Cyclic AMP and calcium in the differential control ofMytilus gill cilia

Summary Lateral (L) cilia ofMytilus gill are activated by serotonin which, in molluscan systems, is known to activate adenylate cyclase. Triton-extracted models of L-cells, arrested at >10^–6 M Ca⁺⁺, are stimulated to beat by the addition of 10^–5 M cAMP while still under Ca⁺⁺ arrest conditions, suggesting that cAMP-activation is not mediated by alterations of Ca⁺⁺ levels. Using isolated, permeabilized cilia, we find, independent of [Ca⁺⁺], that cAMP-dependent protein phosphorylation in L-cilia occurs uniquely and reversibly on three low molecular weight polypeptides of 23,000, 18,000, and 14,000 daltons. Phosphorylation is maximal at cAMP concentrations above 0.5 M. The phosphorylated chains partially coextract at high salt with a 14S dynein fraction and have approximately the same molecular weights as reported for dynein light chains. Such conditions mainly extract the outer dynein arm, about 40% of the Mg⁺⁺-ATPase activity, and a corresponding amount of the cAMP phosphorylated chains. However, the three polypeptides sediment together at 10–11S, clearly separable from the 14S dynein ATPase. Using a gel-overlay technique, we find that calmodulin binds to axonemal polypeptides of L-cilia with molecular weights of 18,000 and 13,000, independent of Ca⁺⁺, while in mixed-population cilia, only a 12,000 dalton chain binds calmodulin, in a Ca⁺⁺ dependent manner. In neither case are calmodulin binding proteins found in the high salt fraction containing the cAMP-dependent phosphorylated chains, indicating that, in spite of some similarity in molecular weight, the cAMP-phosphorylated and calmodulin binding polypeptides are different. Also, double-labeling indicates that only the 18,000 dalton chains co-migrate. These data suggest that serotonin may activate lateral cilia through a cAMP-dependent phosphorylation of a dynein-associated regulatory protein complex, while Ca⁺⁺ may inhibit ciliary movement, independently, by binding to calmodulin associated with a different class of regulatory protein.     

Autoradiographic demonstration of the use of free amino acid by Sargasso Sea zooplankton

An investigation was undertaken to observe whether soft-bodiedsurface zooplankton of nutrient-depleted ocean waters, suchas the Sargasso Sea, can take up and retain dissolved nutrientsin a manner similar to that previously shown for many benthicforms living in richer environments. Freshly collected samplesof 15 species (five phyla) were incubated for 2 h in sea waterto which a small quantity of ¹⁴C-labeled amino acid mixturehad been added. They were then prepared as histological sectionsand examined with stripping film autoradiography. All of theforms showed strong uptake and retention of the tracer in partsof their epidermis, and various specific internal structures.Greatest uptake was seen in tentacles, parapodial paddles, gillsand restricted parts of the guts or gastrovascular cavities.It is concluded that many animals living in depleted ocean watersstill can obtain benefits from dissolved nutrients, either directlyor by symbiotic relationships with microorganisms. These cansupplement the more obvious forms of nutrition and perhaps playimportant roles in sustaining more isolated body parts. Someadditional benefit also may be drived through fluid ingestionas well as ingestion of microorganisms that have taken up dissolvedmaterials.     

