Effect of ammonium ion and extracellular pH on hybridoma cell metabolism and antibody production

The effects of NH(4)Cl addition on batch hybridoma cell growth at different external pH values (pH(e)) were investigated in a bioreactor at constant pH and dissolved oxygen concentration. In agreement with measurements in flasks, changes in pH(e) over the range 6.8-7.6 had minor effects on growth. Addition of 3 mM NH(4)Cl had little effect on cell growth while 10 mM NH(4)Cl caused a substantial growth inhibition, Measurements of the effects of pH(e) and NH(4)Cl concentration on cell metabolism gave similar results for cells grown in flasks in an incubator and in the bioreactor. As pH(e) decreases, the integral cell yield on glucose increases. There is a correlation between the effects of pH(e) on glycolysis and previous measurements of its effects on intracellular pH (pH(i)). Increases in NH(4)Cl concentration were previously determined to decrease pH(i) and are shown here to decrease the integral cell yield on glucose. At all pH(e) values in the absence of NH(4)Cl, glutamine is depleted at the time the maximum cell density is reached. Both pH(e) decreases and NH(4)Cl concentration increases lead to decreases in the integral cell yield on glutamine. Changes in pH(e) and in the NH(4)Cl concentration that cause growth inhibition have no effect on the specific antibody production rate for cells grown in flasks in an incubator or in the bioreactor. Changes in the NH(4)Cl concentration have no effect on the quality of the antibody produced, to a first level of characterization.     

Cation transport by the neuronal K(+)-Cl(-) cotransporter KCC2: thermodynamics and kinetics of alternate transport modes

Both Cs(+) and NH(4)(+) alter neuronal Cl(-) homeostasis, yet the mechanisms have not been clearly elucidated. We hypothesized that these two cations altered the operation of the neuronal K(+)-Cl(-) cotransporter (KCC2). Using exogenously expressed KCC2 protein, we first examined the interaction of cations at the transport site of KCC2 by monitoring furosemide-sensitive (86)Rb(+) influx as a function of external Rb(+) concentration at different fixed external cation concentrations (Na(+), Li(+), K(+), Cs(+), and NH(4)(+)). Neither Na(+) nor Li(+) affected furosemide-sensitive (86)Rb(+) influx, indicating their inability to interact at the cation translocation site of KCC2. As expected for an enzyme that accepts Rb(+) and K(+) as alternate substrates, K(+) was a competitive inhibitor of Rb(+) transport by KCC2. Like K(+), both Cs(+) and NH(4)(+) behaved as competitive inhibitors of Rb(+) transport by KCC2, indicating their potential as transport substrates. Using ion chromatography to measure unidirectional Rb(+) and Cs(+) influxes, we determined that although KCC2 was capable of transporting Cs(+), it did so with a lower apparent affinity and maximal velocity compared with Rb(+). To assess NH(4)(+) transport by KCC2, we monitored intracellular pH (pH(i)) with a pH-sensitive fluorescent dye after an NH(4)(+)-induced alkaline load. Cells expressing KCC2 protein recovered pH(i) much more rapidly than untransfected cells, indicating that KCC2 can mediate net NH(4)(+) uptake. Consistent with KCC2-mediated NH(4)(+) transport, pH(i) recovery in KCC2-expressing cells could be inhibited by furosemide (200 microM) or removal of external [Cl(-)]. Thermodynamic and kinetic considerations of KCC2 operating in alternate transport modes can explain altered neuronal Cl(-) homeostasis in the presence of Cs(+) and NH(4)(+).     

Intracellular pH regulation in colonocytes of rat proximal colon

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.     

