Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water

To investigate whether Na(+) uptake by zebrafish is dependent on NH4(+) excretion, a scanning ion-selective electrode technique was applied to measure Na(+) and NH4(+) gradients at the yolk-sac surface of zebrafish larvae. Low-Na(+) acclimation induced an inward Na(+) gradient (uptake), and a combination of low Na(+) and high NH4(+) induced a larger inward Na(+) gradient. When measuring the ionic gradients, raising the external NH4(+) level (5 mM) blocked NH4(+) excretion and Na(+) uptake; in contrast, raising the external Na(+) level (10 mM) simultaneously enhanced Na(+) uptake and NH4(+) excretion. The addition of MOPS buffer (5 mM), which is known to block NH4(+) excretion, also suppressed Na(+) uptake. These results showed that Na(+) uptake and NH4(+) excretion by larval skin are associated when ambient Na(+) level is low. Knockdown of Rhcg1 translation with morpholino-oligonucleotides decreased both NH4(+) excretion and Na(+) uptake by the skin and Na(+) content of whole larvae. Knockdown of nhe3b translation or inhibitor (5-ethylisopropyl amiloride) treatment also decreased both the NH4(+) excretion and Na(+) uptake. This study provides loss-of-function evidence for the involvement of Rhcg1 and NHE3b in the ammonium-dependent Na(+) uptake mechanism in zebrafish larvae subjected to low-Na(+) water.     

Localization of ammonia transporter Rhcg1 in mitochondrion-rich cells of yolk sac, gill, and kidney of zebrafish and its ionic strength-dependent expression

Members of the Rh glycoprotein family have been shown to be involved in ammonia transport in a variety of species. Here we show that zebrafish Rhcg1, a member of the Rh glycoprotein family, is highly expressed in the yolk sac, gill, and renal tubules. Molecular cloning and characterization indicate that zebrafish Rhcg1 shares 82% sequence identity with the pufferfish ortholog fRhcg1. RT-PCR, combined with in situ hybridization, revealed that Rhcg1 is first expressed in vacuolar-type H(+)-ATPase/mitochondrion-rich cells (vH-MRC) on the yolk sac of larvae at 3 days postfertilization (dpf) and later in vH-MRC-like cells in the gill at 4-5 dpf. Ammonia excretion from zebrafish larvae increased in parallel with the expression of Rhcg1. At larval stages, Rhcg1 mRNA was detected only on the yolk sac and gill; however, the kidney, as well as the gill, becomes a major site of Rhcg1 expression in adults. Using a zebrafish Tol2 transgenic line whose vH-MRC are labeled with green fluorescent protein (GFP) and an antibody against zebrafish Rhcg1, we demonstrate that Rhcg1 is located in the apical regions of 1) vH-MRC on the yolk sac and vH-MRC-like cells (cell population with the expression of Rhcg1 and GFP) in the gill and 2) cells in the renal distal tubule and intercalated cell-like cells in the collecting duct of the kidney. Remarkably, expression of Rhcg1 mRNA at the larval stage was changed by environmental ionic strength. These results suggest that roles of zebrafish Rhcg1 are not solely ammonia secretion to eliminate nitrogen from the gill.     

Ammonia excretion via Rhcg1 facilitates Na⁺ uptake in larval zebrafish, Danio rerio, in acidic water

The involvement of a Na(+)/H(+) exchanger (NHE) in mediating Na(+) uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na(+) uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na(+) uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na(+) uptake in fish reared in acidic water (pH ～ 4.0). A role for NHE in Na(+) uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na(+)/H(+) exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na(+) uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na(+) uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na(+) uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H(+)-ATPase) significantly reduced Na(+) uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na(+) uptake in larval zebrafish in acidic water: one in which Na(+) uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H(+)-ATPase.     

Acid secretion by mitochondrion-rich cells of medaka (Oryzias latipes) acclimated to acidic freshwater

