Intracellular K+ activities in Aplysia gut: effects of transport inhibitors on the Na+ pump

Na+ absorption by the Aplysia californica foregut is affected through an active Na+ transport mechanism located in the basolateral membrane of the epithelial absorptive cells. Since Cl- absorption by the Aplysia gut has been shown to be very different from that demonstrated in vertebrate gut, the present study was undertaken to discern if Na+ transport was also different from that observed in vertebrate preparations. Utilizing microelectrode technique, it was demonstrated that intracellular K+ activity is above electrochemical equilibrium in the Aplysia absorptive cells and that serosal ouabain, Ba2+ or Cd2+ abolished this asymmetry in K+ electrochemical potential. Neither bumetanide nor furosemide had any effect on intracellular K+ activities, mucosal membrane potentials or transepithelial potentials in the Aplysia gut preparation. These results are consistent with the operation of a basolateral Na+/K+ pump.     

Sodium-phosphate symport by Aplysia californica gut

Phosphate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Phosphate transport across apical membranes of vertebrate gastrointestinal epithelia requires sodium; whereas, its transport across the basolateral membrane requires antiport processes involving primarily chloride or bicarbonate. To decipher the phosphate transport mechanism in the foregut apical membrane of the mollusc, Aplysia californica, in vitro short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and phosphate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na+ was enhanced by the presence of phosphate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of phosphate was dependent upon the presence of Na+ and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetande, added to either side, had no effect on transepithelial difference or short-circuit current in the Aplysia gut bathed in a Na2HPO4 seawater medium. However, mucosal arsenate inhibited the net mucosal-to-serosal fluxes of both phosphate and Na+ and the arsenate-sensitive Na+ flux to that of phosphate was 2:1. These results suggest the presence of a Na-PO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.     

Transport energetics of the Na+ pump in Aplysia californica gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na+ transporter (Na+ pump). Increased activity of the Na+ pump, coupled to luminal Na+/AIB symporter activity and basolateral membrane depolarization, changed the Na+ transport energetics across the basolateral membrane to a greater extent than the change in Na+ transport energetics across the luminal membrane.     

Physiology of the tracheal epithelium

The mucosa that lines the airways is covered with a fluid film forming a hypophase between mucus and cell surface. To study the function of this epithelium aims at describing the mechanisms by which fluid is normally produced. Another goal to be pursued consists in looking for the origin of pathological situations, such as cystic fibrosis, in which the functioning of epithelial cell is altered. The elucidation of transport mechanisms present in the apical and in the basolateral membrane results in a conceptual model that illustrates the asymmetrical functioning of epithelial cells. Recent discoveries enlarge our understanding of membrane transport processes; in particular, a concerted, reciprocal regulation of the activity of both membranes was shown to be exerted via the intracellular composition. The tracheal epithelium absorbs Na+ and secretes Cl-. These two transports are active and electrogenic; their sum corresponds approximately to the short-circuit current measured in vitro. Na+ absorption is sensitive to amiloride from the luminal side and also to ouabain added to the serosal compartment. The process is a primary active transport, analogous to that found in amphibian epithelia or in mammalian colon. Cl- secretion is abolished by furosemide (or bumetanide), by ouabain or by Na+ suppression in the serosal incubation solution. The mechanism is a secondary active transport: Cl- influx across the basolateral membrane is coupled to Na+ (probably through Na+, K+, Cl- symport); energy is dissipated by the Na+-K+-ATPase localised in the basolateral membrane. Thus, Na+ is recirculated across that membrane by the pump activity, which maintains a favorable gradient for influx via the symport. Cl- efflux takes place by diffusion through the luminal membrane. This model applies to other epithelia in which Na+-coupled Cl- secretion was shown to take place. It is confirmed by isotopic fluxes measurements and by electrophysiologic properties of the apical and the basolateral membrane. Various agents are known to influence ion transports. In particular Cl- secretion is stimulated by substances that increase the intracellular concentration of cyclic AMP. At the membrane level, the number of active Cl- channels in the apical membrane is primarily controlled, then the basolateral membrane K+ permeability. Yet, species differences are worth to note: the trachea of the cow is barely sensitive to agents that exert a marked action on dog trachea. The tracheal epithelium is used as an experimental model for studying cystic fibrosis, a disease in which the apical membrane is almost devoid of functional Cl- channels, so that Cl- permeability is quite low.(ABSTRACT TRUNCATED AT 400 WORDS)     