Light induces an increase in the pH of and a decrease in the ammonia concentration in the extrapallial fluid of the giant clam Tridacna squamosa

The objective of this study was to examine whether 12 h of light exposure would lead to an increase in the pH of and a decrease in the concentration of total ammonia in the extrapallial fluid of the giant clam Tridacna squamosa. We also aimed to elucidate indirectly whether movements of ammonia and/or protons (H(+)) occurred between the extrapallial fluid and the outer mantle epithelium. The pH of the extrapallial fluid of T. squamosa exposed to 12 h of light was significantly higher than that of clams exposed to 12 h of darkness. Conversely, the total ammonia concentration in the extrapallial fluid of the former was significantly lower than that of the latter. In addition, the glutamine content in the mantle adjacent to the extrapallial fluid of clams exposed to 12 h of light was significantly greater than that of clams exposed to 12 h of darkness. These results suggest that in the extrapallial fluid of T. squamosa exposed to light, NH(3) combined with H(+) as NH(+)(4) and that NH(+)(4) was transported into the mantle and used as a substrate for glutamine formation. Injection of NH(4)Cl into the extrapallial fluid led to an instantaneous increase in the total ammonia concentration therein, but the total ammonia concentration decreased subsequently and returned to the control value within 1 h. This is in support of the proposition that NH(+)(4) could be transported from the extrapallial fluid to the mantle. Injection of HCl into the extrapallial fluid led to an instantaneous decrease in the pH of the extrapallial fluid. However, there was a significant increase in pH within 1 h in light or darkness, achieving a partial recovery toward the control pH value. The increase in pH within this 1-h period in light or darkness was accompanied by a significant decrease in the total ammonia concentration in the extrapallial fluid, which supports the proposition that H(+) could be transported in combination with NH(3) as NH(+)(4). Therefore, our results prompt a reexamination of the previous proposition that the removal of H(+) by NH(3) can facilitate calcification in molluscs in general and an investigation of the relationship between H(+) removal through NH(+)(4) transport and light-enhanced calcification in T. squamosa.     

The Na(+)/H(+) exchanger is a major pH regulator in GABAergic presynaptic nerve terminals synapsing onto rat CA3 pyramidal neurons

The effects of pH(i) on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were studied in mechanically dissociated CA3 pyramidal neurons, by use of ammonium prepulse and whole-cell patch-clamp techniques, under the voltage-clamp condition. NH(4)Cl itself, which is expected to alkalinize pH(i), increased GABAergic mIPSC frequency in a concentration-dependent manner. In contrast, NH(4)Cl decreased mIPSC frequency, either in the presence of 200 microm Cd(2+) or in Ca(2+)-free external solution, suggesting that intraterminal alkalosis decreased GABAergic mIPSC frequency while [NH4(+)] itself may activate Ca(2+) channels by depolarizing the terminal. On the other hand, GABAergic mIPSC frequency was greatly increased immediately after NH(4)Cl removal, a condition expected to acidify pH(i), and recovered to the control level within 2 min after NH(4)Cl removal. This explosive increase in mIPSC frequency observed after NH(4)Cl removal was completely eliminated after depletion of Ca(2+) stores with 1 microm thapsigargin in the Ca(2+)-free external solution, suggesting that acidification increases in intraterminal Ca(2+) concentration via both extracellular Ca(2+) influx and Ca(2+) release from the stores. However, the acidification-induced increase in mIPSC frequency had not recovered by 10 min after NH(4)Cl removal either in the Na(+)-free external solution or in the presence of 10 microm 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a specific Na(+)/H(+) exchanger (NHE) blocker. The present results suggest that NHEs are major intraterminal pH regulators on GABAergic presynaptic nerve terminals, and that the NHE-mediated regulation of pH(i) under normal physiological or pathological conditions might play an important role in the neuronal excitability by increasing inhibitory tones.     