In the present study, medaka embryos were exposed to acidified freshwater (pH 5) to investigate the mechanism of acid secretion by mitochondrion-rich (MR) cells in embryonic skin. With double or triple in situ hybridization/immunocytochemistry, the Na(+)/H(+) exchanger 3 (NHE3) and H(+)-ATPase were localized in two distinct subtypes of MR cells. NHE3 was expressed in apical membranes of a major proportion of MR cells, whereas H(+)-ATPase was expressed in basolateral membranes of a much smaller proportion of MR cells. Gill mRNA levels of NHE3 and H(+)-ATPase and the two subtypes of MR cells in yolk sac skin were increased by acid acclimation; however, the mRNA level of NHE3 was remarkably higher than that of H(+)-ATPase. A scanning ion-selective electrode technique was used to measure H(+), Na(+), and NH(4)(+) transport by individual MR cells in larval skin. Results showed that Na(+) uptake and NH(4)(+) excretion by MR cells increased after acid acclimation. These findings suggested that the NHE3/Rh glycoprotein-mediated Na(+) uptake/NH(4)(+) excretion mechanism plays a critical role in acidic equivalent (H(+)/NH(4)(+)) excretion by MR cells of the freshwater medaka.     

Role of NH3 and NH4+ transporters in renal acid-base transport

Renal ammonia excretion is the predominant component of renal net acid excretion. The majority of ammonia excretion is produced in the kidney and then undergoes regulated transport in a number of renal epithelial segments. Recent findings have substantially altered our understanding of renal ammonia transport. In particular, the classic model of passive, diffusive NH3 movement coupled with NH4+ "trapping" is being replaced by a model in which specific proteins mediate regulated transport of NH3 and NH4+ across plasma membranes. In the proximal tubule, the apical Na+/H+ exchanger, NHE-3, is a major mechanism of preferential NH4+ secretion. In the thick ascending limb of Henle's loop, the apical Na+-K+-2Cl- cotransporter, NKCC2, is a major contributor to ammonia reabsorption and the basolateral Na+/H+ exchanger, NHE-4, appears to be important for basolateral NH4+ exit. The collecting duct is a major site for renal ammonia secretion, involving parallel H+ secretion and NH3 secretion. The Rhesus glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), are recently recognized ammonia transporters in the distal tubule and collecting duct. Rhcg is present in both the apical and basolateral plasma membrane, is expressed in parallel with renal ammonia excretion, and mediates a critical role in renal ammonia excretion and collecting duct ammonia transport. Rhbg is expressed specifically in the basolateral plasma membrane, and its role in renal acid-base homeostasis is controversial. In the inner medullary collecting duct (IMCD), basolateral Na+-K+-ATPase enables active basolateral NH4+ uptake. In addition to these proteins, several other proteins also contribute to renal NH3/NH4+ transport. The role and mechanisms of these proteins are discussed in depth in this review.     

Ammonia excretion and urea handling by fish gills: present understanding and future research challenges

In fresh water fishes, ammonia is excreted across the branchial epithelium via passive NH(3) diffusion. This NH(3) is subsequently trapped as NH(4)(+) in an acidic unstirred boundary layer lying next to the gill, which maintains the blood-to-gill water NH(3) partial pressure gradient. Whole animal, in situ, ultrastructural and molecular approaches suggest that boundary layer acidification results from the hydration of CO(2) in the expired gill water, and to a lesser extent H(+) excretion mediated by apical H(+)-ATPases. Boundary layer acidification is insignificant in highly buffered sea water, where ammonia excretion proceeds via NH(3) diffusion, as well as passive NH(4)(+) diffusion due to the greater ionic permeability of marine fish gills. Although Na(+)/H(+) exchangers (NHE) have been isolated in marine fish gills, possible Na(+)/NH(4)(+) exchange via these proteins awaits evaluation using modern electrophysiological and molecular techniques. Although urea excretion (J(Urea)) was thought to be via passive diffusion, it is now clear that branchial urea handling requires specialized urea transporters. Four urea transporters have been cloned in fishes, including the shark kidney urea transporter (shUT), which is a facilitated urea transporter similar to the mammalian renal UT-A2 transporter. Another urea transporter, characterized but not yet cloned, is the basolateral, Na(+) dependent urea antiporter of the dogfish gill, which is essential for urea retention in ureosmotic elasmobranchs. In ureotelic teleosts such as the Lake Magadi tilapia and the gulf toadfish, the cloned mtUT and tUT are facilitated urea transporters involved in J(Urea). A basolateral urea transporter recently cloned from the gill of the Japanese eel (eUT) may actually be important for urea retention during salt water acclimation. A multi-faceted approach, incorporating whole animal, histological, biochemical, pharmacological, and molecular techniques is required to learn more about the location, mechanism of action, and functional significance of urea transporters in fishes.     

