Differences in plant function in phosphorus- and nitrogen-limited mangrove ecosystems

Mangrove ecosystems can be either nitrogen (N) or phosphorus (P) limited and are therefore vulnerable to nutrient pollution. Nutrient enrichment with either N or P may have differing effects on ecosystems because of underlying differences in plant physiological responses to these nutrients in either N- or P-limited settings. Using a common mangrove species, Avicennia germinans, in sites where growth was either N or P limited, we investigated differing physiological responses to N and P limitation and fertilization. We tested the hypothesis that water uptake and transport, and hydraulic architecture, were the main processes limiting productivity at the P-limited site, but that this was not the case at the N-limited site. We found that plants at the P-deficient site had lower leaf water potential, stomatal conductance and photosynthetic carbon-assimilation rates, and less conductive xylem, than those at the N-limited site. These differences were greatly reduced with P fertilization at the P-limited site. By contrast, fertilization with N at the N-limited site had little effect on either photosynthetic or hydraulic traits. We conclude that growth in N- and P-limited sites differentially affect the hydraulic pathways of mangroves. Plants experiencing P limitation appear to be water deficient and undergo more pronounced changes in structure and function with relief of nutrient deficiency than those in N-limited ecosystems.     

Regulation of growth and polyamine biosynthesis of the ciliated protozoan Tetrahymena thermophila. Effects of L-arginine metabolites and polyamines

Growth of Tetrahymena thermophila in a synthetic nutrient medium with or without the essential amino acid L-arginine was studied in the presence or absence of the arginine metabolites L-citrulline and L-ornithine and the polyamines putrescine, spermidine, and spermine. The effects of the growth conditions on the stimulations of the enzymes of the arginine metabolic and polyamine biosynthetic pathway, arginine deiminase (ADI), citrulline hydrolase (CH), ornithine decarboxylase (ODC), and ornithine-oxo-acid aminotransferase were determined. Tetrahymena cells were unable to grow in the absence of L-arginine and the amino-acid utilization was greatly impaired. None of the metabolites or polyamines was able to substitute for arginine. In the presence of arginine, Tetrahymena cultures grew well and citrulline and ornithine did not alter the growth behaviour in any way. In the presence of putrescine, the lag period was decreased from 3 h to 2 h. Spermidine and spermine acted similar to putrescine but less pronounced. The stimulation of the activity of ADI, the key enzyme of arginine degradation, was absolutely dependent upon the presence of arginine in the medium: in the absence of arginine, the low ADI activity which was present in the cells before inoculation was decreased to zero levels within 30 min. In the presence of arginine, the stimulation of ADI was not altered by citrulline and ornithine but putrescine, spermidine, and spermine decreased ADI-stimulation to half of the control values. The stimulation of CH activity in the presence of arginine was not altered by any added metabolite or polyamine. In the media without arginine, stimulation of CH was greatly reduced, in the presence of ornithine more than in its absence, and even more in the presence of putrescine and spermidine. Stimulation of ODC activity in the presence of arginine was not affected by citrulline and ornithine but in the presence of polyamines it was rapidly decreased to unstimulated levels after an initial ca. 10-fold increase. The "hyperstimulation" of ODC in the absence of free arginine was reduced to normal in the presence of citrulline, the stimulation was decreased even below normal levels in the presence of ornithine and polyamines decreased ODC activity to zero levels. O delta T activity was stimulated more in the presence of arginine than in its absence. In both cases the stimulation was enhanced in the presence of polyamines and only in the absence of arginine--by ornithine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nutrient Addition Differentially Affects Ecological Processes of <Emphasis Type="Italic">Avicennia germinans</Emphasis> in Nitrogen versus Phosphorus Limited Mangrove Ecosystems

Nutrient over-enrichment is a major threat to marine environments, but system-specific attributes of coastal ecosystems may result in differences in their sensitivity and susceptibility to eutrophication. We used fertilization experiments in nitrogen (N)- and phosphorus (P)-limited mangrove forests to test the hypothesis that alleviating different kinds of nutrient limitation may have different effects on ecosystem structure and function in natural systems. We compared a broad range of ecological processes to determine if these systems have different thresholds where shifts might occur in nutrient limitation. Growth responses indicated N limitation in Avicennia germinans (black mangrove) forests in the Indian River Lagoon (IRL), Florida, and P limitation at Twin Cays, Belize. When nutrient deficiency was relieved, A. germinans grew out of its stunted form by increasing wood relative to leaf biomass and shoot length relative to lateral growth. At the P-limited site, P enrichment (+P) increased specific leaf area, N resorption, and P uptake, but had no effect on P resorption. At the N-limited site, +N increased both N and P resorption, but did not alter biomass allocation. Herbivory was greater at the P-limited site and was unaffected by +P, whereas +N led to increased herbivory at the N-limited site. The responses to nutrient enrichment depended on the ecological process and limiting nutrient and suggested that N- versus P-limited mangroves do have different thresholds. +P had a greater effect on more ecological processes at Twin Cays than did +N at the IRL, which indicated that the P-limited site was more sensitive to nutrient loading. Because of this sensitivity, eutrophication is more likely to cause a shift in nutrient limitation at P-limited Twin Cays than N-limited IRL.     