Energetics of potassium ion transport in Aplysia gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na(+)/K(+) transporter (Na(+)/K(+) pump or Na(+)/K (+) -ATPase). This Na(+)/K(+) pump accounts for both the intracellular Na(+) electrochemical potential (micro) being less than the extracelluar Na(+) micro and the intracellular K(+) micro being more than the extracellular K(+ ) micro. Also, K(+) channel activity resides in both luminal and basolateral membranes of the Aplysia foregut epithelial cells. Increased activity of the Na(+)/K(+) pump, coupled to luminal and basolateral membrane depolarization altered the K(+) transport energetics across the basolateral membrane to a greater extent than the alteration in K(+) transport energetics across the luminal membrane. These results suggest that K(+) transport, either into or out of the Aplysia foregut epithelial cells, is rate-limiting at the basolateral membrane.     

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.     

Transapical Na+ electrochemical potential difference and solute accumulation in Aplysia californica enterocytes

Extracellular water averaged 20% of total intestinal water. The sugar, D-galactose, was accumulated by the Aplysia enterocytes. The amino acids glycine, L-alanine and alpha-aminoisobutyric acid (AIB) were all accumulated by the Aplysia enterocytes. The electrochemical potential difference for Na+ across the mucosal membrane of Aplysia enterocytes was energetically adequate in driving D-galactose, L-alanine, glycine or AIB uphill into the Aplysia enterocytes.     

HCO3- transport in basolateral membrane vesicles isolated from rat renal cortex

The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested.     

OCTN3: A Na+-independent L-carnitine transporter in enterocytes basolateral membrane

L-carnitine transport has been measured in enterocytes and basolateral membrane vesicles (BLMV) isolated from chicken intestinal epithelia. In the nominally Na+-free conditions chicken enterocytes take up L-carnitine until the cell to medium L-carnitine ratio is 1. This uptake was inhibited by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, tetraethylammonium (TEA), and betaine. L-3H-carnitine uptake into BLMV showed no overshoot, and it was (i) Na+-independent, (ii) trans-stimulated by intravesicular L-carnitine, and (iii) cis-inhibited by TEA and cold L-carnitine. L-3H-carnitine efflux from L-3H-carnitine preloaded enterocytes was also Na+-independent, and trans-stimulated by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, TEA, and betaine. Both, uptake and efflux of L-carnitine were inhibited by verapamil and unaffected by either extracellular pH or palmitoyl-L-carnitine. RT-PCR with specific primers for the mouse OCTN3 transporter revealed the existence of OCTN3 mRNA in mouse intestine, which was confirmed by in situ hybridization studies. Immunohystochemical analysis showed that OCTN3 protein was mainly associated with the basolateral membrane of rat and chicken enterocytes, whereas OCTN2 was detected at the apical membrane. In conclusion, the results demonstrate for the first time that (i) mammalian small intestine expresses OCTN3 mRNA along the villus and (ii) that OCTN3 protein is located in the basolateral membrane. They also suggest that OCTN3 could mediate the passive, Na+ and pH-independent L-carnitine transport activity measured in the three experimental conditions.     

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.     

