Salt Secretion Rate Research Articles

Structural, developmental and functional analyses of leaf salt glands of mangrove recretohalophyte Aegiceras corniculatum.

Salt secretion is an important strategy used by the mangrove plant Aegiceras corniculatum to adapt to the coastal intertidal environment. However, the structural, developmental and functional analyses on the leaf salt glands, particularly the salt secretion mechanism, are not well documented. In this study, we investigated the structural, developmental and degenerative characteristics and the salt secretion mechanisms of salt glands to further elucidate the mechanisms of salt tolerance of A. corniculatum. The results showed that the salt gland cells have a large number of mitochondria and vesicles, and plenty of plasmodesmata as well, while chloroplasts were found in the collecting cells. The salt glands developed early and began to differentiate at the leaf primordium stage. We observed and defined three stages of salt gland degradation for the first time in A. corniculatum, where the secretory cells gradually twisted and wrinkled inward and collapsed downward as the salt gland degeneration increased and the intensity of salt gland autofluorescence gradually diminished. In addition, we found that the salt secretion rate of the salt glands increased when the treated concentration of NaCl increased, reaching the maximum at 400mM NaCl. The salt-secreting capacity of the salt glands of the adaxial epidermis is significantly greater than that of the abaxial epidermis. The real-time quantitative PCR results indicate that SAD2, TTG1, GL2 and RBR1 may be involved in regulating the development of the salt glands of A. corniculatum. Moreover, Na+/H+ antiporter, H+-ATPase, K+ channel and Cl- channel may play important roles in the salt secretion of salt glands. In sum mary, this study strengthens the understanding of the structural, developmental and degenerative patterns of salt glands and salt secretion mechanisms in mangrove recretohalophyte A. corniculatum, providing an important reference for further studies at the molecular level.

The salt secretion of leaves promotes the competitiveness of Reaumuria soongarica in a desert grassland

BackgroundFor better understanding the mechanism of Reaumuria soongarica community formation in a salt stressed grassland ecosystem, we designed a field experiment to test how leaves salt secretion changes the competitive relationship between species in this plant communities.ResultsAmong the three species (R. soongarica, Stipa glareosa and Allium polyrhizum) of the salt stressed grassland ecosystem, the conductivity of R. soongarica rhizosphere soil was the highest in five soil layers (0–55 cm depth). The high soil conductivity can increase the daily salt secretion rate of plant leaves of R. soongarica. In addition, we found the canopy size of R. soongarica was positively related to the distance from S. glareosa or A. polyrhizum. The salt-tolerance of R. soongarica was significantly higher than the other two herbs (S. glareosa and A. polyrhizum). Moreover, there was a threshold (600 µS/cm) for interspecific competition of plants mediated by soil conductivity. When the soil conductivity was lower than 600 µS/cm, the relative biomass of R. soongarica increased with the soil conductivity increase.ConclusionsThe efficient salt secretion ability of leaves increases soil conductivity under the canopy. This leads the formation of a “saline island” of R. soongarica. Meanwhile R. soongarica have stronger salt tolerance than S. glareosa and A. polyrhizum. These promote the competitiveness of R. soongarica and inhibit interspecies competition advantage of the other two herbs (S. glareosa and A. polyrhizum) in the plant community. It is beneficial for R. soongarica to establish dominant communities in saline regions of desert grassland.

Salt glands play a pivotal role in the salt resistance of four recretohalophyte Limonium Mill. species.