氮磷营养限制影响三角褐指藻光合无机碳利用和碳酸酐酶活性

以海洋硅藻三角褐指藻为实验材料, 研究了不同氮磷比培养对其光合无机碳利用和碳酸酐酶活性的影响, 结果显示三角褐指藻生长速率在N:P=16:1时最大, 高于或低于16:1时明显下降, 表明其最适生长受到氮磷的限制。氮限制(N:P=4:1或1:1)导致叶绿素a含量分别下降30.1% 和47.6%, 磷限制(N:P=64:1或256:1)下降39.1%和52.4%, 但氮或磷限制对叶绿素c含量并没有明显影响。不同营养水平培养对光饱和光合速率具有明显的影响, 与营养充足培养相比, 在严重氮磷限制(N:P=1:1或256:1)培养下光饱和光合速率分别下降39.7%和48.0%, 光合效率与暗呼吸速率也明显下降。在氮磷限制培养下藻细胞pH补偿点明显下降; K0.5CO2值在磷限制下降低30%, 表明磷限制有助于提高细胞对CO2的亲和力, 但氮限制并没有明显影响。在氮磷限制培养的细胞反应液中Fe (CN)63-浓度下降速率较慢, 表明在氮磷限制环境中生长的细胞质膜氧化还原能力明显低于营养充足条件下生长的细胞。氮磷限制也导致胞内、外碳酸酐酶活性明显下降, 其中在氮限制下胞外碳酸酐酶活性分别下降50%和37.5%, 在磷限制下下降22.3%和42.1%。严重的氮(N:P=1:1)或磷(N:P=256:1)限制导致胞内碳酸酐酶活性下降36.5%和42.9%。研究结果表明, 三角褐指藻细胞在氮磷营养限制的环境中, 可以通过调节叶绿素含量、无机碳的利用方式和碳酸酐酶的活性以维持适度的生长。       

INTERACTIONS AMONG PHOSPHATE UPTAKE,PHOTOSYNTHESIS, AND CHLOROPHYLL FLUORESCENCE IN NUTRIENT‐LIMITED CULTURES OF THE CHLOROPHYTE MICROALGA DUNALIELLA TERTIOLECTA1

Phosphate‐limited and phosphate‐sufficient continuous cultures of the marine chlorophyte microalga Dunaliella tertiolecta Butcher were examined for their responses to the addition of phosphate. Phosphate‐limited cultures showed a marked quenching of chl fluorescence following a pulse of phosphate. This response was absent from cells growing under phosphate‐sufficient conditions. Both the extent of fluorescence quenching (where present) and the initial rate of change in quenching were dependent on the concentration of phosphate added to cell suspensions and on the degree of limitation (growth rate in continuous culture). The addition of phosphate also brought about a transient decrease in photosynthetic oxygen evolution and a stimulation in respiration, which were relaxed as the added phosphate was depleted from the external medium. The applicability of using nutrient‐induced fluorescence transients as a tool to identify the nutrient status of phytoplankton populations is discussed.     

Arginine-specific carbamoyl phosphate metabolism in mitochondria of Neurospora crassa. Channeling and control by arginine