Ion transport in the intestine of Gobius niger in both isotonic and hypotonic conditions

Ion transport in the intestine of Gobius niger, a euryhaline teleost, was studied in both isotonic and hypotonic conditions. Isolated tissues, mounted in Ussing chambers and bilaterally perfused with isotonic Ringer solution, developed a serosa negative transepithelial voltage and a short circuit current indicating a net negative current in absorptive direction. Bilateral removal of Cl- and Na+ from the bathing solutions as well as the luminal removal of K+in the presence of Ba2+(10(-3) M) almost abolished both Vt and Isc. Similar results were obtained by adding bumetanide (10(-5)M) to the luminal bath while other inhibitors of Cl- transport mechanisms were ineffective. These observations suggest that salt absorption begins with a coupled entry of Na+, Cl-, and K+ across the apical membrane; a Ba2+inhibitable K+ conductance, demonstrated also by micropuncture experiments, recycles the ion into the lumen. Salt entry into the cell is driven by the operation of the basolateral Na+/K(+)-ATPase since serosal ouabain (10(-4)M) completely abolished both Vt and Isc; this pump also completes the Na(+) absorption. The inhibitory effect of both serosal bumetanide (10(-4)M) and SITS (5 x 10(-4)M) suggests that Cl- would leave the cell via the KCl cotransport, the Cl/HCO3- antiport and/or conductive pathways. Bilateral exposure of tissues to hypotonic media produced a reduction of both the transepithelial voltage and the short circuit current probably due to the activation of homeostatic ionic fluxes involved in cell volume regulation. The results of experiments with both isolated enterocytes and intestine exposed to hypotonic solution suggested that the recovery of cell volume, after the initial cell swelling, involves a parallel opening of K+ and Cl- channels to facilitate net solute and water effluxes from the cell. J. Exp. Zool. 301A:49-62, 2004.     

Cl(-)-HCO3- exchange in rat renal basolateral membrane vesicles

Pathways for HCO3- transport across the basolateral membrane were investigated using membrane vesicles isolated from rat renal cortex. The presence of Cl(-)-HCO3- exchange was assessed directly by 36Cl- tracer flux measurements and indirectly by determinations of acridine orange absorbance changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake compared to Cl- uptake under 100% N2 in the presence of a pH gradient alone. Mediated exchange of Cl- for HCO3- was suggested by the HCO3- gradient-induced concentrative accumulation of intravesicular Cl-. Maneuvers designed to offset the development of ion-gradient-induced diffusion potentials had no significant effect on the magnitude of HCO3- gradient-driven Cl- uptake further suggesting chemical as opposed to electrical Cl(-)-HCO3- exchange coupling. Although basolateral membrane vesicle Cl- uptake was observed to be voltage sensitive, the DIDS insensitivity of the Cl- conductive pathway served to distinguish this mode of Cl- translocation from HCO3- gradient-driven Cl- uptake. No evidence for K+/Cl- cotransport was obtained. As determined by acridine orange absorbance measurements in the presence of an imposed pH gradient (pHo 7.5/pHi 6), a HCO3- dependent increase in the rate of intravesicular alkalinization was observed in response to an outwardly directed Cl- concentration gradient. The basolateral membrane vesicle origin of the observed Cl(-)-HCO3- exchange activity was verified by experiments performed with purified brush-border membrane vesicles. In contrast to our previous observations of the effect of Cl- on HCO3- gradient-driven Na+ uptake suggesting a basolateral membrane Na+-HCO3- for Cl- exchange mechanism, no effect of Na+ on Cl-HCO3- exchange was observed in the present study.     

Ion transport mechanisms in the mesonephric collecting duct system of the toad Bufo bufo: microelectrode recordings from isolated and perfused tubules

It is not clear how and whether terrestrial amphibians handle NaCl transport in the distal nephron. Therefore, we studied ion transport in isolated perfused collecting tubules and ducts from toad, Bufo bufo, by means of microelectrodes. No qualitative difference in basolateral cell membrane potential (Vbl) was observed between tubules and ducts in response to ion substitutions, inhibitor and agonist applications. Cl- substitution experiments indicated a small Cl- conductance in the basolateral membrane. The apical membrane did not have a significant Cl- conductance. Luminal [Na+] steps and amiloride application showed a small apical Na+ conductance. Arginine vasotocin depolarized Vbl. The small apical Na+ conductance indicates that the collecting duct system contributes little to NaCl reabsorption when compared to aquatic amphibians. In contrast, Vbl rapidly depolarized upon lowering of [Na+] in the bath, demonstrating the presence of a Na+-coupled anion transporter. [HCO3-] steps revealed that this transporter is not a Na+-HCO3- cotransporter. Together, our results indicate that a major task of the collecting duct system in B. bufo is not conductive NaCl transport but rather K+ secretion, as shown by our previous studies. Moreover, our results indicate the presence of a novel basolateral Na+-coupled anion transporter, the identity of which remains to be elucidated.     

