Citrate Secretion Research Articles

Combined Effect of Short-Term Water Deficit Stress and Aluminum Toxicity on Citrate Secretion from Soybean Roots

In order to investigate the combined effect of drought stress and aluminum (Al) toxicity on citrate exudation in soybean, hydroponic cultivation with addition of Al and PEG-6000 was conducted to simulate Al-toxic dry soil. Results showed that 2-day exposure of soybeans to 5.5% (w/v) PEG-6000 or 100 µM AlCl3 mainly hindered root growth, while combined exposure to PEG and Al (PEG/Al) reduced both root and shoot growth. Photosynthetic rate of first trifoliolate for the Al-tolerant genotype PI 416937 (PI) was not affected by imposition of 5.5% PEG/100 µM AlCl3 (38–40 h), whereas photosynthetic rate for the Al-sensitive genotype YC was significantly reduced. Based on root fresh weight, Al-induced citrate exudation in the roots of soybean genotypes recovered from pre-treatment with 5.5 or 9% PEG was not altered, but was increased in the plants pretreated with 7% PEG without recovery. After 2 days of recovery from 2-day combined exposure to 5.5% PEG/100 µM AlCl3, the Al-tolerant PI exuded more citrate than its control, but the Al-sensitive YC exuded significantly less citrate than its corresponding control. Split root experiment revealed that Al-induced citrate exudation in one half of the root system was significantly reduced by exposing the other half of the root system to 0.5 mM CaCl2 solution containing 9% PEG or 9% PEG/50 µM AlCl3. However, organic acid secretion was not observed in the half of the root system in the Ca solution when the other part of the root system was exposed to the Ca solution containing 50 µM AlCl3, 9% PEG or 9% PEG/50 µM AlCl3. This suggests that no Al- or drought-induced signals such as ABA are involved in the citrate secretion in soybean.

Zinc and zinc transporters in normal prostate function and the pathogenesis of prostate cancer

Zinc is an essential metal for all cells. It plays a role in a wide variety of physiological and biochemical processes. In the prostate epithelial cell the accumulation of high cellular zinc is a specialized function that is necessary for these cells to carry out the major physiological functions of production and secretion of citrate. The production of citrate and its secretion into prostatic fluid is a differentiated function of the prostate epithelial cells that is apparently important for reproduction. The loss of citrate and zinc accumulation is the most consistent and persistent characteristic of prostate malignancy. This characteristic of prostate cancer indicates that the lost ability of the malignant cells to accumulate zinc and citrate is an important factor in the development and progression of malignancy. The lost ability of the epithelial cells to accumulate zinc and thus to also accumulate citrate is the result of decreased expression of specific zinc uptake transporters. The purpose of this presentation is to review the current understanding of zinc and zinc homeostasis in the prostate and the role of zinc and zinc transporters in the normal function of the prostate and the pathogenesis of prostate cancer.

Modulation of iron on mitochondrial aconitase expression in human prostatic carcinoma cells

The mitochondrial aconitase (mACON) containing a [4Fe-4S] cluster is regarded as the key enzyme for citrate oxidation in the epithelial cells of human prostate. In vitro studies using the human prostatic carcinoma cells, PC-3 cells, found that both hemin and ferric ammonium citrate (FAC) significantly increased mACON enzymatic activity and gene expression. The effect of FAC on mACON was enhanced 2-fold by co-treating with ascorbic acid but blocked by co-treating with iron chelator, deferoxamine mesylate. Hemin treatments blocked 30% of citrate secretion from PC-3 cells but upregualted 2-fold of intracellular ATP biosynthesis. Results from reporter assay by using a cytomegalovirus enhance/promoter driven luciferase mRNA ligated to the iron response element (IRE) of mACON as a reporter construct demonstrated that modulation of FAC on gene translation of mACON gene is dependent on the IRE. Transient gene expression assays indicated that upregulation of mACON gene transcription by FAC may through the putative antioxidant response element (ARE) signal pathway. This study provides the first evidence of the biologic mechanism of human mACON gene translation/transcription and suggests a regulatory link between the energy utilization and the iron metabolism in human prostatic carcinoma cells.