Citrulline is synthesized in mitochondria of Neurospora crassa from ornithine and carbamoyl phosphate. In mycelia grown in minimal medium, carbamoyl phosphate limits citrulline (and arginine) synthesis. Addition of arginine to such cultures reduces the availability of intramitochondrial ornithine, and ornithine then limits citrulline synthesis. We have found that for some time after addition of excess arginine, carbamoyl phosphate synthesis continued. Very little of this carbamoyl phosphate escaped the mitochondrion to be used in the pyrimidine pathway in the nucleus. Instead, mitochondrial carbamoyl phosphate accumulated over 40-fold and turned over rapidly. This was true in ornithine- or ornithine carbamoyltransferase-deficient mutants and in normal mycelia during feedback inhibition of ornithine synthesis. The data suggest that the rate of carbamoyl phosphate synthesis is dependent to a large extent upon the specific activity of the slowly and incompletely repressible synthetic enzyme, carbamoyl-phosphate synthetase A. In keeping with this conclusion, we found that when carbamoyl-phosphate synthetase A was repressed 2-10-fold by growth of mycelia in arginine, carbamoyl phosphate was still synthesized in excess of that used for residual citrulline synthesis. Again, only a small fraction of the excess carbamoyl phosphate could be accounted for by diversion to the pyrimidine pathway. The continued synthesis and turnover of carbamoyl phosphate in mitochondria of arginine-grown cells may allow rapid resumption of citrulline formation after external arginine disappears and no longer exerts negative control on ornithine biosynthesis.     

[15N]aspartate metabolism in cultured astrocytes. Studies with gas chromatography-mass spectrometry.

The metabolism of 2.5 mM-[15N]aspartate in cultured astrocytes was studied with gas chromatography-mass spectrometry. Three primary metabolic pathways of aspartate nitrogen disposition were identified: transamination with 2-oxoglutarate to form [15N]glutamate, the nitrogen of which subsequently was transferred to glutamine, alanine, serine and ornithine; condensation with IMP in the first step of the purine nucleotide cycle, the aspartate nitrogen appearing as [6-amino-15N]adenine nucleotides; condensation with citrulline to form argininosuccinate, which is cleaved to yield [15N]arginine. Of these three pathways, the formation of arginine was quantitatively the most important, and net nitrogen flux to arginine was greater than flux to other amino acids, including glutamine. Notwithstanding the large amount of [15N]arginine produced, essentially no [15N]urea was measured. Addition of NaH13CO3 to the astrocyte culture medium was associated with the formation of [13C]citrulline, thus confirming that these cells are capable of citrulline synthesis de novo. When astrocytes were incubated with a lower (0.05 mM) concentration of [15N]aspartate, most 15N was recovered in alanine, glutamine and arginine. Formation of [6-amino-15N]adenine nucleotides was diminished markedly compared with results obtained in the presence of 2.5 mM-[15N]aspartate.     

Accumulation of putrescine and related amino acids in potassium deficient Sesamum

Sesamum indicum was grown in complete or potassium deficient nutrient solution and amino acids, amines, nitrogen and potassium were determined weekly in the leaves. The incorporation of l-arginine-[U-¹⁴C] into protein was also followed. The interconversions of the amino acids of the ordithine-urea cycle, and their contribution to the formation of amines, were studied in cell-free extracts and intact leaves using labelled amino acids. As the level of potassium in the leaves decreased, the levels of the amino acids ornithine, citrulline and arginine, and of the amines putrescine, N-carbamylputrescine and agmatine increased. Potassium deficiency also reduced the rate of protein synthesis. Putrescine appears to be formed preferentially from citrulline with N-carbamylputrescine as intermediate.     

Effect of inhibitors on ammonium assimilation in Chlorella sorokiniana in light and darkness

In N-sufficient cells of Chlorella sorokiniana Shihira and Krauss strain 211/8K (CCAP of Cambridge University), assimilation of ammonium was strictly dependent on light and CO₂, and was severely inhibited by 100 μ M atrazine or 10 μ M 3-(3,4-dichlorophenyl)-1, l-dimethylurea (DCMU). In N-limited cells, assimilation of NH₄⁺ took place at similar rates in both light and darkness, which were 1.6-fold higher than the rate of light-dependent assimilation by N-sufficient cells. Assimilation by N-limited cells was inhibited by l -methionine- dl -sulfoximine (MSX), but not by atrazine or DCMU.
The rate of photosynthetic O₂ evolution was 2.9±0.9 mmol ml⁻¹ packed cell volume (PCV) h⁻¹ in N-sufficient cells, and 0.64±0.12 mmol ml⁻¹ PCV h⁻¹ in N-limited cells. In the latter resupply of ammonium resulted in a rapid activation by 22%;, followed by a time-dependent increase of the photosynthetic O₂ evolution, which after 12 h reached the same rate as in N-sufficient cells.
Respiratory consumption of oxygen in darkness in N-sufficient and N-limited cells was 0.10±0.03 and 0.11±0.02 mmol ml⁻¹ PCV h⁻¹, respectively. Addition of ammonium was without effect on respiration of N-sufficient cells, but resulted in a 4-fold stimulation of respiration of N-limited cells. Such stimulation took place also in cells treated with DCMU, atrazine, or MSX, and it was also promoted by methylammonium. The stimulation of respiration lasted for several hours.     