A Cl(-) cotransporter selective for NH(4)(+) over K(+) in glial cells of bee retina

There appears to be a flux of ammonium (NH(4)(+)/NH(3)) from neurons to glial cells in most nervous tissues. In bee retinal glial cells, NH(4)(+)/NH(3) uptake is at least partly by chloride-dependant transport of the ionic form NH(4)(+). Transmembrane transport of NH(4)(+) has been described previously on transporters on which NH(4)(+) replaces K(+), or, more rarely, Na(+) or H(+), but no transport system in animal cells has been shown to be selective for NH(4)(+) over these other ions. To see if the NH(4)(+)-Cl(-) cotransporter on bee retinal glial cells is selective for NH(4)(+) over K(+) we measured ammonium-induced changes in intracellular pH (pH(i)) in isolated bundles of glial cells using a fluorescent indicator. These changes in pH(i) result from transmembrane fluxes not only of NH(4)(+), but also of NH(3). To estimate transmembrane fluxes of NH(4)(+), it was necessary to measure several parameters. Intracellular pH buffering power was found to be 12 mM. Regulatory mechanisms tended to restore intracellular [H(+)] after its displacement with a time constant of 3 min. Membrane permeability to NH(3) was 13 microm s(-1). A numerical model was used to deduce the NH(4)(+) flux through the transporter that would account for the pH(i) changes induced by a 30-s application of ammonium. This flux saturated with increasing [NH(4)(+)](o); the relation was fitted with a Michaelis-Menten equation with K(m) approximately 7 mM. The inhibition of NH(4)(+) flux by extracellular K(+) appeared to be competitive, with an apparent K(i) of approximately 15 mM. A simple standard model of the transport process satisfactorily described the pH(i) changes caused by various experimental manipulations when the transporter bound NH(4)(+) with greater affinity than K(+). We conclude that this transporter is functionally selective for NH(4)(+) over K(+) and that the transporter molecule probably has a greater affinity for NH(4)(+) than for K(+).     

Ammonium chloride-induced acidification in renal TALH SVE.1 cells monitored by 31P-NMR.

The aim of this study was to investigate the effect of NH4+ on the intracellular pH in TALH SVE.1 cells derived from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. These cells are specialized to perform NH4+ transport in vivo. Intracellular pH was monitored by 31P-NMR. The steady state intracellular pH (pHi) under standard conditions was 7.24 +/- 0.04 (n = 46). Exposure to NH4Cl resulted in an initial intracellular acidification of the TALH SVE.1 cells, followed by a recovery to the initial steady-state pHi value. The NH4(+)-induced acidification followed saturation kinetics up to 20 mM NH4Cl (delta pHmax = 0.2 pHunits). Half-maximal acidification was observed at 0.6 mmol/l. The intracellular acidification due to NH4Cl exposure was completely inhibited by 0.1 mM of the diuretic bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter. The effect of bumetanide was dose-dependent and a Ki value of 8.10(-7) M was calculated. NH4+ influx via K+ channels or the (Na+ + K+)ATPase could not be detected. pHi recovery to the initial value was caused mainly by amiloride-sensitive Na+/H+ exchange and to a lesser extent by an amiloride-insensitive system, which was not studied in detail. In the presence of bumetanide, pulses of high concentrations of NH4Cl induced small intracellular alkalinizations. From these experiments, an intrinsic buffer capacity (beta i) in TALH SVE.1 cells of 26 +/- 3 mM x pH-1 (pHi = 7.65) was determined. It could also be shown that the TALH SVE.1 cells exhibit maximal 'functional buffer capability' between pHout 6.9 and 7.3. Within these limits the cells can maintain their intracellular pH at a constant level, even though the extracellular pH changes. These data strongly suggest that the Na+/K+/2Cl- cotransporter is the main site of NH4+ entry into rabbit thick ascending limb cells in culture. A high intracellular buffer capacity and potent acid extrusion mechanism cooperate in counteracting the intracellular acidification caused by NH4+ influx into the cell.     