Citrate Secretion Induced by Aluminum Stress May Not be a Key Mechanism Responsible for Differential Aluminum Tolerance of Some Soybean Genotypes

Eighteen soybean genotypes differing in aluminum (Al) tolerance were used to investigate genotypic differences in Al-induced citrate exudation and its role in Al tolerance. Aluminum accumulation and localization in soybean roots were examined by analysis of total Al and hematoxylin staining. Soybean genotypes exhibited a wide range of Al tolerance. Based on relative root elongation, several Al-tolerant genotypes from Brazil such as B1, B10, and B15 were more tolerant than the Al-tolerant PI 416937 (PI) and Perry. All soybeans exuded citrate in response to Al stress, and some Al-sensitive genotypes secreted more citrate than tolerant ones, showing no correlation between the Al tolerance and Al-induced citrate exudation. Further study found that both copper (Cu) and cadmium (Cd) stimulated citrate and malate exudation in soybean, indicating that organic acid secretion is not specifically induced by Al. Aluminum concentrations were significantly higher in 2–3 and 3–4 cm of segments than that in 0–1 and 1–2 cm segments under 15 μM AlCl3. Both the root mature zone and apex were heavily stained by hematoxylin after exposure to 10, 15, or 20 μM AlCl3 (24 h), whereas root elongation zone was not stained. After exposure to 50 μM AlCl3 for 20 min, the Al-tolerant PI was less stained by hematoxylin than the Al-sensitive Young, suggesting that Al accumulation in root apices seem to be an immediate response to Al stress, and related to differential Al sensitivity. Present results suggest that citrate secretion induced by Al stress may not be a key mechanism responsible for the differential Al tolerance of some soybean genotypes and other mechanism(s) conferring Al exclusion should exist and operate immediately after exposure to Al stress.

Differential Al resistance and citrate secretion in the tap and basal roots of common bean seedlings

The common bean root system is composed of several types of root (e.g. tap, basal, and lateral roots), whose physiological functions may be of great difference. However, we do not know if the root system of common bean differs in organic acid secretion and thus aluminium (Al) resistance. In the present study, the tap and basal roots of three common bean genotypes (i.e. G19842, SQ12 and BAT881) from different origins were compared for their citrate secretion and Al resistance. Grown in a simple solution containing 30 µM Al3+ for 24 h, genotype G19842 maintained 75% relative tap root length [RTRL = (tap root length with Al)/(tap root length without Al)], 48% relative basal root length [RBRL = (basal root length with Al)/ (basal root length without Al)], genotype SQ12 maintained 62% RTRL and 57% RBRL, while BAT881 only maintained 31% RTRL and 19% RBRL, indicating differential sensitivity of bean genotypes and root types to Al stress. The amounts of Al‐induced citrate secretion by the tap/basal roots were 9.8/5.1, 8.2/5.9 and 5.4/4.1 nmol cm−2 FR (fresh root) [12 h]−1 for G19842, SQ12 and BAT881, respectively, indicating that both bean genotypes and root types differ in organic acid secretion. In G19842, the root surface area was 25% higher in tap root apex than that in basal root apex, and the amounts of citrate secretion per unit surface area and per root apex were 29 and 62% higher in tap root apex than those in basal root apex, respectively, suggesting that the higher citrate secretion in the tap root apex could be attributed to the larger surface area and the higher secretion activity. Stronger inhibition of Al‐induced citrate secretion in the basal than tap roots by anthracene‐9‐carboxylic acid, an inhibitor of anion channel and K‐252a, a broad range inhibitor of protein kinase may also imply the differences in the activities of anion channels and K‐252a‐sensitive protein kinases on the plasma membrane between the tap and basal roots, resulting in differential citrate secretion. We propose that the higher Al resistance in the tap root than in basal roots might be attributed to both greater number and higher activity of the anion channels in the former, thus allowing more citrate secretion in this root type.