Cellular and molecular basis of increased ammoniagenesis in potassium deprivation

Hypokalemia is associated with increased ammoniagenesis and stimulation of net acid excretion by the kidney in both humans and experimental animals. The molecular mechanisms underlying these effects remain unknown. Toward this end, rats were placed in metabolic cages and fed a control or K(+)-deficient diet (KD) for up to 6 days. Rats subjected to KD showed normal acid-base status and serum electrolytes composition. Interestingly, urinary NH(4)(+) excretion increased significantly and correlated with a parallel decrease in urine K(+) excretion in KD vs. control animals. Molecular studies showed a specific upregulation of the glutamine transporter SN1, which correlated with the upregulation of glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase. These effects occurred as early as day 2 of KD. Rats subjected to a combined KD and 280 mM NH(4)Cl loading (to induce metabolic acidosis) for 2 days showed an additive increase in NH(4)(+) excretion along with an additive increment in the expression levels of ammoniagenic enzymes GA and GDH compared with KD or NH(4)Cl loading alone. The incubation of cultured proximal tubule cells NRK 52E or LLC-PK(1) in low-K(+) medium did not affect NH(4)(+) production and did not alter the expression of SN1, GA, or GDH in NRK cells. These results demonstrate that K(+) deprivation stimulates ammoniagenesis through a coordinated upregulation of glutamine transporter SN1 and ammoniagenesis enzymes. This effect is developed before the onset of hypokalemia. The signaling pathway mediating these events is likely independent of KD-induced intracellular acidosis. Finally, the correlation between increased NH(4)(+) production and decreased K(+) excretion indicate that NH(4)(+) synthesis and transport likely play an important role in renal K(+) conservation during hypokalemia.     

Effect of hypokalemia on renal expression of the ammonia transporter family members, Rh B Glycoprotein and Rh C Glycoprotein, in the rat kidney

Hypokalemia is a common electrolyte disorder that increases renal ammonia metabolism and can cause the development of an acid-base disorder, metabolic alkalosis. The ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), are expressed in the distal nephron and collecting duct and mediate critical roles in acid-base homeostasis by facilitating ammonia secretion. In the current studies, the effect of hypokalemia on renal Rhbg and Rhcg expression was examined. Normal Sprague-Dawley rats received either K(+)-free or control diets for 2 wk. Rats receiving the K(+)-deficient diet developed hypokalemia and metabolic alkalosis associated with significant increases in both urinary ammonia excretion and urine pH. Rhcg expression increased in the outer medullary collecting duct (OMCD). In OMCD intercalated cells, hypokalemia resulted in more discrete apical Rhcg expression and a marked increase in apical plasma membrane immunolabel. In principal cells, in the OMCD, hypokalemia increased both apical and basolateral Rhcg immunolabel intensity. Cortical Rhcg expression was not detectably altered by immunohistochemistry, although there was a slight decrease in total expression by immunoblot analysis. Rhbg protein expression was decreased slightly in the cortex and not detectably altered in the outer medulla. We conclude that in rat OMCD, hypokalemia increases Rhcg expression, causes more polarized apical expression in intercalated cells, and increases both apical and basolateral expression in the principal cell. Increased plasma membrane Rhcg expression in response to hypokalemia in the rat, particularly in the OMCD, likely contributes to the increased ammonia excretion and thereby to the development of metabolic alkalosis.     

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

Although colonic lumen NH(4)(+) levels are high, 15-44 mM normal range in humans, relatively few studies have addressed the transport mechanisms for NH(4)(+). More extensive studies have elucidated the transport of NH(4)(+) in the kidney collecting duct, which involves a number of transporter processes also present in the distal colon. Similar to NH(4)(+) secretion in the renal collecting duct, we show that the distal colon secretory model, T84 cell line, has the capacity to secrete NH(4)(+) and maintain an apical-to-basolateral NH(4)(+) gradient. NH(4)(+) transport in the secretory direction was supported by basolateral NH(4)(+) loading on NKCC1, Na(+)-K(+)-ATPase, and the NH(4)(+) transporter, RhBG. NH(4)(+) was transported on NKCC1 in T84 cells nearly as well as K(+) as determined by bumetanide-sensitive (86)Rb-uptake. (86)Rb-uptake and ouabain-sensitive current measurement indicated that NH(4)(+) is transported by Na(+)-K(+)-ATPase in these cells to an equal extent as K(+). T84 cells expressed mRNA for the basolateral NH(4)(+) transporter RhBG and the apical NH(4)(+) transporter RhCG. Net NH(4)(+) transport in the secretory direction determined by (14)C-methylammonium (MA) uptake and flux occurred in T84 cells suggesting functional RhG protein activity. The occurrence of NH(4)(+) transport in the secretory direction within a colonic crypt cell model likely serves to minimize net absorption of NH(4)(+) because of surface cell NH(4)(+) absorption. These findings suggest that we rethink the present limited understanding of NH(4)(+) handling by the distal colon as being due solely to passive absorption.     