Sodium-Sulfate Symport by Aplysia californica Gut

Sulfate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Sulfate transport can be coupled to proton, sodium symport or antiport processes involving chloride or bicarbonate. It had previously been observed in Aplysia gut that sulfate was actively absorbed. To understand the mechanism for this transport, short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and sulfate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na(+) was enhanced by the presence of sulfate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of sulfate was dependent upon the presence of Na(+) and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetanide, added to either side, had no effect on transepithelial potential difference or short-circuit current in the Aplysia gut bathed in a Na2SO4 seawater medium. However, mucosal thiosulfate inhibited the net mucosal-to-serosal fluxes of both sulfate and Na(+) and the thiosulfate-sensitive Na(+) flux to that of sulfate was 2:1. These results suggest the presence of a Na-SO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.     

Electrolyte transport in the epithelium of pulmonary segments of normal and cystic fibrosis lung.

The epithelium of pulmonary segments from trachea to aveoli actively transports electrolytes and allows osmotic movement of water to maintain the ionic environment in the airway lumen. Models of airway absorption and secretion depict the operation of transporters localized to apical or basolateral membrane. In many epithelia, a variety of electrolyte transporters operate in different combinations to produce absorption or secretion. This also applies to pulmonary epithelium of the large airways (trachea, main-stem bronchi), bronchioles, and alveoli. Na+ absorption occurs in all three pulmonary segments but by different transporters: apical Na+ channels in large airways and bronchioles; Na+/H+ exchange and Na+ channels in adult alveoli. The Na+ channels in each pulmonary segment share a sensitivity to amiloride, a potent inhibitory of epithelial Na+ channels. Fetal alveoli display spontaneous Cl- secretion, as do the large airways of some mammals, such as dog and bovine trachea. Cl- channels differ in conductance properties and in regulation by intracellular second messengers, osmolarity, and voltage mediate stimulated Cl- secretion. Electroneutral carriers, such as NaCl(K) cotransport, Cl-/HCO3- exchange, and Na+/HCO3- exchange, operate in large airways and alveoli during absorption and secretion. Abnormal ion transport in airways of cystic fibrosis (CF) patients is manifest as a reduced Cl- conductance and increased Na+ conductance. Isolation of the CF gene and identification of its product CFTR now allow investigations into the basic defect. Intrinsic to these investigations is the development of systems to study the function of CFTR and its relation to electrolyte transporters and their regulation.     