A macrophage cell model for pH and volume regulation

A whole-cell model of a macrophage (mphi) is developed to simulate pH and volume regulation during a NH4Cl prepulse challenge. The cell is assumed spherical, with a plasma membrane that separates the cytosolic and extracellular bathing media. The membrane contains background currents for Na+, K+ and Cl-, a Na(+)-K+ pump, a V-type H(+)-extruder (V-ATPase), and a leak pathway for NH4+. Cell volume is controlled by instantaneous osmotic balance between cytosolic and extracellular osmolytes. Simulations reveal that the mphi model can mimic alterations in measured pH(i) and cell volume (Vol(i)) data during and after delivery of an ammonia prepulse, which induces an acid load within the cell. Our analysis indicates that there are substantial problems in quantifying transporter-mediated H+ efflux solely from experimental observations of pH(i) recovery, as is commonly done in practice. Problems stemming from the separation of effects arise, since there is residual NH4+ dissociation to H+ inside the mphi during pH(i) recovery, as well as, proton extrusion via the V-ATPase. The core assumption of conventional measurement techniques used to estimate the H+ extrusion current (I(H)) is that the recovery phase is solely dependent on transporter-mediated H+ extrusion. However, our model predictions suggest that there are major problems in using this approach, due to the complex interactions between I(H), NH3/NH4+ buffering and NH3/NH4+ efflux during the active acid extrusion phase. That is, the conventional buffer capacity-based I(H) estimation must also take into account the perturbation that a prepulse challenge brings to the cytoplasmic acid buffer itself. The importance of this whole-cell model of mphipH(i) and volume regulation lies in its potential for extension to the characterization of several other types of non-excitable cells, such as the microglia (brain macrophage) and the T-lymphocyte.     

Interaction and inhibitory effect of ammonium cation in the oxygen evolving center of photosystem II

Photosynthetic O(2) evolution takes place at the Mn cluster in photosystem II (PSII) by oxidation of water. It has been proposed that ammonia, one of water analogues, functions as an inhibitor of O(2) evolution at alkaline pH. However, the detailed mechanism of inhibition has not been understood yet. In this study, we investigated the mechanism of ammonia inhibition by examining the NH(4)Cl-induced inhibition of O(2) evolution in a wide pH range (pH 5.0-8.0) and by detecting the interaction site using Fourier transform infrared (FTIR) spectroscopy. In addition to intact PSII membranes from spinach, PSII membranes depleted of the PsbP and PsbQ extrinsic proteins were used as samples to avoid the effect of the release of these proteins by salt treatments. In both types of samples, oxygen evolution activity decreased by approximately 40% by addition of 100 mM NH(4)Cl in the range of pH 5.0-8.0. The presence of inhibition at acidic pH without significant pH dependence strongly suggests that NH(4)(+) cation functions as a major inhibitor in the acidic pH region, where neutral NH(3) scarcely exists in the buffer. The NH(4)Cl treatment at pH 6.5 and 5.5 induced prominent changes in the COO(-) stretching regions in FTIR difference spectra upon the S(1) → S(2) transition measured at 283 K. The NH(4)Cl concentration dependence of the amplitude of the spectral changes showed a good correlation with that of the inhibition of O(2) evolution. From this observation, it is proposed that NH(4)(+) cation interacts with carboxylate groups coupled to the Mn cluster as direct ligands or proton transfer mediators, causing inhibition of the O(2) evolving reaction.     

Internal pH can regulate Ca2+ uptake and the acrosome reaction in sea urchin sperm

