Component Of Influx Research Articles

Adaptation of the material balance of a gas deposit

The aim of the researching is to study the application of the material balance method with modern technologies to create a useful gas reservoir model with water influx. The basic equations of material balance are analyzed taking into account the Mendeleev-Clapeyron law for gases. The analysis was performed taking into account the water influx component according to Fetkovich's equations. The paper highlights the problems of identifying the parameters of the material balance model as material balance model doesn’t include the geological structure of deposits. The calculation was done by material balance models created on Mathcad and VBA Excel. The analysis and examples given in the article testify to the expediency of applying the method of material balance with VBA Excel to clarify gas reserves, including those with a water-driven or mixed mode of development and obtain an adequate model of the reservoir.

Vasoconstriction triggered by hydrogen sulfide: Evidence for Na+,K+,2Cl-cotransport and L-type Ca2+ channel-mediated pathway

ObjectivesThis study examined the dose-dependent actions of hydrogen sulfide donor sodium hydrosulphide (NaHS) on isometric contractions and ion transport in rat aorta smooth muscle cells (SMC). MethodsIsometric contraction was measured in ring aortas segments from male Wistar rats. Activity of Na+/K+-pump and Na+,K+,2Cl-cotransport was measured in cultured endothelial and smooth muscle cells from the rat aorta as ouabain-sensitive and ouabain-resistant, bumetanide-sensitive components of the 86Rb influx, respectively. ResultsNaHS exhibited the bimodal action on contractions triggered by modest depolarization ([K+]o=30mM). At 10−4 M, NaHS augmented contractions of intact and endothelium-denuded strips by ~ 15% and 25%, respectively, whereas at concentration of 10−3M it decreased contractile responses by more than two-fold. Contractions evoked by 10−4M NaHS were completely abolished by bumetanide, a potent inhibitor of Na+,K+,2Cl-cotransport, whereas the inhibition seen at 10−3M NaHS was suppressed in the presence of K+ channel blocker TEA. In cultured SMC, 5×10−5M NaHS increased Na+,K+,2Cl- - cotransport without any effect on the activity of this carrier in endothelial cells. In depolarized SMC, 45Ca influx was enhanced in the presence of 10−4M NaHS and suppressed under elevation of [NaHS] up to 10−3M. 45Ca influx triggered by 10−4M NaHS was abolished by bumetanide and L-type Ca2+ channel blocker nicardipine. ConclusionsOur results strongly suggest that contractions of rat aortic rings triggered by low doses of NaHS are mediated by activation of Na+,K+,2Cl-cotransport and Ca2+ influx via L-type channels.

Bioelectrochemical Catalysis Reversal Reaction in Purification and Resources of Mineral Processing Flue Gas Via BECR

The Bio-electrochemical catalysis reactor (BECR) is employed to purify SOx, NOx, carbon dioxide and other pollutants from mineral processing flue gases in mineral processing. They are converted into sulphur and nitrogen and high added-value biofuels via biological agent of seperating air/pollutants and bioelectrocatalysis reversal process (BECRP). Biofuel producing bacteria and BECR reactors are coupled to a system, and the BECRP is utilized and realized to purify mineral processing flue gas and convert carbon dioxide biofuel in the reactors. The metabolic products, mineral processing flue gas components of influx and exhaust and other processing parameters are analysed by using online Micro-GC, HPLC-MS, and etc. The influences of microbial metabolic pathway of the BECRP and low voltage loading on the bacteria and the electrode reaction mechanism in the electrode process are investigated. The multi-variable bioelectrochemical transition matrix and optimal parameters are established to improve the flue gas purification efficiency and the conversion rate of carbon dioxide.

Intestinal permeability and P-glycoprotein-mediated efflux transport of ticagrelor in Caco-2 monolayer cells.

Ticagrelor is the unique reversible oral antiplatelet drug commercialized today. During this study, the intestinal permeability of ticagrelor and its potential P-glycoprotein (P-gp)-mediated active transport were assessed. To this end, bidirectional transport of ticagrelor was performed across Caco-2 (human epithelial colorectal adenocarcinoma) monolayer model in the presence and absence of potent P-gp inhibitor valspodar. Ticagrelor presented an apical-basolateral apparent permeability coefficient (Papp ) of 6.0 × 10-6 cm/s. On the other hand, mean efflux ratio (ER) of 2.71 was observed for ticagrelor describing a higher efflux permeability compared to the influx component. Valspodar showed a significant inhibitory effect on the efflux of ticagrelor suggesting involvement of P-gp in its oral disposition. Co-incubation of the P-gp inhibitor decreased the efflux Papp of ticagrelor from 1.60 × 10-5 to 1.13 × 10-5 cm/s and decreased its ER by 70%. Results suggest a modest active transport of ticagrelor by P-gp across the Caco-2 cell monolayer. The co-administration of ticagrelor with a P-gp inhibitor seems altogether unlikely to have an extended impact on pharmacokinetics of ticagrelor and cause bleeding events in patients.

