCC1 activity (by altering the Na+ gradient), these data support the involvement of additional pathways for basolateral NH4+ loading (such as RhBG) as described above since bumetanide or ouabain resulted in only an ~40 or ~33% reduction in NH4+ flux. In our study, bumetanide had little effect on transepithelial MA transport. It should be noted, as well, that Handlogten et al. (12) showed minimal MA uptake on Na+-K+-ATPase or NKCC1 in mIMCD-3 cells. Our data support a role for NKCC1 in retarding the movement of NH4+ from the apical compartment to the basolateral compartment, perhaps acting as an NH3/NH4+ scavenger by rapidly internalizing NH4+ present in the basolateral vicinity of the crypt compartment.

Ammonium was transported by both Na+-K+-ATPase and NKCC1 [as has been described for other epithelial transport models of ammonium secretion (30, 38, 40, 43, 45, 47)], as well as RhBG and RhCG. Under asymmetric flux conditions at pH 7.4, both MA and NH4+ transepithelial fluxes were greater in the secretory compared with absorptive direction. Whereas MA uptake and flux (RhBG/RhCG mediated) were unaffected by forskolin, NH4+ flux was stimulated by cAMP, presumably because of increased NKCC1 activity. In vivo, luminal pH is more acidic (pH of ~5.5 in humans); this would further favor net secretion under physiological pH conditions because relative luminal acidity favors luminal NH4+ over NH3 and thus minimizes passive backdiffusion of NH3 through the apical membrane. The redundancy in mechanisms for basolateral ammonium loading in both kidney and intestine suggests the importance of maintaining ammonium secretory capacity. Indeed, predicted distal tubular acidosis or hyperammonemia was absent in RhBG KO mice (5, 6).

An important caveat to our study is the observation that native colonic crypt cells of certain mammals appear to exhibit low apical permeability to ammonia (NH3) (2, 18, 42, 44). In contrast, the apical membrane of T84 cells has a more readily apparent NH3 permeability (16). It should be noted that the higher apical NH3 permeability in T84 cells would impair their ability to secrete NH4+ against a concentration gradient and thus might underestimate the ability of intact crypts to secrete NH4+ or maintain a large luminal-to-serosal NH4+ gradient. Moreover, it is unknown whether cultured crypt epithelial lines differ, in general, from native crypts with respect to apical ammonium permeability; certainly, there may be considerable species, segmental, or cellular heterogeneity.

Several models for NH4+ secretion in other tissues (see introduction) include ion trapping of NH4+ at the luminal surface. In particular, colonic H+-K+-ATPase along with an apical K+ channel have been shown to be important participants for mammalian IMCD (30). Although it is well established that native colonic mucosa contains both c-H+-K+-ATPase and a ROMK1-like apical K+ channel (22), both appear to be absent from the T84 cell line (36). The lack of these two apical transporters (cH+-K+-ATPase and K+ channel) in T84 cells thus also limits their ability to secrete NH4+ against a concentration gradient or maintain a large luminal-to-serosal NH4+ gradient.

Due to the nature of the ammonium assay (signal-to-noise) and the NH3 permeability of T84 apical membrane, it is difficult to measure NH4+ transport against a sizeable apical [NH4+]. An alternative approach would be the use of the stable nonradioactive isotope of N (15N) in tracer fluxes, although it is questionable whether this approach would enhance accuracy of transepithelial flux assays. As an indirect alternative, we determined secretory MA flux against an apical-to-basolateral NH4+ gradient (Fig. 5). The rationale is that NH4+ is transported by RhBG and RhCG to a greater extent than is MA; thus NH4+ acts competitively against MA transport on RhGs (Fig. 2 and Ref. 12). We found that 20 mM cis NH4+ inhibits uptake of MA by about half. Thus it is reasonable that trans NH4+, if sensed by the secretory machinery, should also inhibit the transepithelial flux of MA. However, secretory direction MA flux was not significantly altered by the trans addition of up to 100 mM NH4+. This observation suggests that NH4+ secretion (in this case estimated by 14C-MA tracer) can occur against a physiologically relevant apical-to-basolateral NH4+ gradient. These results underestimate the secretory capacity since MA is not transported by NKCC1 or Na+-K+-ATPase, whereas NH4+ would be loaded into the cell by these transporters. T84 cells were also capable of maintaining an apical-to-basolateral NH4+ gradient (Figs. 9 and 11). The initial loss of basolateral compartment NH4+ may represent a combination of cellular NH4+ sequestering and/or metabolism; however, this does not negate the fact that T84 cells can maintain an appreciable apical-to-basolateral gradient.

Taken together, our data suggest that ammonium transport in the secretory direction can occur in T84 model crypt epithelia and that RhG proteins, NKCC1, and, to some extent, Na+-K+-ATPase participate in basolateral loading of NH4+ into the cell. We speculate that a secretory mechanism may be present in natural colonic mucosa to counter ammonium absorptive pathways and thus limit the amount of NH4+ reaching the portal circulation. Further elucidation of the mechanisms involved in colonic NH4+ transport and regulation may be useful in developing new targets for more effective therapeutic treatment of hyperammonemia.


   ACKNOWLEDGMENTS
 
We thank Corryn A. Morris for administrative assistance on this project. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases DK-051630 to J. B. Matthews and by the Epithelial Pathobiology Group.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Roger T. Worrell, Univ. of Cincinnati, Dept. of Surgery, Molecular and Cellular Physiology, 3125 Eden Ave., Vontz Center for Molecular Studies, Cincinnati, OH 45219-0581 (e-mail: Roger.Worrell{at}uc.edu)

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.


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