Nitrogen or phosphorus limitation in rich fens? - Edaphic differences explain contrasting results in vegetation development after fertilization

Background and aims

Many rich fens are threatened by high nutrient inputs, but the literature is inconsistent with respect to the type of nutrient limitation and the influence of edaphic characteristics.

Methods

We performed experiments with N- and P-fertilization in three endangered rich fen types: floating fen with Scorpidium scorpioides, non-floating fen with Scorpidium cossonii, floodplain fen with Hamatocaulis vernicosus. In addition, K-fertilization was carried out in the floodplain fen.

Results

The floodplain fen showed no response to P-addition, but N- and K-addition led to grass encroachment and decline of moss cover and species richness. In contrast, in the P-limited floating fen with S. scorpioides, P-addition led to increased vascular plant production at the expense of moss cover. Scorpidium scorpioides, however, also declined after N-addition, presumably due to ammonium toxicity. The fen with S. cossonii took an intermediate position, with NP co-limitation. These striking contrasts corresponded with edaphic differences. The N-limited fen showed low Ca:Fe ratios and labile N-concentrations, and high concentrations of plant-available P and Fe-bound P. The P-limited fen showed an opposite pattern with high Ca:Fe ratios and labile N-concentrations, and low P-concentrations.

Conclusions

This implies that edaphic characteristics dictate the nature of nutrient limitation, and explain contrasting effects of N- and P-eutrophication in different fens.     

Nutrient limitations in an extant and drained poor fen: implications for restoration

In a species-rich poor fen (Caricetum nigrae) and a species-poor drained fen, the difference in nutrient limitation of the vegetation was assessed in a full-factorial fertilization experiment with N, P and K. The results were compared to the nutrient ratios of plant material and to chemical analysis of the topsoil. A rewetting experiment with intact sods was carried out in the glasshouse and the results are discussed in view of restoration prospects of drained and degraded peatlands. In the undrained poor fen the above-ground biomass yield was N-limited while the vegetation of the drained fen was K-limited. Experimental rewetting of intact turf samples, taken in the drained site, did not change the biomass yield or the type of nutrient limitation. It was concluded that mire systems which have been subjected to prolonged drainage are inclined to pronounced K-deficiency, probably due to washing out of potassium and harvesting the standing crop. This may hamper restoration projects in degraded peat areas where nature conservation tries to restore species-rich vegetation types with a high nature value.     

Ammonium accumulation in relation to growth of rice cell suspension under phosphate deprivation

Suspension-cultured rice cells growth was markedly inhibited and ammonium content increased when rice cells were deprived of phosphate. When rice cells were cultured at increasing concentrations of ammonium chloride, ammonium content increased, however, no significant inhibition of cell growth was observed. Addition of D-arginine, an inhibitor of putrescine biosynthesis, resulted in a complete recovery of growth in rice cells under phosphate deprivation, but did not decrease the content of ammonium. Our results indicate that the growth inhibition induced by phosphate deprivation is not associated with ammonium accumulation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of urea and amino acids of the ornithine cycle on the biomass accumulation and amino acids synthesis by Candida guilliermondii]

When assimilating urea, arginine, ornithine and citrulline as the sole source of nitrogen, C. guilliermondii shows a higher economic coefficient of biomass accumulation (54.2, 59.7, 40.6% respectively) as compared with ammonium sulphate whose coefficient is 35.6%. Nitrogen sources exert a significant influence on the content of essential amino acids in the alcohol soluble fraction of cell biomass. For instance, urea and arginine are responsible for the accumulation of ornithine (220 and 480 mug/100 mg abs. dry weight), arginine (470 and 587 mug), aspartic acid (220 mug), glutamic acid (520 and 444 mug), alanine (460 and 500 mug), whereas ammonium sulphate provides an accumulation of serine--52 mug, glycine--57 mug, gamma-aminobutyric acid--480 mug, phenyl alanine--96 mug and leucine--96 mug.     