Calcium Sparklets in Intact Mammalian Skeletal Muscle Fibers Expressing the Embryonic CaV1.1 Splice Variant

The embryonic splice variant of the voltage-gated L-type calcium channel (CaV1.1e) displays an altered voltage-dependence and gating kinetics as compared to that expressed in adult skeletal muscle. Because the adult CaV1.1a only opens slowly at strong depolarizations, its contribution as a source of calcium influx during action potentials is negligible. In contrast, calcium influx through the embryonic CaV1.1e substantially contributes to depolarization-induced calcium transients in fetal muscles and in cultured myotubes. In a genetically modified mouse (CaV1.1αE29), which exclusively expresses the embryonic CaV1.1e variant also in adult muscle the calcium influx component is maintained throughout life. Utilizing this mouse model, calcium release events - calcium sparklets - were recorded in enzymatically isolated, intact adult skeletal muscle fibers from the m. flexor digitorum longus using the fluorescent calcium probe fluo-8 and the fast confocal scanner (ZeissLive) in the x-y mode. While control animals did not display such events, CaV1.1αE29 mice spontaneously generated sparklets with a frequency of 9.2∗10-4±6∗10-4 Hz/μm2 (19 fibers; mean±SEM). The role of external calcium as the trigger was tested by either removing calcium from the external solution or by the application of 5μM nizoldipine to block the calcium current through CaV1.1e. Both interventions resulted in a complete loss of the events. Identified sparklets (n=311) were characterized by an average amplitude (αF/F0) of 0.287±0.005, a full-width at half-maximum of 3.05±0.05 µm, and duration of 235±4 ms, clearly different from the properties of calcium sparks on saponin-permeabilized adult mammalian skeletal muscle fibers. These findings indicate that the sustained expression of the CaV1.1e splice variant gives rise to spontaneous calcium entry events (sparklets) in adult muscle fibers and that their properties are distinct from calcium sparks arising from ryanodine receptors. Support: OTKA-NN107765, FWF P23479, W1101, TAMOP-4.2.4.A./2-11-1-2012-0001.

Differential Effects of the Gβ5-RGS7 Complex on Muscarinic M3 Receptor–Induced Ca2+ Influx and Release

The G protein β subunit Gβ5 uniquely forms heterodimers with R7 family regulators of G protein signaling (RGS) proteins (RGS6, RGS7, RGS9, and RGS11) instead of Gγ. Although the Gβ5-RGS7 complex attenuates Ca(2+) signaling mediated by the muscarinic M3 receptor (M3R), the route of Ca(2+) entry (i.e., release from intracellular stores and/or influx across the plasma membrane) is unknown. Here, we show that, in addition to suppressing carbachol-stimulated Ca(2+) release, Gβ5-RGS7 enhanced Ca(2+) influx. This novel effect of Gβ5-RGS7 was blocked by nifedipine and 2-aminoethoxydiphenyl borate. Experiments with pertussis toxin, an RGS domain-deficient mutant of RGS7, and UBO-QIC {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4- methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone (9CI)}, a novel inhibitor of Gq, showed that Gβ5-RGS7 modulated a Gq-mediated pathway. These studies indicate that Gβ5-RGS7, independent of RGS7 GTPase-accelerating protein activity, couples M3R to a nifedipine-sensitive Ca(2+) channel. We also compared the action of Gβ5-RGS7 on M3R-induced Ca(2+) influx and release elicited by different muscarinic agonists. Responses to Oxo-M [oxotremorine methiodide N,N,N,-trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn-1-ammonium iodide] were insensitive to Gβ5-RGS7. Pilocarpine responses consisted of a large release and modest influx components, of which the former was strongly inhibited whereas the latter was insensitive to Gβ5-RGS7. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride] was the only compound whose total Ca(2+) response was enhanced by Gβ5-RGS7, attributed to, in part, by the relatively small Ca(2+) release this partial agonist stimulated. Together, these results show that distinct agonists not only have differential M3R functional selectivity, but also confer specific sensitivity to the Gβ5-RGS7 complex.