Root-zone acidity and nitrogen source affects Typha latifolia L. growth and uptake kinetics of ammonium and nitrate

The NH(4)(+) and NO(3)(-) uptake kinetics by Typha latifolia L. were studied after prolonged hydroponics growth at constant pH 3.5, 5.0, 6.5 or 7.0 and with NH(4)(+) or NO(3)(-) as the sole N-source. In addition, the effects of pH and N source on H(+) extrusion and adenine nucleotide content were examined. Typha latifolia was able to grow with both N sources at near neutral pH levels, but the plants had higher relative growth rates, higher tissue concentrations of the major nutrients, higher contents of adenine nucleotides, and higher affinity for uptake of inorganic nitrogen when grown on NH(4)(+). Growth almost completely stopped at pH 3.5, irrespective of N source, probably as a consequence of pH effects on plasma membrane integrity and H(+) influx into the root cells. Tissue concentrations of the major nutrients and adenine nucleotides were severely reduced at low pH, and the uptake capacity for inorganic nitrogen was low, and more so for NO(3)(-)-fed than for NH(4)(+)-fed plants. The maximum uptake rate, V(max), was highest for NH(4)(+) at pH 6.5 (30.9 micro mol h(-1) g(-1) root dry weight) and for NO(3)(-) at pH 5.0 (31.7 micro mol h(-1) g(-1) root dry weight), and less than 10% of these values at pH 3.5. The affinity for uptake as estimated by the half saturation constant, K((1/2)), was lowest at low pH for NH(4)(+) and at high pH for NO(3)(-). The changes in V(max) and K((1/2)) were thus consistent with the theory of increasing competition between cations and H(+) at low pH and between anions and OH(-) at high pH. C(min) was independent of pH, but slightly higher for NO(3)(-) than for NH(4)(+) (C(min)(NH(4)(+)) approximately 0.8 mmol m(-3); C(min)(NO(3)(-)) approximately 2.8 mmol m(-3)). The growth inhibition at low pH was probably due to a reduced nutrient uptake and a consequential limitation of growth by nutrient stress. Typha latifolia seems to be well adapted to growth in wetland soils where NH(4)(+) is the prevailing nitrogen compound, but very low pH levels around the roots are very stressful for the plant. The common occurrence of T. latifolia in very acidic areas is probably only possible because of the plant's ability to modify pH-conditions in the rhizosphere.     

NH4+-stimulated and -inhibited components of K+ transport in rice (Oryza sativa L.)

The disruption of K(+) transport and accumulation is symptomatic of NH(4)(+) toxicity in plants. In this study, the influence of K(+) supply (0.02-40 mM) and nitrogen source (10 mM NH(4)(+) or NO(3)(-)) on root plasma membrane K(+) fluxes and cytosolic K(+) pools, plant growth, and whole-plant K(+) distribution in the NH(4)(+)-tolerant plant species rice (Oryza sativa L.) was examined. Using the radiotracer (42)K(+), tissue mineral analysis, and growth data, it is shown that rice is affected by NH(4)(+) toxicity under high-affinity K(+) transport conditions. Substantial recovery of growth was seen as [K(+)](ext) was increased from 0.02 mM to 0.1 mM, and, at 1.5 mM, growth was superior on NH(4)(+). Growth recovery at these concentrations was accompanied by greater influx of K(+) into root cells, translocation of K(+) to the shoot, and tissue K(+). Elevating the K(+) supply also resulted in a significant reduction of NH(4)(+) influx, as measured by (13)N radiotracing. In the low-affinity K(+) transport range, NH(4)(+) stimulated K(+) influx relative to NO(3)(-) controls. It is concluded that rice, despite its well-known tolerance to NH(4)(+), nevertheless displays considerable growth suppression and disruption of K(+) homeostasis under this N regime at low [K(+)](ext), but displays efficient recovery from NH(4)(+) inhibition, and indeed a stimulation of K(+) acquisition, when [K(+)](ext) is increased in the presence of NH(4)(+).     