Limonium Mill. plants are typical recretohalophytes, as they withstand salt stress by secreting excess salt onto the leaf surface through salt glands. However, little is known on the salinity thresholds of these plants and the function of salt glands in salt tolerance. Here, we investigated the salinity thresholds of salt tolerance of the Limonium species L. aureum (Linn.) Hill, L. gmelinii (Willd.) Kuntze, L. otolepis (Schrenk) Kuntze and L.sinuatum (L.) Mill grown with various concentrations of NaCl. The salinity thresholds of L. otolepis, L. aureum, L. sinuatum and L. gmelinii were 300, 350, 400 and 420mm NaCl, respectively. Correlation analysis indicated that total dry weight, chlorophyll content and intercellular CO2 concentration were highly positively correlated with the total fresh weights of all four Limonium species and could therefore be used as indicators of plant salt tolerance. Furthermore, as the salt gland density on the leaf surface increased, the rate of salt secretion per salt gland also increased, allowing more Na+ to be secreted from the plant. Redundancy discriminant analysis indicated that salt gland density, Na+ content and Na+ secretion rate per salt gland were positively correlated with salt concentration. These observations support the notion that salt glands play important roles in the adaptation of Limonium species to high salinity conditions.

Exogenous melatonin enhances salt secretion from salt glands by upregulating the expression of ion transporter and vesicle transport genes in Limonium bicolor

BackgroundSalt, a common environmental stress factor, inhibits plant growth and reduces yields. Melatonin is a pleiotropic molecule that regulates plant growth and can alleviate environmental stress in plants. All previous research on this topic has focused on the use of melatonin to improve the relatively low salt tolerance of glycophytes by promoting growth and enhancing antioxidant ability. It is unclear whether exogenous melatonin can increase the salt tolerance of halophytes, particularly recretohalophytes, by enhancing salt secretion from the salt glands.ResultsTo examine the mechanisms of melatonin-mediated salt tolerance, we explored the effects of exogenous applications of melatonin on the secretion of salt from the salt glands of Limonium bicolor (a kind of recretohalophyte) seedlings and on the expression of associated genes. A pretreatment with 5 μM melatonin significantly improved the growth of L. bicolor seedlings under 300 mM NaCl. Furthermore, exogenous melatonin significantly increased the dry weight and endogenous melatonin content of L. bicolor. In addition, this treatment reduced the content of Na+ and Cl− in leaves, but increased the K+ content. Both the salt secretion rate of the salt glands and the expression level of genes encoding ion transporters (LbHTK1, LbSOS1, LbPMA, and LbNHX1) and vesicular transport proteins (LbVAMP721, LbVAP27, and LbVAMP12) were significantly increased by exogenous melatonin treatment. These results indicate that melatonin improves the salt tolerance of the recretohalophyte L. bicolor via the upregulation of salt secretion by the salt glands.ConclusionsOur results showed that melatonin can upregulate the expression of genes encoding ion transporters and vesicle transport proteins to enhance salt secretion from the salt glands. Combining the results of the current study with previous research, we formulated a novel mechanism by which melatonin increases salt secretion in L. bicolor. Ions in mesophyll cells are transported to the salt glands through ion transporters located at the plasma membrane. After the ions enter the salt glands, they are transported to the collecting chamber adjacent to the secretory pore through vesicle transport and ions transporter and then are secreted from the secretory pore of salt glands, which maintain ionic homeostasis in the cells and alleviate NaCl-induced growth inhibition.

Salt glands of recretohalophyte Tamarix under salinity: Their evolution and adaptation.

Here, we studied the evolution of salt glands in 11 species of Tamarix and determined their role in adaptation to saline environments by measuring the effect of NaCl on plant growth and salt gland characteristics. Cluster analysis divided Tamarix species into three types (types I–III) according to salt‐gland characteristics. A phylogenetic tree based on ITS sequences indicated an evolutionary relationship consistent with the geographical distribution of Tamarix. We measured growth under different NaCl conditions (0, 100, 200, and 300 mM) for 40 days in three species (T. gallica, T. ramosissima, and T. laxa) representing the three Tamarix types. With increasing NaCl concentration, the biomass of all species was significantly reduced, especially that of T. gallica. Salt secretion ability and salt‐gland density showed similar trends in three types. The order of salt tolerance was type I > type II > type III. We conclude that during Tamarix adaptation to salinity, salt‐gland evolution followed two directions: one increasing salt‐gland density, and the other increasing salt secretion rate per salt‐gland. This study provides a basis for potential mechanisms of recretohalophyte adaptation to salinity.