Molecular mapping of a gene responsible for Al-activated secretion of citrate in barley.

Aluminium (Al) toxicity is an important limitation to barley (Hordeum vulgare L.) on acid soil. Al-resistant cultivars of barley detoxify Al externally by secreting citrate from the roots. To link the genetics and physiology of Al resistance in barley, genes controlling Al resistance and Al-activated secretion of citrate were mapped. An analysis of Al-induced root growth inhibition from 100 F2 seedlings derived from an Al-resistant cultivar (Murasakimochi) and an Al-sensitive cultivar (Morex) showed that a gene associated with Al resistance is localized on chromosome 4H, tightly linked to microsatellite marker Bmag353. Quantitative trait locus (QTL) analysis from 59 F4 seedlings derived from an F3 plant heterozygous at the region of Al resistance on chromosome 4H showed that a gene responsible for the Al-activated secretion of citrate was also tightly linked to microsatellite marker Bmag353. This QTL explained more than 50% of the phenotypic variation in citrate secretion in this population. These results indicate that the gene controlling Al resistance on barley chromosome 4H is identical to that for Al-activated secretion of citrate and that the secretion of citrate is one of the mechanisms of Al resistance in barley. The identification of the microsatellite marker associated with both Al resistance and citrate secretion provides a valuable tool for marker-assisted selection of Al-resistant lines.

Nitroprusside stimulates mitochondrial aconitase gene expression through the cyclic adenosine 3′,5′‐monosphosphate signal transduction pathway in human prostate carcinoma cells

Mitochondrial aconitase (mACON), an iron-requiring enzyme, is a major target of nitric oxide (NO) in cells, which causes the oxidant-mediated disruption of the [4Fe-4S] prosthetic group of the enzyme. In this study, the effect of NO on mACON enzymatic activity and gene expression were investigated. Three NO generators, sodium nitroprusside (SNP), S-nitoso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN) were used to determine the regulation of mACON enzymatic activity by NO. The effect of SNP on mACON, which modulates citrate secretion and cellular bioenergetics in PC-3 cells, was investigated by determining the effect of SNP on mACON gene expression using Western blot and transient gene expression assays. SNP upregulated mACON enzymatic activity and gene expression in PC-3 cells. However, treating cells with other NO generators, SNAP and SIN, resulted in decreased mACON enzymatic activity. The addition of ascorbic acid to the SNP treatment resulted in a decrease in mACON enzymatic activity and gene expression. Our results showed that both SNP and dibutyryl-cAMP increased the mACON promoter activity 2-fold while the effect was blocked by adding H-89. Mutation of the cAMP response element (CRE) to the AGAGCT abolished the activating effects of SNP and dibutyryl-cAMP on mACON promoter activity. These results establish the function of nitroprusside as a signaling molecule for mACON gene expression through the cAMP signal transduction pathway in human prostatic carcinoma cells.

Studies on the mechanism of aluminum tolerance in pea (Pisum sativum L.) using aluminum-tolerant cultivar ‘Alaska’ and aluminum-sensitive cultivar ‘Hyogo’