Kinetics of amino acid uptake by ectomycorrhizal roots

It is well established that ectomycorrhizal fungi can use amino acids as nitrogen and carbon sources, but data on the kinetic properties of amino acid uptake systems of ectomycorrhizal systems are scarce. Using ¹⁴C-labelled compounds we have determined the kinetics of uptake of amino acids by excised ectomycorrhizal roots for a range of distinct mycorrhizal types from three tree species, beech, spruce, and pine. All mycorrhizal types examined took up amino acids via high-affinity transport systems ( K _M values ranging from 19 to 233 mmol m^–3). A comparative analysis of kinetic parameters for uptake of amino acids and the ammonium analogue methylammonium showed that ectomycorrhizal roots have similar or even higher affinities (lower K _M values) for the amino acids, indicating that absorption of these organic forms of nitrogen (N) can contribute significantly to total N uptake by ectomycorrhizal plants. Analysis of amino acid uptake by ectomycorrhizal roots collected along a European north/south gradient of increasing mineral N pollution from northern Sweden to south Germany revealed no obvious trend in the uptake capabilities for amino acids by ectomycorrhizal roots in relation to the location of the sampling site on this gradient. Rather, the fungal species forming a particular morphotype was the factor determining uptake kinetics. It can therefore be deduced that the species composition of the fungal community will contribute significantly to the functional diversity of a population of mycorrhizal roots.     

Multiple pathways for amino acid transport inMytilus gill

Summary The epidermal tissues of marine mussels can accumulate amino acids from surrounding sea water. In the present study, gill tissue isolated from the California coastal mussel,Mytilus californianus, was used in conjunction with intact, actively pumping mussels to study epidermal transport processes. There appeared to be at least four pathways for this uptake: i) a -neutral pathway which transports taurine; ii) an -acidic pathway specific for substrates such as aspartate; iii) an -neutral pathway having a general specificity for this class of compound, but which also accepts the basic amino acid, lysine; and iv) a second -neutral pathway, also of broad specificity, capable of accepting the imino acid, proline, as a substrate. Replacement of Na in sea water with choline reduced uptake of leucine, taurine, aspartate, and proline by more than 95%, and reduced lysine uptake by 75%, suggesting that Na-independent pathways play no significant role in epidermal transport in the gill. Isolated gill tissue was used to estimate the maximum transport capacities (J _max's) of the pathways, which ranged from approximately 5 to 25 mol/(g·hr). Apparent Michaelis constants (K _t ^*'s) of the epidermal transporters were estimated using a convection-diffusion model introduced previously (Wright and Secomb, Am J Physiol 247:R346–R355, 1984). TheseK _t ^*'s ranged from 1 to 5 M. The characteristics of the epidermal transporters are such that they can play a significant role in both animal nutrition and in the reacumulation of endogenous amino acids lost from surface cells through passive diffusion.Abbreviation ASW artificial sea water     

Diel rhythmicity in amino acid uptake by Prochlorococcus

The marine cyanobacterium Prochlorococcus , the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light-synchronized axenic Prochlorococcus (PCC9511 strain) culture and ³⁵S-methionine and ³H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >10⁴ times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G₂ cell cycle stage were consistently 2.2 times higher than those of cells at the G₁ stage. Furthermore, S+G₂ cells upregulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday to 42% at dusk of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations.     

Mechanical sensitivity and cell coupling in the ciliated epithelial cells ofMytilus edulis gill

Summary Transmission electron microscopy has not provided strong evidence for gap junctions inMytilus edulis gill tissue, in spite of extensive physiological evidence for coupled ciliary arrest in lateral cells and coupled activation in abfrontal cells. To investigate the kinds and relative distribution of cell junctions and also to determine whether ciliary membrane particle differences exist in these two types of oppositely mechanically sensitive cells, we analyzed the structure of these and two other ciliated cell types (frontal and laterofrontal) by freeze-fracture replication. Gap junctions occur in all four ciliated cell types, but they are relatively small and of variable morphology, often consisting of elongate, winding complexes of membrane particles. Statistically, such structures rarely would be recognized as gap junctions in thin sections. Gap junctions appear to be most abundant between the highly coupled abfrontal cells, minimal between laterofrontal cells, and not evident in the epithelial cells that separate coupled ciliated cell types. The ciliary necklaces of the mechanically activated abfrontal cilia are typically 4- or 5-stranded while those of the remaining three cell types are mainly 3-stranded. In developing gill tips, ciliated cells have abundant gap junctions and newly formed cilia have a full complement of necklace particles. Nascent lateral cilia are not mechanically sensitive, indicating that the acquisition of mechanosensitivity does not correlate with the presence of ciliary necklace or other membrane particles. Lateral and laterofrontal cells become sensitive to neurotransmitters soon after the appearance of the latter during development, but mechanosensitivity of both lateral and abfrontal cells arises substantially later.     