The lb23 mutant of recretohalophyte Limonium bicolor (Bag.) Kuntze with 20-, 24-, 28- and 32-cell salt glands shows elevated salt secretion

Salt glands are specialized epidermal salt secretion structures on the surfaces of recretohalophytes leaves. In wild-type plants of the recretohalophyte Limonium bicolor (Bag.) Kuntze, salt glands comprise 16 cells each and appear as a blue ring under fluorescence microscopy, with four strong autofluorescence points. Here, we describe the lb23 mutant, which showed four additional types of salt glands, with five, six, seven and eight strong autofluorescence points. These salt glands comprised 20, 24, 28 and 32 cells, respectively, with correspondingly increased numbers of secretory, accessory, inner cup and outer cup cells. The cell arrangement and cell types within the salt glands in the mutant were unaltered from those of the wild type. For example, salt glands with five strong autofluorescence points had five secretory cells, five accessory cells, five inner cup cells and five outer cup cells. Compared with wild type, the abnormal salt glands with five, six, seven and eight strong autofluorescence points showed higher salt secretion rates per salt gland. Thus, the increase in salt gland cell numbers resulted in markedly increased salt secretion. This L. bicolor mutant provides an important resource for research on the molecular mechanism of salt gland development.

K+ accumulation in the cytoplasm and nucleus of the salt gland cells of Limonium bicolor accompanies increased rates of salt secretion under NaCl treatment using NanoSIMS

Recretohalophytes with specialized salt-secreting structures (salt glands) can secrete excess salts from plant, while discriminating between Na+ and K+. K+/Na+ ratio plays an important role in plant salt tolerance, but the distribution and role of K+ in the salt gland cells is poorly understood. In this article, the in situ subcellular localization of K and Na in the salt gland of the recretohalophyte Limonium bicolor Kuntze is described. Samples were prepared by high-pressure freezing (HPF), freeze substitution (FS) and analyzed using NanoSIMS. The salt gland of L. bicolor consists of sixteen cells. Higher signal strength of Na+ was located in the apoplast of salt gland cells. Compared with control, 200mM NaCl treatment led to higher signal strength of K+ and Na+ in both cytoplasm and nucleus of salt gland cells although K+/Na+ ratio in both cytoplasm and nucleus were slightly reduced by NaCl. Moreover, the rate of Na+ secretion per salt gland of L. bicolor treated with 200mM NaCl was five times that of controls. These results suggest that K+ accumulation both in the cytoplasm and nucleus of salt gland cells under salinity may play an important role in salt secretion, although the exact mechanism is unknown.

Ion homeostasis in a salt-secreting halophytic grass.

Salinity adversely affects plant growth and development, and disturbs intracellular ion homeostasis, resulting in cellular toxicity. Plants that tolerate salinity, halophytes, do so by manifesting numerous physiological and biochemical processes in coordination to alleviate cellular ionic imbalance. The present study was undertaken to analyse the salt tolerance mechanism in Aeluropus lagopoides (L.) trin. Ex Thw. (Poaceae) at both physiological and molecular levels. Plants secreted salt from glands, which eventually produced pristine salt crystals on leaves and leaf sheaths. The rate of salt secretion increased with increasing salt concentration in the growth medium. Osmotic adjustment was mainly achieved by inorganic osmolytes (Na(+)) and at 100 mM NaCl no change was observed in organic osmolytes in comparison to control plants. At 300 mM NaCl and with 150 mM NaCl + 150 mM KCl, the concentration of proline, soluble sugars and amino acids was significantly increased. Transcript profiling of transporter genes revealed differential spatial and temporal expressions in both shoot and root tissues in a manner synchronized towards maintaining ion homeostasis. In shoots, AlHKT2;1 transcript up-regulation was observed at 12 and 24 h in all the treatments, whereas in roots, maximum induction was observed at 48 h with K(+) starvation. The HAK transcript was relatively abundant in shoot tissue with all the treatments. The plasma membrane Na(+)/H(+) antiporter, SOS1, and tonoplast Na(+)/H(+) antiporter, NHX1, were found to be significantly up-regulated in shoot tissue. Our data demonstrate that AlHKT2;1, HAK, SOS1, NHX1 and V-ATPase genes play a pivotal role in regulating the ion homeostasis in A. lagopoides.