The egg jelly-induced acrosome reaction of sea urchin sperm is accompanied by intracellular alkalinization and Ca2+ entry. We have previously shown that in the absence of egg jelly, NH4Cl, which increases intracellular pH (pHi), induces Ca2+ uptake and the acrosome reaction in sperm of the sea urchin, Strongylocentrotus purpuratus. Here we show that at a constant concentration of NH4Cl (20 mM) in seawater, sperm react less as external pH is lowered from the normal 8 to 7.25. The pH dependence of the NH4Cl response is not very sensitive to temperatures between 12 and 17 degrees C. NH4Cl (15-50 mM) stimulates Ca2+ uptake and acrosome reactions in sperm suspended in Na+-free seawater, a condition known to inhibit the inductive effect of jelly. Jelly does not further stimulate Ca2+ uptake of sperm preincubated in NH4Cl, indicating that once the permeability to Ca2+ is increased by raising the pHi, the jelly has no further effect. We have used the membrane potential-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide to follow the membrane potential change that occurs when NH4Cl is added. Depolarization (25 mV) is associated with the acrosome reaction when either the natural inducer, egg jelly, or NH4Cl is added to sperm. Response to both inducers is inhibited under conditions known to abolish the acrosome reaction, i.e., low-pH seawater and nisoldipine. These results indicate that the NH4Cl-induced depolarization that accompanies the reaction is probably due to the opening of channels that allow Ca2+ to enter the cell and not to the depolarization by NH4+ ions. High-K+ seawater, which depolarizes sperm, and tetraethylammonium, a K+ channel blocker, inhibit the jelly-induced depolarization and the acrosome reaction, but do not inhibit NH4Cl-induced changes. It has already been shown that nigericin promotes Ca2+ entry and the acrosome reaction in sea urchin sperm. We found that the action of this ionophore depends on the pH of normal seawater. In the absence of external Na+ (replaced by choline), nigericin does not induce the reaction and does not stimulate Ca2+ uptake.     

ATP-driven,Na(+)-independent inward Cl- pumping in neuroblastoma cells

In immature neurones, the steady-state intracellular Cl- concentration [Cl-](i) is generally higher than expected for passive distribution, and this is believed to be due to Na(+)-K(+)-2Cl(-) co-transport. Here, we show that N2a neuroblastoma cells, incubated in HEPES-buffered NaCl medium maintain a [Cl-](i) around 60 mm, two- to threefold higher than expected for passive distribution at a membrane potential of - 49 mV. When the cells were transferred to a Cl(-) -free medium, [Cl-](i) decreased quickly (t(1/2) < 5 min), suggesting a high Cl- permeability. When the intracellular ATP concentration was reduced to less than 1 mm by metabolic inhibitors, the initial rate of (36) Cl- uptake was strongly inhibited (60-65%) while steady-state [Cl-](i) decreased to 24 mm, close to the value predicted from the Nernst equilibrium. Moreover, after reduction of [ATP](i) and [Cl-](i) by rotenone, the subsequent addition of glucose led to a reaccumulation of Cl-, in parallel with ATP recovery. Internal bicarbonate did not affect Cl- pumping, suggesting that Cl-/HCO(3)(-) exchange does not significantly contribute to active transport. Likewise, Na(+) -K(+) -2Cl(-) co-transport also appeared to play a minor role: although mRNA for the NKCC1 form of the co-transporter was detected in N2a cells, neither the initial rate of (36)Cl- uptake nor steady-state [Cl-](i) were appreciably decreased by 10 microm bumetanide or replacement of external Na(+) by choline. These results suggest that a highly active ATP-dependent mechanism, distinct from Na(+) -K(+) -2Cl(-) co-transport, is responsible for most of the inward Cl- pumping in N2a cells.     

Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li(+) and (86)Rb(+), with secondarily increased (86)Rb(+) influx sensitive to ouabain and to bumetanide. Increased RhAG-associated (14)C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li(+), (86)Rb(+), and (14)C-MA were pharmacologically distinct, and Li(+) uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH(4)(+) and Gd(3+). RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH(3)/NH(4)(+), but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA(+)). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH(4)Cl, but MA/MA(+) elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li(+) substitution or bath addition of 5 mM NH(4)Cl or MA/MA(+). These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH(3)/NH(4)(+) and MA/MA(+); 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA(+) transport, and decreased NH(3)/NH(4)(+)-associated depolarization; and 3) RhAG transports NH(3)/NH(4)(+) and MA/MA(+) by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.     