Modulation of amino acid uptake by TGF-beta in lung myofibroblasts

Hormones such as insulin, growth factors, and cell stress stimulate system A amino acid transporter. Transforming growth factor-beta (TGF-beta) stimulates amino acid uptake thereby inducing cell proliferation, cellular hypertrophy, and matrix synthesis. Insulin appears to activate amino acid in smooth muscle cells via a phosphatidylinositol 3-kinase (PI3-kinase)-dependent pathway. We examine the effect and interaction of TGF-beta, insulin, and PI3-kinase activity on amino acid uptake in human lung myofibroblasts. TGF-beta treatment induced large increases in system A activity and a small delayed increase in the phosphorylation of protein kinase B, also termed phospho-Akt. In contrast, insulin induced small increases in system A activity and large increases in phospho-Akt levels. LY294002, a PI3-kinase inhibitor, blocked the TGF-beta-induced amino acid uptake only partially, but completely blocked TGF-beta-induced Akt phosphorylation. Moreover, the level of phospho-Smad3 was found to be high even when LY294002 blocked TGF-beta-induced phospho-Akt levels. Inhibition of PI3-kinase activity resulted in increase in Km, consistent with a major change in transporter activity without change in transporter number. The PI3-kinase inhibitor also did not change the amino acid transporter 2 (ATA2) mRNA levels. Taken together, these results suggest that TGF-beta induced Smad-3 and amino acid uptake through a PI3-kinase independent pathway.     

Kinetic analysis of insulin action on amino acid uptake by isolated chick embryo heart cells

1. Isolated chick embryo heart cells were used to investigate the mode of action of insulin on the transport of three naturally occurring amino acids: l-proline, l-serine and glycine. Initial velocities of uptake were measured over a period of 5min with an 80-fold range of amino acid concentration. Corrections for amino acid diffusion, incorporation into protein and conversion into carbon dioxide were introduced. 2. The uptake processes approximated Michaelis-Menten kinetics within definite ranges of amino acid concentrations. A single transport system for proline and at least two transport systems for serine and glycine were detected. 3. The kinetic effects of insulin on transport systems for the amino acids tested were consistent with an acceleration of the maximal velocity of the process, without substantial changes in substrate concentration for half-maximal transport velocity. 4. These hormonal effects were not essentially altered by the corrections for amino acid incorporation into protein and conversion into carbon dioxide.     

Effect of ammonia on amino acid uptake by brain microvessels

NH+4 ions, at a concentration (0.25 mM) similar to that found in the plasma of patients with hepatic encephalopathy, cause, in vitro, a significant stimulation of the uptake by brain microvessels of large neutral amino acids, without any effect on the uptake of alpha-methylaminoisobutyric acid, glutamic acid, or lysine. Such a stimulation occurs essentially through an increase of the maximal transport capacity (Vmax) of the saturable component. It is apparently mediated by the intracellular formation of glutamine, which is then exchanged, through the L-system of transport, for large neutral amino acids such as leucine, phenylalanine, or tyrosine. At higher concentrations (greater than or equal to 0.5 mM), NH+4 ions cause also a decrease of carrier affinity for neutral amino acids, which counteracts the stimulatory effect on their uptake.     

Cystine and dibasic amino acid uptake by opossum kidney cells

The characteristics of the uptake of L-cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which is found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystine/glutamate transport system present in LLC-PK1 cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high-affinity saturable process which is independent of medium sodium concentration. The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cystine with a ratio greater than 100:1 for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in human cystinuria.     