Abstract 113: Streptococcal Serum Opacity Factor Activity Diverts Human High Density Lipoprotein (HDL)-Cholesteryl Esters (CE) to the LDL-Receptor Pathway in Human Huh7 Hepatocytes

Plasma concentrations of HDL-cholesterol (C) are negatively correlated with atherosclerotic cardiovascular disease. Nevertheless, current evidence suggests that this correlation is not axiomatic and that some forms of HDL are dysfunctional and atherogenic. Moreover, clinical trials of interventions that raise HDL-C have not been uniformly successful suggesting that mechanisms by which HDL-C is increased determine its anti atherogenic potency. Additionally, mice overexpressing the HDL receptor, SR-BI, have lower plasma HDL-C concentrations and less atherosclerosis. Thus, new strategies that reduce HDL-C while promoting reverse cholesterol transport (RCT) may have therapeutic value. Serum opacity factor (SOF) disrupts HDL and forms three products_lipid-free apo A-I, a CE-depleted remnant neo HDL, and a cholesteryl ester-rich microemulsion (CERM) containing apo E and the CE of up to ~400,000 HDL particles. Hepatic CE uptake in Huh7 and HepG2 cells is faster when delivered by CERM than from the parent HDL, and cleared in vitro , in part, via the LDL-receptor (LDLR). We investigated the therapeutic potential of SOF in promoting RCT by comparing the uptake of HDL-, LDL- and CERM-CE in the final RCT steps_hepatocyte uptake, CE metabolism to cholesterol and bile salts, and their secretion and transfer to bile. HDL and LDL with [ 14 C]CE were labeled with [ 14 C]CE by incubation with lipoprotein deficient serum and the lipoproteins recovered by ultracentrifugation. CERM-labeled with [ 14 C]CE was obtained from SOF activity vs. HDL labeled with [ 14 C]CE. Incorporation efficiencies for HDL and LDL were 67% and 52% while [ 14 C]CE specific activities were 3.93 x 10 -4 and 3.04 x 10 -4 mCi/mg CE, respectively. [ 14 C]Metabolites from the hepatic uptake of HDL, LDL, and CERM were quantified by a two-solvent TLC system that resolves CE, C, and the major bile salts. The [ 14 C]CE of all three particles was taken up by cells and converted to free cholesterol with half-times of 2.7, 3.8, and 3.7 h respectively from HDL, CERM and LDL. Rates of bile salt synthesis and secretion are currently being investigated. These data show that SOF diverts potentially toxic HDL-CE to the hepatic LDLR pathway where it is hydrolyzed.

The influence of genes regulating transmembrane transport of Na+ on the salt resistance of Aeluropus lagopoides.

Plantlets of Aeluropus lagopoides (Linn.) Trin. Ex Thw. were grown at different NaCl concentrations (26, 167, 373 and 747mM) for 3, 7 and 15 days; their growth, osmotic adjustment, gas exchange, ion compartmentalisation and expression of various genes related to Na+ flux was studied. Plantlets showed optimal growth in non-saline (control; 26mM NaCl) solutions, whereas CO2/H2O gas exchange, leaf water concentration and water use efficiency decreased under all salinity treatments, accompanied by increased leaf senescence, root ash, sodium content and leaf osmolality. A decrease in malondialdehyde (MDA) content with time was correlated with Na+ accumulation in the leaf apoplast and a concomitant increase in Na+ secretion rate. A. lagopoides accumulated a higher concentration of Na+ in root than in leaf vacuoles, corresponding with higher expression of V-NHX and lower expression of PM-NHX in root than leaf tissue. It appears that V-ATPase plays a vital role during Na+ transport by producing an electromotive force, driving ion transport. Leaf calcium increased with increasing salinity, with more rapid accumulation at high salinity than at low salinity, indicating a possible involvement of Ca2+ in maintaining K+:Na+ ratio. Our results suggest that A. lagopoides successfully compartmentalised Na+ at salinities up to 373mM NaCl by upregulating the gene expression of membrane linked transport proteins (V-NHX and PM-NHX). At higher salinity (747mM NaCl), a reduction in the expression of V-NHX and PM-NHX in leaves without any change in the rate of salt secretion, is a possible cause of the toxicity of NaCl.