The mechanism of aluminum (Al) tolerance was investigated in an Al-tolerant cultivar (Alaska) and an Alsensitive cultivar (yo o) of pea (Pisum sativum L.) seedlings. Al sen sitivity was evaluated as the degree of root elongation inhibition in a calcium solution. Fifty percent inhibition of the elongation was observed after 24h treatment with 5 µM Al in Alaska and with less than 2.5 µM Al in Hyogo. At 10 µM Al, both cultivars exhibited more than 70% inhibition, At 2.5 µM and 5 µM, Al triggered citrate secretion in both cultivars to the same extent, and the levels of Al accumulation in root apices were similar in both celti vare, indicating that the difference in Al tolerance observed in these cultivars was not re lated to the exclusion of Al with organic acid. Al treatment enhanced the production of reactive oxygen species (ROS) specifically in the elongation zone in both cultivars. Al dose and time course experiments indicated the existence of a strong positive correlation be tweera the root elongation inhibition and ROS production in both cultivars. At 10 pµM, Al inhibited the respiration rate in the root apex by 20% in Alaska and 30% in Hyogo but not significantly at lower concentrations. In the absence of Al, the ATP content in the root apex was 2 times higher in Alaska than in Hyogo. With the increase in Al concentration, the AT content in Alaska decreased and reached the same level in Hyogo at 5 µM Al, while the AT content in Hyogo did not change. These findings suggest that Al-triggered ROS production is a key factor for the root elongation inhibition in both cultivars, and the possible involvement of high ATP content in Alaska in the protection mechanism against ROS production and root elongation inhibition under Al stress.

Modulation of mitochondrial aconitase on the bioenergy of human prostate carcinoma cells

A bioenergetic theory of prostate malignancy proposed that normal citrate-producing prostate epithelial cell become citrate-oxidizing cells, in which mitochondrial aconitase (mACON) is not limiting, providing the energy required for the onset and progression of malignancy and metastasis. However, no direct evidence has been approved to support the hypothesis. A full-length cDNA encoding human skeletal muscle mACON cDNA was cloned and sequenced. mACON cDNA contains 19-bp 5 ′ untranslated region, a 2343-bp coding segment, and 376-bp 3 ′ untranslated region. This precursor enzyme contains mitochondrial targeting sequence of 27 amino acid residues and mature enzyme of 753 amino acids residues. A human anti-mACON overexpression vector containing the 1171-bp mACON cDNA fragment in the reverse orientation was stable transfected into human prostate carcinoma cells, PC-3 and DU145 cells. Results showed that mACON antisense blocked 40–60% mACON expression and enzymatic activity which induced decrease in the intracellular ATP biosynthesis but increase citrate secretion in the human prostate carcinoma cells. mACON antisense-transfected cells have lower cell proliferation ratio than the mock of DNA-transfected cells. Our study demonstrated the key role of the mACON in the cellular bioenergy and cell proliferation of human prostate carcinoma cells.

Citrate Secretion Induced by Aluminum Stress May Not be a Key Mechanism Responsible for Differential Aluminum Tolerance of Some Soybean Genotypes

Abstract Eighteen soybean genotypes differing in aluminum (Al) tolerance were used to investigate genotypic differences in Al-induced citrate exudation and its role in Al tolerance. Aluminum accumulation and localization in soybean roots were examined by analysis of total Al and hematoxylin staining. Soybean genotypes exhibited a wide range of Al tolerance. Based on relative root elongation, several Al-tolerant genotypes from Brazil such as B1, B10, and B15 were more tolerant than the Al-tolerant PI 416937 (PI) and Perry. All soybeans exuded citrate in response to Al stress, and some Al-sensitive genotypes secreted more citrate than tolerant ones, showing no correlation between the Al tolerance and Al-induced citrate exudation. Further study found that both copper (Cu) and cadmium (Cd) stimulated citrate and malate exudation in soybean, indicating that organic acid secretion is not specifically induced by Al. Aluminum concentrations were significantly higher in 2–3 and 3–4 cm of segments than that in 0–1 and 1–2 cm segments under 15 μM AlCl3. Both the root mature zone and apex were heavily stained by hematoxylin after exposure to 10, 15, or 20 μM AlCl3 (24 h), whereas root elongation zone was not stained. After exposure to 50 μM AlCl3 for 20 min, the Al-tolerant PI was less stained by hematoxylin than the Al-sensitive Young, suggesting that Al accumulation in root apices seem to be an immediate response to Al stress, and related to differential Al sensitivity. Present results suggest that citrate secretion induced by Al stress may not be a key mechanism responsible for the differential Al tolerance of some soybean genotypes and other mechanism(s) conferring Al exclusion should exist and operate immediately after exposure to Al stress.