The initial kinetics of NH3/NH4(+) efflux from L3 Teladorsagia circumcincta

The initial rate of NH(3)/NH(4)(+) accumulation in a medium containing L(3) Teladorsagia circumcincta was 0.18-0.6 pmol h(-1) larva(-1), which increased linearly with larval density. However it appeared that the larva-generated external concentration of NH(3)/NH(4)(+) did not exceed about 130 μM. The rate of NH(3)/NH(4)(+) accumulation increased with temperature between 4 °C and 37 °C, declined with increasing pH or increasing external NH(3)/NH(4)(+) concentration and was not significantly affected by the concentration of the phosphate buffer or by exsheathing the larvae. We infer from these data that the efflux of NH(3)/NH(4)(+) is a diffusive process and that the secreted or excreted NH(3)/NH(4)(+) is generated enzymatically rather than dissociating from the surface of the nematode. The enzymatic source of the NH(3)/NH(4)(+) is yet to be identified. Since the concentration of NH(3)/NH(4)(+) in the rumen and abomasum is higher than 130 μM, it is unlikely that T. circumcincta contributes to it, but NH(3)/NH(4)(+) may be accumulated from the rumen fluid by the nematode.     

High-affinity potassium transport in barley roots. Ammonium-sensitive and -insensitive pathways

In an attempt to understand the process mediating K(+) transport into roots, we examined the contribution of the NH(4)(+)-sensitive and NH(4)(+)-insensitive components of Rb(+) transport to the uptake of Rb(+) in barley (Hordeum vulgare L.) plants grown in different ionic environments. We found that at low external Rb(+) concentrations, an NH(4)(+)-sensitive component dominates Rb(+) uptake in plants grown in the absence of NH(4)(+), while Rb(+) uptake preferentially occurs through an NH(4)(+)-insensitive pathway in plants grown at high external NH(4)(+) concentrations. A comparison of the Rb(+)-uptake properties observed in roots with those found in heterologous studies with yeast cells indicated that the recently cloned HvHAK1 K(+) transporter may provide a major route for the NH(4)(+)-sensitive component. HvHAK1 failed to complement the growth of a yeast strain defective in NH(4)(+) transport, suggesting that it could not act as an NH(4)(+) transporter. Heterologous studies also showed that the HKT1 K(+)/Na(+)-cotransporter may act as a pathway for high-affinity Rb(+) transport sensitive to NH(4)(+). However, we found no evidence of an enhancement of Rb(+) uptake into roots due to Na(+) addition. The possible identity of the systems contributing to the NH(4)(+)-insensitive component in barley plants is discussed.     

A kinetic study of the gill (Na+, K+)-ATPase, and its role in ammonia excretion in the intertidal hermit crab, Clibanarius vittatus

To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na(+), K(+), NH(4)(+) and ATP of (Na(+), K(+))-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg(2+), Na(+) and K(+) concentrations, two well-defined ATP hydrolyzing sites were revealed. ATP was hydrolyzed at the high-affinity sites at a maximum rate of V=19.1+/-0.8 U mg(-1) and K(0.5)=63.8+/-2.9 nmol L(-1), obeying cooperative kinetics (n(H)=1.9); at the low-affinity sites, hydrolysis obeyed Michaelis-Menten kinetics with K(M)=44.1+/-2.6 mumol L(-1) and V=123.5+/-6.1 U mg(-1). Stimulation by Na(+) (V=149.0+/-7.4 U mg(-1); K(M)=7.4+/-0.4 mmol L(-1)), Mg(2+) (V=132.0+/-5.3 U mg(-1); K(0.5)=0.36+/-0.02 mmol L(-1)), NH(4)(+) (V=245.6+/-9.8 U mg(-1); K(M)=4.5+/-0.2 mmol L(-1)) and K(+) (V=140.0+/-4.9 U mg(-1); K(M)=1.5+/-0.1 mmol L(-1)) followed a single saturation curve and, except for Mg(2+), obeyed Michaelis-Menten kinetics. Under optimal ionic conditions, but in the absence of NH(4)(+), ouabain (K(I)=117.3+/-3.5 mumol L(-1)) and orthovanadate inhibited up to 67% of the ATPase activity. The inhibition studies performed suggest the presence of F(0)F(1), V- and P-ATPases, but not Na(+)-, K(+)- or Ca(2+)-ATPases as contaminants in the gill microsomal preparation. (Na(+), K(+))-ATPase activity was synergistically modulated by NH(4)(+) and K(+). At 20 mmol L(-1) K(+), a maximum rate of V=290.8+/-14.5 U mg(-1) was seen as NH(4)(+) concentration was increased up to 50 mmol L(-1). However, at fixed NH(4)(+) concentrations, no additional stimulation was found for increasing K(+) concentrations (V=135.2+/-4.1 U mg(-1) and V=236.6+/-9.5 U mg(-1) and for 10 and 30 mmol L(-1) NH(4)(+), respectively). This is the first report to detail ionic modulation of gill (Na(+), K(+))-ATPase in C. vittatus, revealing an asymmetrical, synergistic stimulation of the enzyme by K(+) and NH(4)(+), as yet undescribed for other (Na(+), K(+))-ATPases, and should provide a better understanding of NH(4)(+) excretion in pagurid crabs.     