Nitrogen supply effects on productivity and potential leaf litter decay of Carex species from peatlands differing in nutrient limitation

We investigated the effect of increased N-supply on productivity and potential litter decay rates of Carex species, which are the dominant vascular plant species in peatlands in the Netherlands. We hypothesized that: (1) under conditions of N-limited plant growth, increased N-supply will lead to increased productivity but will not affect C:N ratios of plant litter and potential decay rates of that litter; and (2) under conditions of P-limited plant growth, increased N-supply will not affect productivity but it will lead to lower C:N ratios in plant litter and thereby to a higher potential decay rate of that litter. These hypotheses were tested by fertilization experiments (addition of 10 g N m^-2 year^-1) in peatlands in which plant growth was N-limited and P-limited, respectively. We investigated the effects of fertilization on net C-fixation by plant biomass, N uptake, leaf litter chemistry and potential leaf litter decay. In a P-limited peatland, dominated by Carex lasiocarpa, there was no significant increase of net C-fixation by plant biomass upon enhanced N-supply, although N-uptake had increased significantly compared with the unfertilized control. Due to the N-fertilization the C:N ratio in the plant biomass decreased significantly. Similarly, the C:N ratio of leaf litter produced at the end of the experiment showed a significant decrease upon enhanced N-supply. The potential decay rate of that litter, measured as CO₂-evolution from the litter under aerobic conditions, was significantly increase upon enhanced N-supply. In a N-limited peatland, dominated by C. acutiformis, the net C-fixation by plant biomass increased with increasing N-supply, whereas the increase in N-uptake was not significant. The C:N ratio of both living plant material and of dead leaves did not change in response to N-fertilization. The potential decay rate of the leaf litter was not affected by N-supply. The results agree with our hypotheses. This implies that atmospheric N-deposition may affect the CO₂-sink function of peatlands, but the effect is dependent on the nature of nutrient limitation. In peatlands where plant growth is N-limited, increased N-supply leads to an increase in the net accumulation of C. Under conditions of P-limited plant growth, however, the net C-accumulation will decrease, because productivity is not further increased, whereas the amount of C lost through decomposition of dead organic matter is increased. As plant growth in most terrestrial ecosystems is N-limited, increased N-supply will in most peatlands lead to an increase of net C-accumulation.     

Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium

Dundas, Ian E. D. (University of Illinois, Urbana), and H. Orin Halvorson. Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium. J. Bacteriol. 91:113-119. 1966.-Arginine was shown to be essential for growth of Halobacterium salinarium strain 1 in a chemically defined medium. Citrulline was the only compound which could substitute for arginine without affecting growth. Resting cells of H. salinarium converted arginine to citrulline and citrulline to ornithine. Cells grown in an arginine-free medium with C(14)-ureido-labeled citrulline incorporated the isotope mainly into the arginine of their proteins. The enzymes arginine desimidase and ornithine transcarbamylase were found and studied in cell-free extracts of H. salinarium. Experiments indicated that arginine was degraded in H. salinarium by arginine desimidase to citrulline, and that citrulline was further degraded by ornithine transcarbamylase to carbamyl phosphate and ornithine. Synthesis of arginine from citrulline seems to occur via the formation of argininosuccinic acid.     

Reduced digestibility of UV-B stressed and nutrient-limited algae by Daphnia magna

Daphnia magna was fed the green alga Selenastrum capricornutum, cultured under four different growth conditions: (1) phosphorus limitation, (2) nitrogen limitation, (3) UV-B irradiation, and (4) no nutrient limitation, no UV-B irradiation. Contrary to non-limited algal cells, nutrient-limited cells were not efficiently assimilated. Especially, P-limited cells passed through the gut mostly intact, while N-limited cells were partly assimilated. Also, algae exposed to moderate doses of UV-B radiation (0.3 mW cm^–2 of UV₃₁₂) were less efficiently assimilated after being grazed. Digestibility of the algae decreased with increased UV-B exposure time. Nutrient-limited and UV-B stressed algal cells increased in volume and became granular in appearance. These changes in the algal cells, combined with changed cell wall properties, most probably reduced their digestibility.     