Expression and cellular localization of Na,K-ATPase isoforms in the rat ventral prostate.

To determine the expression and plasma membrane domain location of isoforms of Na,K-ATPase in the rat ventral prostate. Ventral prostate glands from adult male rats were dissected, cryosectioned (7 micro m) and attached to poly-l-lysine coated glass slides. The sections were then fixed in methanol and subjected to indirect immunofluorescence and immunoperoxidase procedures using a panel of well-characterized monoclonal and polyclonal antibodies raised against known Na,K-ATPase subunit isoforms. Immunofluorescence micrographs were digitally captured and analysed by image analysis software. There was expression of Na,K-ATPase alpha1, beta1, beta2 and beta3 subunit isoforms in the lateral and basolateral plasma membrane domains of prostatic epithelial cells. The alpha1 isoform was abundant but there was no evidence of alpha2, alpha3 or gamma isoform expression in epithelial cells. The alpha3 isoform was not detected, but there was a relatively low level of alpha2 isoform expression in the smooth muscle and stroma. Rat prostate Na,K-ATPase consists of alpha1/beta1, alpha1/beta2 and alpha1/beta3 isoenzymes. These isoform proteins were located in the lateral and basolateral plasma membrane domains of ventral prostatic epithelial cells. The distribution and subcellular localization of Na,K-ATPase is different in rodent and human prostate. Basolateral Na,K-ATPase probably contributes to the establishment of transepithelial ionic gradients that are a prerequisite for the uptake of metabolites by secondary active transport mechanisms and active citrate secretion.

An intracellular mechanism of aluminum tolerance associated with high antioxidant status in cultured tobacco cells

An aluminum (Al) tolerance mechanism, together with oxidative stress tolerance, was investigated in an Al tolerant cell line (ALT301) and the parental Al sensitive cell line (SL) of tobacco. During Al exposure in a simple calcium solution for 24 h, Al triggered the evolution of a reactive oxygen species (ROS) in SL much higher than ALT301 [Plant Physiol. 128 (2002) 63]. Under the conditions, Al enhanced comparable rates of citrate secretion from both cell lines to the same extent. Al enhanced the gene expression of manganese superoxide dismutase (MnSOD) in both cell lines, but at a significantly higher rate in SL than in ALT301, and also enhanced the enzyme activity of MnSOD in both cell lines to nearly the same level. These results suggest that the extracellular chelation of Al with organic acids and MnSOD is not involved in the mechanism of Al tolerance of ALT301. ALT301 contained ascorbate (ASA) and glutathione (GSH) levels that were higher than SL under normal growth conditions. During 24 h of post-Al treatment culture in growth medium, but not during 24-h Al exposure in a simple Ca 2+ solution, lipid peroxidation was enhanced in SL much higher than in ALT301, and the average SL amounts of ASA and GSH were exhausted compared to ALT301. Pre-loading of ASA prior to Al treatment improved the growth of SL during the post-Al treatment culture. ALT301 also exhibited cross-tolerance to H 2O 2, Fe 2+ and Cu 2+. Under these oxidant exposures, ALT301 contained lower levels of intracellular H 2O 2 or lipid peroxides, and maintained higher amounts of ASA and GSH than SL. Taken together, we conclude that the accumulation of Al in cells enhances the peroxidation of lipids exclusively under growing conditions, and that the higher content of ASA and GSH in ALT301 than in SL seems to be in part responsible for the tolerance mechanism of ALT301 to Al by protecting cells from either lipid peroxidation or H 2O 2 commonly enhanced by Al or other oxidants.

Al-induced efflux of organic acid anions is poorly associated with internal organic acid metabolism in triticale roots.