Characteristics of amino acid uptake in barley

Plants have the ability to take up organic nitrogen (N) but this has not been thoroughly studied in agricultural plants. A critical question is whether agricultural plants can acquire amino acids in a soil ecosystem. The aim of this study was to characterize amino acid uptake capacity in barley (Hordeum vulgare L.) from a mixture of amino acids at concentrations relevant to field conditions. Amino acids in soil solution under barley were collected in microlysimeters. The recorded amino acid composition, 0–8.2 μM of l-Serine, l-Glutamic acid, Glycine, l-Arginine and l-Alanine, was then used as a template for uptake studies in hydroponically grown barley plants. Amino acid uptake during 2 h was studied at initial concentrations of 2–25 μM amino acids and recorded as amino acid disappearance from the incubation solution, analysed with HPLC. The uptake was verified in control experiments using several other techniques. Uptake of all five amino acids occurred at 2 μM and below. The concentration dependency of the uptake rate could be described by Michaelis–Menten kinetics. The affinity constant (K _m) was in the range 19.6–33.2 μM. These K _m values are comparable to reported values for soil micro-organisms.     

Intact amino acid uptake by northern hardwood and conifer trees

Empirical and modeling studies of the N cycle in temperate forests of eastern North America have focused on the mechanisms regulating the production of inorganic N, and assumed that only inorganic forms of N are available for plant growth. Recent isotope studies in field conditions suggest that amino acid capture is a widespread ecological phenomenon, although northern temperate forests have yet to be studied. We quantified fine root biomass and applied tracer-level quantities of U–¹³C₂–¹⁵N-glycine, ¹⁵NH₄ ⁺ and ¹⁵NO₃ ⁻ in two stands, one dominated by sugar maple and white ash, the other dominated by red oak, beech, and hemlock, to assess the importance of amino acids to the N nutrition of northeastern US forests. Significant enrichment of ¹³C in fine roots 2 and 5 h following tracer application indicated intact glycine uptake in both stands. Glycine accounted for up to 77% of total N uptake in the oak–beech–hemlock stand, a stand that produces recalcitrant litter, cycles N slowly and has a thick, amino acid-rich organic horizon. By contrast, glycine accounted for only 20% of total N uptake in the sugar maple and white ash stand, a stand characterized by labile litter and rapid rates of amino acid production and turnover resulting in high rates of mineralization and nitrification. This study shows that amino acid uptake is an important process occurring in two widespread, northeastern US temperate forest types with widely differing rates of N cycling.     

A GC-MS method for determination of amino acid uptake by plants

In this study, we present a rapid, robust and sensitive method for quantification of plant amino acid uptake using universally (U) (¹³C, ¹⁵N)-labelled amino acids and gas chromatography-mass spectrometry (GC-MS). Amino acids were analysed as their tert -butyldimethylsilyl (tBDMS) derivatives and displayed detection limits in the range 10–100 fmol on column, depending on the amino acid. The technique allows for simultaneous detection and quantification of both unlabelled and isotopically labelled species of amino acids. This makes simple quantification of plant amino acid uptake from an isotopically labelled source possible. The analytical variation was low, concerning total amino acid concentrations (relative standard deviation, rsd , less than 5.3%) as well as enrichment of U-¹³C, ¹⁵N-labelled glycine (Gly), arginine (Arg) and glutamic acid (Glu) ( rsd <2.1%). An application of the GC-MS method was conducted on non-mycorrhizal Pinus sylvestris roots supplied with U-¹³C, ¹⁵N-labelled amino acids. Intact, labelled amino acids were traced in root extracts. This provided conclusive evidence of plant root uptake of intact amino acids. Uptake rates of the three amino acids Gly, Glu and Arg in the range 0.5–37.9 μmol g⁻¹ dry weight h⁻¹ were recorded. These rates are comparable with those recorded in earlier studies of amino acid uptake, using other methods, as well as uptake rates measured for nitrate and ammonium.