Effects of salinity and nitric oxide donor sodium nitroprusside (SNP) on development and salt secretion of salt glands of Limonium bicolor

NO, as a signaling molecule, is involved in abiotic stresses. Limonium bicolor seedlings were treated with 200 mM NaCl combined with 0.05 mM SNP for 20 days to study the effects of NO on development and salt-secretion rates of salt glands. It was shown that the total number of salt glands on adaxial surfaces under condition of 200 mM NaCl containing 0.05 mM SNP treatment increased significantly compared with that under 200 mM NaCl treatment. Na+ secretion rate per leaf under 200 mM NaCl containing 0.05 mM SNP was significantly higher than that under 200 mM NaCl without SNP. However, there was no significant difference in salt-secretion rate of individual salt glands between 200 mM NaCl containing 0.05 mM SNP treatment and 200 mM NaCl treatment. Although there was no significant difference in salt-secretion rate of individual glands, Na+ concentration in the leaves treated with 200 mM NaCl solution containing SNP was significantly lower than that treated with 200 mM NaCl solution. Treatment with 200 mM NaCl solution containing SNP caused a remarkable increase in Na+ concentration in salt glands. Obviously, the efficiency of the secretion process per gland was enhanced by adding SNP to NaCl. The results showed NO may enhance the salt secretion by inducing more dermatogen cells to develop into salt glands and by enhancing the efficiency of the secretion process per gland.

Short-term and diurnal patterns of salt secretion by Tamarix ramosissima and their relationships with climatic factors

Tamarisk (Tamarix spp.) is an invasive shrub or tree species in the United States. Glands within Tamarix leaves secrete salt that may increase surface soil salinity and consequently result in an unviable ecosystem for native riparian tree species. To increase our understanding of the effects of invasive Tamarix on the ecosystem and its native riparian vegetation, we investigated salt secretion of a stand of Tamarix ramosissima in the Las Vegas Wash, Nevada. Short-term and diurnal salt secretion rates were monitored in the stand from September to October 2009. Soil samples were collected beneath the canopy so that water and salinity conditions could be determined. We used weather data from a Las Vegas database in conjunction with soil temperature data collected for this study. The major cation secreted by salt glands was sodium. Diurnal variation in the salt secretion rate had a maximum during the day, suggesting that salt secretion is dependent on salt transportation to leaves. Variations in the salt secretion rates were mostly influenced by solar radiation, suggesting that salt secretion in T. ramosissima would be enhanced by light in natural conditions.

Nitric oxide enhances salt secretion and Na+ sequestration in a mangrove plant, Avicennia marina, through increasing the expression of H+-ATPase and Na+/H+ antiporter under high salinity