The secretion of organic acid anions from roots has been identified as a mechanism of resistance to Al. However, the process leading to the secretion of organic acid anions is poorly understood. The effect of Al on organic acid metabolism was investigated in two lines of triticale (xTriticosecale Wittmark) differing in Al-induced secretion of malate and citrate and in Al resistance. The site of Al-induced secretion of citrate and malate from a resistant line was localized to the root apices (terminal 5 mm). The levels of citrate (root apices and mature root segments) and malate (mature segments only) in roots increased during exposure to Al, but similar changes were observed in both triticale genotypes. The in vitro activities of four enzymes involved in malate and citrate metabolism (citrate synthase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and NADP-isocitrate dehydrogenase) were similar for sensitive and resistant lines in both root apices and mature root segments. The response of these enzymes to pH did not differ between tolerant and sensitive lines or in the presence and absence of Al. Moreover, cytoplasmic and vacuolar pH were not affected by exposure to Al in either line. Together, these results indicate that the Al-dependent efflux of organic acid anions from the roots of triticale is not regulated by their internal levels in the roots or by the capacity of the root cells to synthesize malate and citrate.

Effect of phosphorus deficiency on aluminium‐induced citrate exudation in soybean (Glycine max)

Aluminium (Al) toxicity or phosphorus (P) deficiency can induce exudation of organic acids from the roots of some plants, which is believed to be a tolerance mechanism against Al toxicity or P deficiency. In the present study, the effect of P deficiency on Al‐induced citrate exudation was investigated in three soybean varieties differing in low‐P tolerance. P starvation alone failed to induce secretion of organic acids from all three soybean varieties. However, P deficiency altered Al‐induced citrate exudation over time, showing a complex interaction. Short × term P starvation (4 days) produced up to 50% increase in Al‐induced citrate secretion, while longer‐term (10 days) starvation reduced Al‐induced citrate secretion to trace amounts. However, after a further 1 day in complete nutrient solution for recovery, Al‐induced citrate exudation from the recovered roots was approximately 6 times higher than that from the continuously P‐starved plants, but still approximately 3.6 times lower than that from the P‐sufficient control. With increasing P or Al supply, Al‐induced citrate exudation increased, while Al accumulation in soybean roots decreased in parallel with the decrease of P supply. The photosynthetic rate, stomatal conductance and transpiration were decreased by P deficiency, whereas the intracellular CO2 concentration was increased. These findings indicate that P nutrition has a significant effect on Al‐induced citrate exudation and Al accumulation in soybean root apices.

EXUDATION OF CITRATE IN COMMON BEAN IN RESPONSE TO ALUMINUM STRESS

ABSTRACT Difference in aluminum (Al) tolerance in plant genotypes might be used to identify or develop plants for remediation of high Al soils. In this study, two common bean cultivars (Phaseolus vulgaris L. “G19842” and “ZPV”), which differ in Al toxicity were grown hydroponically to study the exudation of Al-chelating substances in response to Al stress. When exposed to Al, Al uptake per unit of dry weight in G19842 was much lower than that in ZPV, and secreted citrate in G19842 was double of that in ZPV. A dose-response experiment indicated that amount of secreted citrate in both G19842 and ZPV increased with increasing external Al concentrations which ranged from 0 to 50 Âμmol L−1, while ranged from 50 to 80 Âμmol L−1, secreted citrate decreased with increasing external Al concentrations. The maximum of secreted citrate was at 12 h after initiation of Al treatment. The secreted citrate induced by P deficiency was less than that by Al stress of 50 Âμmol L−1. Addition of exogenous citrate to Al-containing solution ameliorated the toxic effect of Al more significantly in ZPV than in G19842. Exposure to 50 Âμmol L−1 of lanthanum (La3+) solution did not induce the exudation of citrate. Anthracene-9-carboxylic acid (A-9-C), an anion channel inhibitor inhibited the secretion of citrate by 25–36%. All these findings suggest that the exudation of citrate in common bean is an important physiological response, which constitutes a mechanism of resistance to Al stress.