NH(4)Cl inhibition of acid secretion: possible involvement of an apical K(+) channel in bullfrog oxyntic cells

This study was undertaken to determine the mechanism by which ammonium chloride (NH(4)Cl) inhibits stimulated acid secretion in the bullfrog gastric mucosa. To this end, four possible pathways of inhibition were studied: 1) blockade of basolateral K(+) channel, 2) blockade of ion transport activity, 3) neutralization of secreted H(+) in the luminal solution, or 4) ATP depletion. Addition of nutrient 10 mM NH(4)Cl (calculated NH(3) concentration = 92.5 microM and NH(4)(+) concentration = 9.91 mM) inhibited acid secretion within 30 min. Inhibition of acid secretion did not occur by blockade of basolateral K(+) channel activity or ion transport activity or by neutralization of the luminal solution. Although ATP depletion occurred in the presence of NH(4)Cl, the magnitude of ATP depletion in 30 min was not sufficient to inhibit stimulated acid secretion. By comparing the effect of NH(4)Cl on the resistance of inhibited or stimulated tissues, we demonstrate that NH(4)Cl acts specifically on stimulated tissues. We propose that NH(4)Cl blocks activity of an apical K(+) channel present in stimulated oxyntic cells. Our data suggest that the activity of this channel is important for the regulation of acid secretion in bullfrog oxyntic cells.     

Kinetics of biofilm nitrification

The reaction rates (r(NH(4) (+) ) and r(NO(2) (-) )) in the two-step nitrification reaction were measured in a fluidized-sand-bed biofilm reactor under a range of steady-state conditions with respect to bulk NH(4) (+), NO(2) (-), and O(2) concentrations. It was shown from theory and experiment that under low NH(4) (+) concentration conditions, if the O(2)/NH(4) (+) concentration ratio in the bulk liquid is less than the stoichiometric coefficient (3.4 mg/mg), then oxygen will be rate limiting. In all experiments r(NO(2) (-) ) decreased more than r(NH(4) (+) ) under low oxygen conditions. This resulted in high NO(2) (-) effluent concentrations under low residence time conditions. The influence of the oxygen penetration effects on the relative values of r(NH(4) (+) ) and r(NO(2) (-) ) was experimentally shown to be caused either by the Nitrobacter location in the inner biofilm regions or by a K(m) effect for oxygen. Theoretical support of these findings was provided by a differential diffusion-reaction model which was used to simulate the experimental results.     

Intracellular pH regulation in hepatocytes isolated from three teleost species.

The mechanisms of intracellular pH (pH(i)) regulation were studied in hepatocytes isolated from three species of teleost: rainbow trout (Oncorhynchus mykiss), black bullhead (Ameiurus melas) and American eel (Anguilla rostrata). Intracellular pH was monitored over time using the pH-sensitive fluorescent dye BCECF in response to acid loading under control conditions and in different experimental media containing either low Na(+) or Cl(-) concentrations, the Na(+)-H(+) exchanger blocker amiloride or the blocker of the V-type H(+)-ATPase, bafilomycin A(1). In trout and bullhead hepatocytes, recovery to an intracellular acid load occurred principally by way of a Na(+)-dependent amiloride-sensitive Na(+)-H(+) exchanger. In eel hepatocytes, the Na(+)-H(+) exchanger did not contribute to recovery to an acid load though evidence suggests that it is present on the cell membrane and participates in the maintenance of steady-state pH(i). The V-type H(+)-ATPase did not participate in recovery to an acid load in any species. A Cl(-)-HCO(3)(-) exchanger may play a role in recovery to an acid load in eel hepatocytes by switching off and retaining base that would normally be tonically extruded. Thus, it is clear that hepatocytes isolated from the three species are capable of regulating pH(i), principally by way of a Na(+)-H(+) exchanger and a Cl(-)-HCO(3)(-) exchanger, but do not exploit identical mechanisms for pH(i) recovery. J. Exp. Zool. 284:361-367, 1999.     

Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent

Normal human colonic luminal (NH(4)(+)) concentration ([NH(4)(+)]) ranges from approximately 10 to 100 mM. However, the nature of the effects of NH(4)(+) on transport, as well as NH(4)(+) transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH(4)(+) on cAMP-stimulated Cl(-) secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH(4)(+) had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH(4)(+) reduced current within 5 min to 61 +/- 4% in the presence of 25 mM HCO(3)(-). Current inhibition was not simply due to an increase in extracellular K(+)-like cations, in that the current magnitude was 95 +/- 5% with 10 mM apical K(+) and 46 +/- 3% with 10 mM apical NH(4)(+) relative to that with 5 mM apical K(+). We previously demonstrated that inhibition of Cl(-) secretion by basolateral NH(4)(+) occurs in HCO(3)(-)-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH(4)(+) inhibition of current in HCO(3)(-) buffer did not show anomalous mole fraction behavior and followed the absolute [NH(4)(+)] in K(+)-NH(4)(+) mixtures, where K(+) concentration + [NH(4)(+)] = 10 mM. The apical NH(4)(+) inhibitory effect was not prevented by 100 microM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH(4)(+) inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 microM DIDS, 100 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 microM niflumic acid, suggesting a role for NH(4)(+) action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO(3)(-) dependence of apical NH(4)(+) inhibition of secretion is due to the action of NH(4)(+) on an apical anion exchanger.     

The effect of pH and various cations on the GTP hydrolysis of rice heterotrimeric G-protein alpha subunit expressed in Escherichia coli

Previously, we reported the biochemical properties of RGA1 that is expressed in Escherichia coli (Seo et al., 1997). The activities of RGA1 that hydrolyzes and binds guanine nucleotide were dependent on the MgCl(2) concentration. The steady state rate constant (k(cat) ) for GTP hydrolysis of RGA1 at 2 mM MgCl(2) was 0.0075 +/- 0.0001 min(-1). Here, we examined the effects of pH and cations on the GTPase activity. The optimum pH at 2 mM MgCl(2) was approximately 6.0; whereas, the pH at 2 mM NH(4)Cl was approximately 4.0. The result from the cation dependence on the GTPase (guanosine 5'-triphosphatase) activity of RGA1 under the same condition showed that the GTP hydrolysis rate (k(cat)= 0.0353 min(-1)) under the condition of 2 mM NH(4)Cl at pH 4.0 was the highest. It corresponded to about 3.24-fold of the k(cat) value of 0.0109 min(-1) in the presence of 2 mM MgCl(2) at pH 6.0.     

Matrix free Ca2+ in isolated chromaffin vesicles

Isolated secretory vesicles from bovine adrenal medulla contain 80 nmol of Ca2+ and 25 nmol of Mg2+ per milligram of protein. As determined with a Ca2+-selective electrode, a further accumulation of about 160 nmol of Ca2+/mg of protein can be attained upon addition of the Ca2+ ionophore A23187. During this process protons are released from the vesicles, in exchange for Ca2+ ions, as indicated by the decrease of the pH in the incubation medium or the release of 9-aminoacridine previously taken up by the vesicles. Intravesicular Mg2+ is not released from the vesicles by A23187, as determined by atomic emission spectroscopy. In the presence of NH4Cl, which causes the collapse of the secretory vesicle transmembrane proton gradient (delta pH), Ca2+ uptake decreases. Under these conditions A23187-mediated influx of Ca2+ and efflux of H+ cease at Ca2+ concentrations of about 4 microM. Below this concentration Ca2+ is even released from the vesicles. At the Ca2+ concentration at which no net flux of ions occurs the intravesicular matrix free Ca2+ equals the extravesicular free Ca2+. In the absence of NH4Cl we determined an intravesicular pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 microM. From this value and the known pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 microM was calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles determined in the presence of NH4Cl.(ABSTRACT TRUNCATED AT 250 WORDS)