Modulation of nitric oxide (NO) on ion homeostasis, by enhancing salt secretion in the salt glands and Na(+) sequestration into the vacuoles, was investigated in a salt-secreting mangrove tree, Avicennia marina (Forsk.) Vierh. The major results are as follows: (i) under 400mM NaCl treatment, the application of 100µM sodium nitroprusside (SNP), an NO donor, significantly increased the density of salt crystals and salt secretion rate of the leaves, along with maintaining a low Na(+) to K(+) ratio in the leaves. (ii) The measurement of element contents by X-ray microanalysis in the epidermis and transversal sections of A. marina leaves revealed that SNP (100µM) significantly increased the accumulation of Na(+) in the epidermis and hypodermal cells, particularly the Na(+) to K(+) ratio in the salt glands, but no such effects were observed in the mesophyll cells. (iii) Using non-invasive micro-test technology (NMT), both long-term SNP (100µM) and transient SNP (30µM) treatments significantly increased net Na(+) efflux in the salt glands. On the contrary, NO synthesis inhibitors and scavenger reversed the effects of NO on Na(+) flux. These results indicate that NO enhanced salt secretion by increasing net Na(+) efflux in the salt glands. (iv) Western blot analysis demonstrated that 100µM SNP stimulated protein expressions of plasma membrane (PM) H(+)-ATPase and vacuolar membrane Na(+)/H(+) antiporter. (v) To further clarify the molecular mechanism of the effects of NO on enhancing salt secretion and Na(+) sequestration, partial cDNA fragments of PM H(+)-ATPase (HA1), PM Na(+)/H(+) antiporter (SOS1) and vacuolar Na(+)/H(+) antiporter (NHX1) were isolated and transcriptional expression of HA1, SOS1, NHX1 and vacuolar H(+)-ATPase subunit c (VHA-c1) genes were analyzed using real-time quantitative polymerase chain reaction. The relative transcript abundance of the four genes were markedly increased in 100µM SNP-treated A. marina. Moreover, the increase was reversed by NO synthesis inhibitors and scavenger. Taken together, our results strongly suggest that NO functions as a signal in salt resistance of A. marina by enhancing salt secretion and Na(+) sequestration, which depend on the increased expression of the H(+)-ATPase and Na(+)/H(+) antiporter.

Ca 2+ significantly enhanced development and salt-secretion rate of salt glands of Limonium bicolor under NaCl treatment

The divalent cation, Ca 2+, plays crucial roles in plant growth, development and stress resistance. Limonium bicolor seedlings were treated with 200 mM NaCl combined with three levels of Ca 2+ (0 mM, 5 mM and 20 mM) for 15 days to study the effects of Ca 2+ on development and salt-secretion rates of salt glands. It was shown that the 4th leaf areas of L. bicolor seedlings under 20 mM Ca 2+ treatment were significantly higher than those under 0 mM and 5 mM Ca 2+ treatments. The total number and the densities of salt glands per leaf increased markedly with increased Ca 2+ concentrations. The diameters of salt glands increased by 59% and 63% as Ca 2+ concentration increased from zero to 5 mM and 20 mM, respectively. Under 20 mM Ca 2+ treatment, the salt-secretion rate per leaf was obviously higher than that treated with 5 mM Ca 2+, but there was no significant difference in the salt-secretion rates per salt gland between the two groups. Under 0 mM Ca 2+ treatment, leaf-cell membrane permeability increased significantly, which led to serious leakage of ions and a significant increase in Na + loss rate. The results showed that the increase of Ca 2+ concentration markedly enhanced development and salt-secretion rates of salt glands in the leaves of L. bicolor, the increase of salt secretion per leaf is due to the efficiency of the secretion process per salt gland and the number of salt glands, the salt-secretion rates per salt gland have a relationship with the diameters of salt glands.

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

Peroxisome proliferator-activated receptor delta (PPARdelta) is involved in regulation of energy homeostasis. Activation of PPARdelta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary cholesterol secretion, nor by reduced cholesterol absorption. To test the hypothesis that PPARdelta activation leads to stimulation of transintestinal cholesterol efflux (TICE), we quantified it by intestine perfusions in FVB mice treated with PPARdelta agonist GW610742. To exclude the effects on cholesterol absorption, mice were also treated with cholesterol absorption inhibitor ezetimibe or ezetimibe/GW610742. GW601742 treatment had little effect on plasma lipid levels but stimulated both fecal neutral sterol excretion ( approximately 200%) and TICE ( approximately 100%). GW610742 decreased intestinal Npc1l1 expression but had no effect on Abcg5/Abcg8. Interestingly, expression of Rab9 and LIMPII, encoding proteins involved in intracellular cholesterol trafficking, was increased upon PPARdelta activation. Although treatment with ezetimibe alone had no effect on TICE, it reduced the effect of GW610742 on TICE. These data show that activation of PPARdelta stimulates fecal cholesterol excretion in mice, primarily by the two-fold increase in TICE, indicating that this pathway provides an interesting target for the development of drugs aiming at the prevention of atherosclerosis.