Aluminum-induced secretion of both citrate and malate in rye

Aluminum (Al)-resistant mechanisms responsible for Al-induced secretion of organic acids are poorly understood. In this study, we characterized the Al-induced secretion of both citrate and malate from rye (Secale cereale L. cv. King). Secretion of organic acids increased with increasing concentration (10, 30 and 50 μM) and duration of Al treatments. Neither phosphorous (P) deficiency up to 15 days nor addition of 50μM lanthanum, 50 μM lead, 10 μM cadmium, or 200 μM manganese caused secretion of organic acids, suggesting that this secretion was a specific response to Al stress. Aluminum activated citrate synthase, the main enzyme for the synthesis of citrate, but its activation occurred only in the root tip. The elongation of roots of an Al-sensitive cultivar of wheat (Tritium aestivum L. cv. Scout 66) was not inhibited by 50 μM Al in the presence of externally applied 50 μM citrate or 400 μM malate. The secretion of citrate and malate from intact rye roots exposed to 50 μM Al corresponded to 31.3 ± 1.7 μM and 11.5 ± 2.5 μM, respectively, in the rhizosphere based on an assumption of a 2 mm thick unstirred layer around root tips. This result indicated that Al-resistance in rye was achieved by the Al-induced synthesis of citrate in root apices followed by Al-induced specific secretion of citrate from root tips.

Characterization of aluminium‐induced citrate secretion in aluminium‐tolerant soybean (Glycine max) plants

Recently, we showed that secretion of citrate in an aluminium (Al) tolerant cultivar soybean (Glycine max) (cv. Suzunari) is a specific response to Al stress [Yang et al. (2000) Physiol Plant 110: 72–77]. Here we investigated the intrinsic mechanisms behind the secretion of citrate induced by Al. The amount of citrate secreted during the 24‐h Al treatment period increased with increasing concentration of Al (0–70 μM). We analysed citrate secretion basically under 3 conditions: (1) by varying light‐exposure, (2) with intact or excised shoots and (3) by using a divided chamber technique. Further, the content of organic acids in the tissue and the activity of enzymes involved in organic acid metabolism were analysed and evaluated. The results indicate that high rate of citrate secretion in soybean requires a 4‐h induction period. Al had a continuous effect on the citrate secretion when Al was removed from the treatment solution. Citrate secretion increased steadily under exposure to continuous light. However, when the shoots were excised the citrate secretion rate dropped to 3–6 times that of their control counterparts. Results of root manipulation experiments revealed that citrate secretion required the direct contact of Al. In other words, only the Al‐treated root portions secreted citrate. All these observations suggest that the shoots play a role in Al‐induced citrate secretion. Although shoots may not supply citrate for the secretion upon Al treatment, it seems that they may provide the carbon source and/or energy for citrate synthesis in the root. On the other hand, the root organic acid content (1‐cm apex) indicated that malate might contribute to citrate secretion by keeping the balance between citrate synthesis and release in the root apices. Quantification of enzymes involved in organic acid metabolism showed only a 16% increase in citrate synthase activity upon Al treatments (6 h) with no differences in other enzymes. Hence, we could not rule out completely the potential contribution of citrate from shoots and the results are discussed in the light of shoots contributing either energy or citrate itself for enhanced citrate secretion in the Al‐tolerant plant roots.

Strong inhibition of crystal–cell attachment by pediatric urinary macromolecules: a close relationship with high urinary citrate secretion