Na+-dependent and Na+-independent mechanisms for inorganic phosphate uptake in Trypanosoma rangeli

BackgroundTrypanosoma rangeli is dependent on the presence of exogenous orthophosphate (Pi) for maximal growth and ecto-phosphatase activity is responsible for Pi supply under low Pi. Here we investigated the mechanisms of Pi uptake. MethodsWe investigated the kinetics of 32Pi transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:Pi cotransporter and Na+-ATPase. ResultsT. rangeli grown under limiting Pi transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a Pi transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of 32Pi influx by 80–95%. The H+-ATPase inhibitor, bafilomycin A1, inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of 32Pi to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high Pi had a lower Pi influx than that found in those grown in low Pi, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by Pi starvation. ConclusionsTwo modes of Pi transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for Pi acquisition during development of T. rangeli. General significanceThis study provides the first description of the mechanism of Pi transport across the plasma membrane of trypanosomatids.

ATP Dependent Efflux Transporters ABCB1 and ABCG2 Are Unlikely to Impact the Efficacy, or Mediate Resistance to the Tyrosine Kinase Inhibitor, Ponatinib

Abstract 2745Ponatinib is a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) currently in a pivotal phase 2 clinical trial. Ponatinib (PON) was specifically designed to target both native and all mutant forms of BCR-ABL, including T315I. The phase I study of oral ponatinib in patients with refractory CML/ALL or other hematologic malignancies recently reported that 66% and 53% of patients with CP-CML achieved MCyR and CCyR respectively (Cortes et al., ASH 2011 abstract #210).While extensive modelling experiments in BaF3 cells have been performed characterising in vitro response to ponatinib, little is known about the interactions of this drug and drug transporters that impact the response of other tyrosine kinase inhibitors (TKIs). To explore this we have examined both the degree of in vitro kinase inhibition mediated by ponatinib in BCR-ABL+ cell lines, and the intracellular uptake and retention (IUR) of ponatinib achieved. The IC50 was determined by assessing the reduction in %p-Crkl in response to increasing concentrations of ponatinib in vitro. The IUR assay was performed as previously using [14-C]-ponatinib. To determine the role of ABCB1 and ABCG2, both previously implicated in the transport of other TKIs, IC50 analysis was performed on K562 cells, and variants; ABCB1 overexpressing K562-DOX and ABCG2 overexpressing K562-ABCG2. As shown in Table 1, in contrast to the results previously observed with imatinib (IM), nilotinib (NIL) and dasatinib (DAS) there was no significant difference in the IC50ponatinib between these three cell lines, suggesting neither ABCB1 nor ABCG2 play a major role in ponatinib transport. Furthermore, the addition of either the ABCB1 and ABCG2 inhibitor pantoprazole, or the multidrug resistance (MDR) inhibitor cyclosporin did not result in a significant change in the IC50ponatinib in any of the cell lines tested. In contrast the addition of either pantoprazole or cyclosporin resulted in a significant reduction in IC50IM, IC50NIL. and IC50DAS of K562-DOX cells, supporting the notion that these TKIs interact with ABCB1.Table 1:The IC50 of ponatinib (compared to IM, NIL and DAS) in K562 cells and the over-expressing variants DOX and ABCG2 in the presence of the ABC inhibitors pantoprazole and cyclosporin. n=5. *p<0.05IC50% reduction in IC50+ pantoprazole+ cyclosporinPON (nM)IM (μM)NIL (nM)DAS (nM)PONIMNILDASPONIMNILDASK5627.793751111544*NA−107NA2DOX7.919*598*100*1018*63*1655*88*ABCG26.4730025*6NATo further examine the effect of ABC transporters on ponatinib efflux we have determined the IUR of [14-C]-ponatinib in K562, DOX and ABCG2 cell lines. We demonstrate no significant difference in the IUR between these cell lines at 37°C (n=6) (K562 vs DOX p=0.6; K562 vs ABCG2 p=0.37 and DOX vs ABCG2 p=0.667 at 2uM respectively). Temperature dependent IUR experiments reveal a significant reduction in the ponatinib IUR at 4°C compared to 37°C in K562 cells (n=6) (p=0.008), DOX cells (p=0.004) and ABCG2 cells (p=0.002) supporting the likely involvement of an ATP/temperature dependent, and yet to be determined, component of ponatinib influx. There was no significant difference in the IUR between these cell lines at 4°C (p=0.824, p=0.7 and p=0.803 respectively). Importantly, these data are consistent with the IC50ponatinib findings. If ATP dependent efflux pumps (ABCB1 and ABCG2) were actively transporting ponatinib, a significant decrease in IUR in DOX and ABCG2 at 37°C compared to K562 cells would be expected, but is not observed here. Analysis of ponatinib IUR in the prototypic ABCB1 over-expressing CEM-VBL100 cells, and their parental, ABCB1 null counterparts (CCRF-CEM) further confirmed these findings. The IUR in VBL100 cells was significantly higher than that observed in CEM’s (p<0.001; n=5), providing further evidence that ponatinib was not being exported from the cell actively via ABCB1.These data suggest that the transport of ponatinib is, at least in part, temperature-dependent indicating a yet to be determined ATP transporter may be involved in the transport of ponatinib into leukaemic cells. Importantly, this data suggests that ponatinib is unlikely to be susceptible to resistance via the major ATP efflux transporters (ABCB1 or ABCG2) that have been previously demonstrated to significantly impact the transport of, and mediate resistance to other clinically available TKIs. Disclosures:White:BMS: Honoraria, Research Funding; Novartis Pharmaceuticals: Honoraria, Research Funding. Clackson:ARIAD: Employment. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees.