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.     

Responses to reversed NH3 and NH4+ gradients in a teleost (Ictalurus punctatus), an elasmobranch (Raja erinacea), and a crustacean (Callinectes sapidus):evidence for NH4+/H+ exchange in the teleost and the elasmobranch

Ammonia excretion rates of channel catfish, Ictalurus punctatus, little skate (Raja erinacea), and blue crab (Callinectes sapidus) were measured in experimental regimes which permitted simultaneous assessment of the partial pressure gradients for nonionized NH3 and the chemical concentration gradients of NH4+. Under conditions of low external ammonia, the average ammonia excretion was +295 microM kg-1 h-1 for catfish, +149 microM kg-1 h-1 for blue crabs, and +59 microM kg-1 h1 for skates with partial pressure gradients of +72.5 mu Torr, +413 mu Torr, and +24.4 mu Torr, respectively; and [NH4+] gradients of +189 microM l-1, +643 microM l-1, and +107 microM l-1 (positive indicating greater from inside to medium). When the external ammonia was increased to 1.15 mM l-1, both gradients were reversed, and the net ammonia movement was initially from the external water into all three species. In the catfish the inward movement ceased, however, and ammonia excretion eventually resumed in the face of reversed gradients of both NH3 partial pressure and [NH4+]. Unidirectional Na+ influx, indicative of a Na+/NH4+ exchange, did not increase. The ammonia data, changes in titratable acidity, and net apparent H+ efflux were all consistent with a linked extrusion of internal NH4+ for external H+. Incorporation of such an exchange into a computer simulation model of the ammonia equilibrium and exchange system duplicated the experimental data. Other hypotheses failed to match experimental data, or failed to predict internal ammonia levels lower than outside. In the crab, internal ammonia levels rose rapidly to concentrations and partial pressures above the external medium until excretion was reestablished, with no evidence of maintenance of a reversed gradient. In the skate, internal concentrations rose appreciably in the first hour and continued to rise for 6-8 h, with no resumption of ammonia excretion. The interspecies differences appear to be due at least partly to differences in ammonia permeability of the gills.     

Kinetics and fates of ammonia, urea, and uric acid during oocyte maturation and ontogeny of the Atlantic halibut (Hippoglossus hippoglossus L.)

Considering that amino acids constitute an important energy fuel during early life of the Atlantic halibut (Hippoglossus hippoglossus L.), it is of interest to understand how the nitrogenous end products are handled. In this study we focused on the kinetics and fates of ammonia, urea and uric acid. The results showed that ammonia (T(Amm): NH(3)+NH(4)(+)), and urea-N contents increased during final oocyte maturation. Urea-N excretion dominated the total nitrogenous end product formation in early embryos. Later, yolk T(Amm) levels increased in embryos and ammonia excretion was low. In the last part of the embryonic stage T(Amm) accumulation dominated, and was apparently due to yolk storage. Around hatching, the larval body tissues (larva with yolk-sac removed) accounted for 68% of whole animal urea-N accumulation, while T(Amm) levels increased predominately by yolk accumulation. Afterwards, ammonia excretion dominated and uric acid accumulation accounted for less than 1%. Urea, synthesised either through the ornithine-urea cycle, argininolysis or uricolysis, accounted for approximately 8% of total nitrogenous end product formation in yolk-sac larvae. The results suggested that a sequence occurred regarding which nitrogenous end products dominated and how they were handled. Urea excretion dominated in early embryos (<7 dPF), followed by yolk ammonia accumulation (7-12 dPF), and finally, ammonia excretion dominated in later embryonic and yolk-sac larval stages (>12 dPF).