Objectives. To investigate other reasons for the low incidence of pediatric urolithiasis, we evaluated the difference in the crystal–cell adhesion inhibitory activity of urinary macromolecules (UMMs) between children and adults. We also evaluated whether citrates influence the above inhibitory activity, because citrates are important in pediatric urine. Methods. Urine samples were collected from children and healthy male adults during a 24-hour period, and urinary components with a molecular weight of 3 kDa or greater were extracted as UMMs to compare their inhibitory activity during the adhesion of calcium oxalate monohydrate crystals to cultured Madin-Darby canine kidney cells between children and adults. Subsequently, various concentrations of citrates were added to adult UMMs to evaluate the changes in the crystal–cell adhesion inhibitory activity of UMMs. Results. Pediatric UMMs more strongly inhibited the adhesion of calcium oxalate monohydrate crystals to cultured Madin-Darby canine kidney cells at a concentration of 0.1 mg/mL compared with adult UMMs. In addition, pediatric UMMs contained higher proportions of fibronectin and glycosaminoglycans, both of which exhibit crystal–cell adhesion inhibitory activity. When citrates were added to adult UMMs, the crystal–cell adhesion inhibitory activity of UMMs was increased in a dose-dependent manner. However, citrates alone did not result in any differences in the inhibitory activity at any of the three different concentrations. Conclusions. We speculate that the incidence of pediatric urolithiasis is low because pediatric UMMs more potently inhibit the adhesion of calcium oxalate crystals to renal tubular cells or because the higher proportion of citrates in pediatric urine enhances the crystal–cell adhesion inhibitory activity of UMMs in a dose-dependent manner.

Magnesium ameliorates aluminum rhizotoxicity in soybean by increasing citric acid production and exudation by roots.

Superior effectiveness of Mg over Ca in alleviating Al rhizotoxicity cannot be accounted for by predicted changes in plasma membrane Al3+ activity. The influence of Ca and Mg on the production and secretion of citrate and malate, and on Al accumulation by roots was investigated with soybean genotypes Young and PI 416937 which differ in Al tolerance. In the presence of a solution Al3+ activity of 4.6 microM, citrate and malate concentrations of tap root tips of both genotypes increased with additions of either Ca up to 3 mM or Mg up to 50 microM. Citrate efflux rate from roots exposed to Al was only enhanced with Mg additions and exceeded malate efflux rates by as much as 50-fold. Maximum citrate release occurred within 12 h after adding Mg to solution treatments. Adding 50 microM Mg to 0.8 mM CaSO4 solutions containing Al3+ activities up to 4.6 microM increased citrate concentration of tap root tips by 3- to 5-fold and root exudation of citrate by 6- to 9-fold. Plants treated with either 50 microM Mg or 3 mM Ca had similar reductions in Al accumulation at tap root tips, which coincided with the respective ability of these ions to relieve Al rhizotoxicity. Amelioration of Al inhibition of soybean root elongation by low concentrations of Mg in solution involved Mg-stimulated production and efflux of citrate by roots.

Isoforms of Na+, K+-ATPase in human prostate; specificity of expression and apical membrane polarization.

The cellular distribution of Na+, K+-ATPase subunit isoforms was mapped in the secretory epithelium of the human prostate gland by immunostaining with antibodies to the alpha and beta subunit isoforms of the enzyme. Immunolabeling of the alpha1, beta1 and beta2 isoforms was observed in the apical and lateral plasma membrane domains of prostatic epithelial cells in contrast to human kidney where the alpha1 and beta1 isoforms of Na+, K+-ATPase were localized in the basolateral membrane of both proximal and distal convoluted tubules. Using immunohistochemistry and PCR we found no evidence of Na+, K+-ATPase alpha2 and alpha3 isoform expression suggesting that prostatic Na+, K+-ATPase consists of alpha1/beta1 and alpha1/beta2 isozymes. Our immunohistochemical findings are consistent with previously proposed models placing prostatic Na+, K+-ATPase in the apical plasma membrane domain. Abundant expression of Na+, K+-ATPase in epithelial cells lining tubulo-alveoli in the human prostate gland confirms previous conclusions drawn from biochemical, pharmacological and physiological data and provides further evidence for the critical role of this enzyme in prostatic cell physiology and ion homeostasis. Na+, K+-ATPase most likely maintains an inwardly directed Na+ gradient essential for nutrient uptake and active citrate secretion by prostatic epithelial cells. Na+, K+-ATPase may also regulate lumenal Na+ and K+, major counter-ions for citrate.