SirT1 Regulates Adipose Tissue Inflammation

OBJECTIVEMacrophage recruitment to adipose tissue is a reproducible feature of obesity. However, the events that result in chemokine production and macrophage recruitment to adipose tissue during states of energetic excess are not clear. Sirtuin 1 (SirT1) is an essential nutrient-sensing histone deacetylase, which is increased by caloric restriction and reduced by overfeeding. We discovered that SirT1 depletion causes anorexia by stimulating production of inflammatory factors in white adipose tissue and thus posit that decreases in SirT1 link overnutrition and adipose tissue inflammation.RESEARCH DESIGN AND METHODSWe used antisense oligonucleotides to reduce SirT1 to levels similar to those seen during overnutrition and studied SirT1-overexpressing transgenic mice and fat-specific SirT1 knockout animals. Finally, we analyzed subcutaneous adipose tissue biopsies from two independent cohorts of human subjects.RESULTSWe found that inducible or genetic reduction of SirT1 in vivo causes macrophage recruitment to adipose tissue, whereas overexpression of SirT1 prevents adipose tissue macrophage accumulation caused by chronic high-fat feeding. We also found that SirT1 expression in human subcutaneous fat is inversely related to adipose tissue macrophage infiltration.CONCLUSIONSReduction of adipose tissue SirT1 expression, which leads to histone hyperacetylation and ectopic inflammatory gene expression, is identified as a key regulatory component of macrophage influx into adipose tissue during overnutrition in rodents and humans. Our results suggest that SirT1 regulates adipose tissue inflammation by controlling the gain of proinflammatory transcription in response to inducers such as fatty acids, hypoxia, and endoplasmic reticulum stress.

Increasing the Supply of Women Physicians in Rural Areas: Outcomes of a Medical School Rural Program

Women physicians are less likely then men to practice in rural areas. With women representing an increasing proportion of physicians, there is concern that this could exacerbate the rural physician shortage. The Physician Shortage Area Program (PSAP) of Jefferson Medical College (JMC) is one of a small number of medical school rural programs shown to be successful in addressing the rural physician shortage; however, little is known about their specific impact on women. For 2394 physicians from the 1992 to 2002 JMC graduating classes, the 2007 practice location and specialty for PSAP and non-PSAP graduates were obtained from the Jefferson Longitudinal Study. The relative likelihood of PSAP versus non-PSAP graduates practicing in rural areas was determined for women and men and compared. Women PSAP graduates were more than twice as likely as non-PSAP women to practice in rural areas (31.7% vs 12.3%; relative risk, 2.6; 95% CI, 1.6-4.2). This was similar to the PSAP outcomes for men (51.8% vs 17.7%; relative risk, 2.9, 95% CI, 2.2-3.9; relative risk ratio, 0.9, 95% CI, 0.5-1.5). PSAP outcomes were also similar for women and men practicing rural family medicine and rural primary care. These results provide support that medical school rural programs have the potential to help address the serious shortage of women physicians in rural areas, thereby increasing access to care for those living there.

Activation of P2Y1 purinergic receptors (P2Y1Rs) induced calcium influx in rat supraoptic neurons (SON) through stretch inactivated cation (SIC) channels