Membrane localization of H+ and HCO3- transporters in the rat pancreatic duct

The pancreatic duct secretes alkaline fluid that is rich in HCO3- and poor in Cl-. The molecular mechanisms that mediate ductal secretion and are responsible for the axial gradients of Cl- and HCO3- along the ductal tree are not well understood because H+ and HCO3- transport by duct cells have not been characterized or localized. To address these questions, we microdissected the intralobular, main, and common segments of the rat pancreatic duct. H+ and HCO3- transporters were characterized and localized by following intracellular pH while perfusing the bath and the lumen of the ducts. In intralobular ducts, Na(+)-dependent and amiloride-sensitive recovery from acid load in the absence of HCO3- was used to localize a Na+/H+ exchanger to the basolateral membrane (BLM). Modification of Cl- gradients across the luminal (LM) and BLM in the presence of HCO3- showed the presence of Cl- /HCO3- exchangers on both membranes of intralobular duct cells. Measurement of the effect of Cl- on one side of the membrane on the rate and extent of pHi changes caused by removal and addition of Cl- to the opposite side suggested that both exchangers are present in the same cell. In the presence of HCO3-, intralobular duct cells used three separate mechanisms to extrude H+: (a) BLM-located Na+/H+ exchange, (b) Na(+)-independent vacuolar-type H+ pump, and (c) BLM-located, Na(+)- dependent, amiloride-insensitive, and 4',4'-diisothiocyanatostilbene- 2,2'-disulfonic acid sensitive mechanism, possibly a Na(+)-dependent HCO3- transporter. The main and common segments of the duct displayed similar mechanisms and localization of H+ and HCO3- transporters to the extent studied in the present work. In addition to the transporters found in intralobular ducts, the main and common ducts showed Na+/H+ exchange activity in the LM. Three tests were used to exclude a significant luminal to basolateral Na+ leak as the cause for an apparent luminal Na+/H+ exchange in an HCO3- secreting cells: (a) addition of amiloride and removal of Na+ from the LM had a profound effect on Na+/H+ exchange activity on the BLM and vice versa; (b) inhibition of all transporters in the BLM by bathing the duct in the inert hydrocarbon Fluorinert FC-75 did not prevent cytosolic acidification caused by removal of luminal Na+; and (c) luminal Na+ did not activate the basolateral Na(+)-dependent HCO3- transporter. An Na(+)-independent, bafilomycin-sensitive H+ pumping activity was marginal in the absence of HCO3-.(ABSTRACT TRUNCATED AT 400 WORDS)     

The cellular mechanism of active chloride secretion in vertebrate epithelia: studies in intestine and trachea

The cellular mechanism of active chloride secretion, as it is manifested in the intestine and trachea, appears to possess the following elements: (1)NaCl cl-transport across the basolateral membrane; (2) Cl- accumulation in the cell above electrochemical equilibrium due to the Na+ gradient; (3) a basolateral Na+-K+ pump that maintains the Na+ gradient; (4) a hormone-regulated Cl- permeability in the apical membrane; (5) passive Na/ secretion through a paracellular route, driven by the transepithelial potential difference; and (6) an increase in basolateral membrane K+ permeability occurring in conjunction with an increase in Na+-K+ pump rate. Electrophysiological studies in canine trachea support this model. Adrenalin, a potent secretory stimulus in that tissue, increases apical membrane conductance through a selective increase in Cl- permeability. Adrenalin also appears to increase basolateral membrane K+ permeability. Whether or not adrenalin also increases paracellular Na+ permeability is unclear. Some of the testable implications of the above secretion model are discussed.     

Ammonium effects on colonic Cl- secretion: anomalous mole fraction behavior

A significant amount of ammonium (NH4+) is absorbed by the colon. The nature of NH4+ effects on transport and NH4+ transport itself in colonic epithelium is poorly understood. The goal of this study was to elucidate the effects of NH4+ on cAMP-stimulated Cl- secretion in the colonic cell line T84. In HEPES-buffered solutions, application of basolateral NH4+ resulted in a reduced level of Cl- secretory current. The effect of NH4+ appears to occur by at least three mechanisms: 1) basolateral membrane depolarization, 2) a competitive effect with K+, and 3) a long-term (>20 min) increase in transepithelial resistance (TER). The competitive effect with K+ exhibits anomalous mole fraction behavior. Transepithelial current relative to that in 10 mM basolateral K+ was inhibited 15% by 10 mM NH4+ alone and by 30% with a mixture of 2 mM K+ and 8 mM NH4+. A mole fraction mix of 2 mM K+:8 mM NH4+ produced a greater inhibition of basolateral membrane K+ current than pure K+ or NH4+ alone. Similar anomalous behavior was also observed for inhibition of bumetanide-sensitive 36Cl- uptake, e.g., Na+-K+-2Cl- -cotransporter (NKCC-1). No anomalous effect was observed on Na+-K+-ATPase current. Both NKCC-1 and Na+-K+-ATPase activity were elevated in 10 mM NH4+ with respect to 10 mM K+. The effect on TER did not exhibit anomalous mole fraction behavior. The overall effect of basolateral NH4+ on cAMP-stimulated transport is dependent on the [K+]o /[NH4+]o ratio at the basolateral membrane, where o is outside of the cell.