Arginine Catabolism by Leishmania donovani Promastigotes

ABSTRACT Leishmania donovani promastigotes were grown to late log phase, washed and resuspended in iso-osmotic buffer containing L-arginine, and the rate of urea formation was then measured under various conditions. Addition of glucose or mannose activated urea formation, whereas 2-deoxyglucose inhibited and 6-deoxyglucose had no effect. Addition of alanine or of α -aminoisobutyrate inhibited urea formation, alanine causing a greater inhibition than α -aminoisobutyrate. Addition of leucine, proline, glycine, or lysine had no effect on urea formation. The presence of glutamate also increased the rate of urea formation from arginine, but to a lesser extent than did glucose. The presence of both glucose and alanine caused no net change in urea formation, whereas the inhibitory effect of alanine exceeded the activating effect of glutamate, so that a small inhibition in the rate of urea formation occurred in the presence of both alanine and glutamate. Cells grown to 3-day stationary phase had a markedly reduced rate of arginine catabolism to urea, but the activating effect of glucose and the inhibitory effect of alanine were qualitatively similar to their effects on late log phase cells. Addition of water to cells suspended in buffer also inhibited urea formation, but this appeared to be due primarily to the release of alanine caused by the hypo-osmotic stress. Addition of mannitol to cells suspended in buffer caused a small inhibition of arginine catabolism. Addition of dibutyrylcyclic AMP, 3',5'-cyclic GMP, phorbol myristic acid, or A23187 had no effect on the rate of urea formation from arginine. It is suggested that the effects of glucose and 2-deoxyglucose on arginine catabolism depend largely upon the nature of their metabolites, whereas the effects of the various amino acids examined depend largely on the extent to which they interfere with or enhance arginine transport into the cells.     

Effect of natural amino acids on ethanol oxidation in isolated rat hepatocytes]

The effects of the various naturally occurring amino acids on ethanol oxidation in hepatocytes from starved rats was systematically studied. In order to minimize the non ADH pathways, the ethanol concentration used was 4 mmol/litre, the amino acids being added at the same concentration. In hepatocytes from fasted rats, alanine, arginine, asparagine, aspartate, citrulline, cysteine, glutamate, glutamine, glycine, histidine, hydroxyproline, ornithine and serine increase significantly ethanol consumption. The stimulatory effect of glutamine being much less pronounced than the asparagine one and proline being devoid of action, the influence of ammonium chloride addition on ethanol consumption in the presence of these amino acids was studied. Ammonium chloride determines an enhancement of ethanol oxidation in these conditions, the results showing no apparent correlation between intracellular glutamate concentration and ethanol oxidation rate, contrarily to previous data. In hepatocytes from fed rats, only alanine, asparagine, cysteine, glycine, hydroxyproline, ornithine and serine increase ethanol oxidation, although to a lesser extent than in cells from starved rats.     

INTERACTIONS BETWEEN UV‐B EXPOSURE AND PHOSPHORUS NUTRITION. I. EFFECTS ON GROWTH,PHOSPHATE UPTAKE,AND CHLOROPHYLL FLUORESCENCE1

The interactive effects of P starvation and exposure to UV radiation on growth rates, quantum efficiency of PSII electron transport, and P‐uptake capacity of the chlorophyte microalga Dunaliella tertiolecta Butcher are presented. Ultraviolet radiation did not in itself cause marked changes in growth rate, though it did induce changes in the effective quantum yield of PSII. Depriving cells of phosphate resulted in significant changes in all parameters examined. The decline of growth rate and fluorescence parameters after P starvation was significantly faster in the presence of UV radiation. Ultraviolet radiation also stimulated the magnitude of the transient changes in chl fluorescence (nutrient‐induced fluorescence transient) exhibited by P‐starved cells after resupply of that nutrient.     

Caveolar localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with endothelial nitric oxide synthase.

Although normal intracellular levels of arginine are well above the K(m), and should be sufficient to saturate nitric oxide synthase in vascular endothelial cells, nitric oxide production can, nonetheless, be stimulated by exogenous arginine. This phenomenon, termed the "arginine paradox," has suggested the existence of a separate pool of arginine directed to nitric oxide synthesis. In this study, we demonstrate that exogenous citrulline was as effective as exogenous arginine in stimulating nitric oxide production and that citrulline in the presence of excess intracellular and extracellular arginine further enhanced bradykinin stimulated endothelial nitric oxide production. The enhancement of nitric oxide production by exogenous citrulline could therefore be attributed to the capacity of vascular endothelial cells to efficiently regenerate arginine from citrulline. However, the regeneration of arginine did not affect the bulk intracellular arginine levels. This finding not only supports the proposal for a unique pool of arginine, but also suggested channeling of substrates that would require a functional association between nitric oxide production and arginine regeneration. To support this proposal, we showed that nitric oxide synthase, and the enzymes involved in arginine regeneration, argininosuccinate synthase and argininosuccinate lyase, cofractionated with plasmalemmal caveolae, a subcompartment of the plasma membrane. Overall, the results from this study strongly support the proposal for a separate pool of arginine for nitric oxide production that is defined by the cellular colocalization of enzymes involved in nitric oxide production and the regeneration of arginine.