Ezetimibe prevents cholesterol gallstone formation in mice

Intestinal cholesterol absorption may influence gallstone formation and its modulation could be a useful therapeutic strategy for gallstone disease (GSD). Ezetimibe (EZET) is a cholesterol-lowering agent that specifically inhibits intestinal cholesterol absorption. To test whether EZET can prevent gallstone formation in mice. Gallstone-susceptible C57BL/6 inbred mice were fed control and lithogenic diets with or without simultaneous EZET administration. Lithogenic diet increased biliary cholesterol content and secretion, and induced sludge or gallstone formation in 100% of the animals. EZET administration reduced intestinal cholesterol absorption by 90% in control animals and by 35% in mice receiving the lithogenic diet. EZET prevented the appearance of cholesterol crystals and gallstones. In addition, mice fed the lithogenic diet plus EZET exhibited a 60% reduction in biliary cholesterol saturation index. Of note, EZET treatment caused a significant increase in bile flow (+50%, P<0.01) as well as bile salt, phospholipid and glutathione secretion rates (+60%, +44% and +100%, respectively, P<0.01), which was associated with a moderately increased expression of hepatic bile salt transporters. In addition, relative expression levels of Nieman-Pick C1 like 1 (NPC1L1) in the enterohepatic axis in humans were assessed. Expression levels of NPC1L1 were 15- to 30-fold higher in the duodenum compared with the liver at transcript and protein levels, respectively, suggesting preferential action of EZET on intestinal cholesterol absorption in humans. In a murine model of GSD, EZET prevented gallstone formation by reducing intestinal cholesterol absorption and increasing bile salt-dependent and -independent bile flow. EZET could be useful in preventing GSD disease in susceptible patients.

Salinity effects on leaf ion composition and salt secretion rate in Avicennia germinans (L.) L.

The effect of salinity on leaf water relations, and rate and ionic composition of leaf secretion was investigated in plants of Avicennia germinans growing under controlled salinity conditions. Increases in salinity from 0 to 940 mol NaCl m-3 reduced the predawn water potential from -0.56 to -4.16 MPa and the solute potential from -2.27 to -4.48 MPa, whereas the pressure potential remained positive in all treatments. Compared to the control, at 940 mol NaCl m-3 Na+ and Cl- concentrations of leaf sap increased 19 and 7 times, respectively. With the addition of salt the main ions accumulated in leaves were Na+ and Cl- whereas plants grown without salt accumulated K+ in leaves at levels similar to those of Na+ with salt treatments. Secretion rates of Na+ and Cl- ions increased up to the highest salinity treatment, amounting to 59 and 46 times respectively, compared to control plants. The increase in secretion rate contributed to counteract the increase of salt concentration in the leaf. In the treatments receiving salts, the Na+/K+ ratio of leaf sap ranged from 4 to 10, whereas that of the secretion ranged from 24 to 56. The ratio of Cl- vs the rest of anions remained around 3 in the leaf sap of salt-treated plants whereas it varied between 96 and 243 in the secretion. With increasing salinity higher secretion rates maintained the internal ion leaf concentration relatively low. The selective character of the secretion contributes to maintain favourable Na+/K+ and Cl-/anion ratios in the leaf cells.

Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats

Human milk contains prebiotic oligosaccharides, which stimulate the growth of intestinal bifidobacteria and lactobacilli. It is unclear whether the prebiotic capacity of human milk contributes to the larger bile salt pool size and the more efficient fat absorption in infants fed human milk compared with formula. We determined the effect of prebiotic oligosaccharides on bile salt metabolism in rats. Rats were fed a control diet or an isocaloric diet containing a mixture of galactooligosaccharides (GOS), long-chain fructooligosaccharides (lcFOS), and acidified oligosaccharides (AOS) for 3 wk. We determined synthesis rate, pool size, and fractional turnover rate (FTR) of the primary bile salt cholate by using stable isotope dilution methodology. We quantified bile flow and biliary bile salt secretion rates through bile cannulation. Prebiotic intervention resulted in significant changes in fecal and colonic flora: the proportion of lactobacilli increased 344% (P < 0.01) in colon content and 139% (P < 0.01) in feces compared with the control group. The number of bifidobacteria also increased 366% (P < 0.01) in colon content and 282% in feces after the prebiotic treatment. Furthermore, pH in both colon and feces decreased significantly with 1.0 and 0.5 pH point, respectively. However, despite this alteration of intestinal bacterial flora, no significant effect on relevant parameters of bile salt metabolism and cholate kinetics was found. The present data in rats do not support the hypothesis that prebiotics naturally present in human milk contribute to a larger bile salt pool size or altered bile salt pool kinetics.

The phosphatidylethanolamine N-methyltransferase pathway is quantitatively not essential for biliary phosphatidylcholine secretion

The phosphatidylethanolamine N-methyltransferase (PEMT) pathway of phosphatidylcholine (PC) biosynthesis is not essential for the highly specific acyl chain composition of biliary PC. We evaluated whether the PEMT pathway is quantitatively important for biliary PC secretion in mice under various experimental conditions. Biliary bile salt and PC secretion were determined in mice in which the gene encoding PEMT was inactivated (Pemt(-/-)) and in wild-type mice under basal conditions, during acute metabolic stress (intravenous infusion of the bile salt tauroursodeoxycholate), and during chronic metabolic stress (feeding a taurocholate-containing diet for 1 week). The activity of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of PC biosynthesis via the CDP-choline pathway, and the abundance of multi-drug-resistant protein 2 (Mdr2; encoded by the Abcb4 gene), the canalicular membrane flippase essential for biliary PC secretion, were determined. Under basal conditions, Pemt(-/-) and wild-type mice exhibited similar biliary secretion rates of bile salt and PC ( approximately 145 and approximately 28 nmol/min/100 g body weight, respectively). During acute or chronic bile salt administration, the biliary PC secretion rates increased similarly in Pemt(-/-) and control mice. Mdr2 mRNA and protein abundance did not differ between Pemt(-/-) and wild-type mice. The cytidylyltransferase activity in hepatic lysates was increased by 20% in Pemt(-/-) mice fed the basal (bile salt-free) diet (P < 0.05). We conclude that the biosynthesis of PC via the PEMT pathway is not quantitatively essential for biliary PC secretion under acute or chronic bile salt administration.

The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice

The main player in biliary cholesterol secretion is the heterodimeric transporter complex, ABCG5/ABCG8, the function of which is necessary for the majority of sterols secreted into bile. It is not clear whether the primary step in this process is flopping of cholesterol from the inner to the outer leaflet of the canalicular membrane, with desorption by mixed micelles, or decreasing of the activation energy required for cholesterol desorption from the outer membrane leaflet. In this study, we investigated these mechanisms by infusing Abcg8(+/+), Abcg8(+/-), and Abcg8(-/-) mice with hydrophilic and hydrophobic bile salts. In Abcg8(-/-) mice, this failed to substantially stimulate biliary cholesterol secretion. Infusion of the hydrophobic bile salt taurodeoxycholate also resulted in cholestasis, which was induced in Abcg8(-/-) mice at a much lower infusion rate compared with Abc8(-/-) and Abcg8(+/-) mice, suggesting a reduced cholesterol content in the outer leaflet of the canalicular membrane. Indeed, isolation of canalicular membranes revealed a reduction of 45% in cholesterol content under these conditions in Abcg8(-/-) mice. Our data support the model that ABCG5/ABCG8 primarily play a role in flopping cholesterol (and sterols) from the inner leaflet to the outer leaflet of the canalicular membrane.