Activation of P2Y1Rs contributes to the ATP‐induced increase in intracellular calcium ([Ca++]i) in SON. Although activation of P2Y1Rs induced release of Ca++stores presumably as a result of IP3 production, it also induced a small Ca++ influx, possibly through plasma membrane IP3 receptors or modulation of other ion channels such as osmo‐sensitive SIC channels. Current studies evaluated the role of SIC channels in P2Y1R induced Ca++ influx by directly monitoring [Ca++]i changes using live cell calcium imaging. Hypothalamo‐neurohypophyseal explants were loaded with the Ca++‐sensitive dye, fura 2AM, and the change in [Ca++]i in SON was determined by ratiometric analysis of fluorescence emitted by excitation at 340 and 380 nm. Activation of P2Y1Rs by 2‐methyl‐thio‐ADP (2‐MeSADP, 100 μM) induced a large increase in [Ca++]i. The amplitude of the increase was reduced by ruthenium red (RR, 10 μM) and gadolinium (Gd, 100 μM), both of which block mechano‐sensitive channels including the SIC channels. Furthermore, the decay after peak response was much faster in the presence of RR and Gd. These results indicate that activation of P2Y1Rs may modulate SIC channels, thus inducing a small component of influx of extra‐cellular Ca++, in addition to release of Ca++ from intracellular stores. Further studies are ongoing to clarify the underlying mechanisms and functional importance of the Ca++ influx.Supported by AHA SDG 0735329N.

Cadmium uptake by roots: Contribution of apoplast and of high- and low-affinity membrane transport systems

The aim was to measure the respective contributions of apoplast and symplast to the Cd root uptake and to explain the linear component of the symplastic absorption. Two plants were used, maize ( Zea mays L.) and two ecotypes of alpine pennycress ( Noccaea caerulescens (J. Presl & C. Presl) F.K. Mey.), with contrasted abilities to accumulate Cd. Their roots were exposed to labelled Cd solutions of increasing concentrations. Root Cd was physico-chemically fractioned to obtain the exchanged apoplastic, non-exchanged apoplastic and symplastic pools. For both species, the proportion of Cd retained by the cell walls increased with Cd concentration in the exposure solution (ranging from 0.05 to 50 μmol L −1), from approximately 30% to 90% of the total root Cd. This was modeled using Freundlich isotherms. The non-exchanged apoplastic Cd was negligible at the highest exposure concentrations, but reached almost 30% of the total root uptake at the lowest ones. The symplastic influx in roots of both species fitted a Michaelis-Menten function associated with a linear one. The linear component of the symplastic influx could reflect absorption through a low-affinity transport system (LATS). The strong adsorption of Cd on root apoplast might act as a driving force to extract the metal from the soil, compete with the symplastic absorption and contribute to the amount of element taken up by the plant, at least in its roots.

Ussing's conundrum and the search for transport mechanisms in plants

Plant transport physiologists have developed a range of models describing the movement of ions across cell membranes. However, while substantial progress has been made towards providing precise descriptions of the mechanisms underlying these fluxes, important instances remain in which the prevailing models cannot account for repeated observations, particularly in terms of energy transformations. As we shall show, disagreements with experimental findings may entail a revision of the proposed models, similarly to what has been required in animal transport studies (Ussing, 1994; see below). We present, as a key example, the futile cycling of sodium under toxic conditions (see Britto & Kronzucker, 2006, and discussed later), and show that unidirectional flux magnitudes measured by several groups, including our own, cannot be explained energetically by current models. We attempt to explain these observations by proposing alternative mechanisms of Na + transport across the root-cell plasma membrane. The influx/efflux cycle of Na + in plants has been attributed to the sophisticated activity of distinct transport proteins in

TRPV4 channels contribute to ATP‐stimulated calcium influx in endothelial cells

Objective: We examined the role of TRPV4 channels in ATP‐stimulated Ca2+ influx in endothelial cells (ECs) of the carotid artery (CA).Methods: Rat CAs were harvested, cut into segments, and organ cultured for three days with TRPV4 siRNA to selectively knockdown TRPV4 protein. Mock transfection and scrambled siRNA controls were performed in parallel. Knockdown was confirmed by real‐time PCR, Western, and immunofluorescence. Intracellular free Ca2+ concentration was measured (fura‐2) in response to ATP in ECs isolated from organ cultured arteries.Results: TRPV4 mRNA and protein were significantly knocked down in whole CA in the TRPV4 siRNA group. EC‐specific expression of TRPV4 from organ cultured arteries was also significantly reduced in the siRNA group compared with control (immunofluorescence). Next, endothelial Ca2+ responses to 4α‐PDD (a TRPV4 channel opener) and ATP were evaluated in mock and TRPV4 siRNA transfected arteries. The EC Ca2+ increase to 4α‐PDD was significantly reduced (~50%) following TRPV4 knockdown, demonstrating significant functional loss of TRPV4 channels. Finally, stimulation with ATP revealed a significant reduction in the sustained Ca2+ influx component whereas peak Ca2+ responses were unchanged.Conclusion: These results demonstrate that ATP‐stimulated Ca2+ influx in CA endothelial cells is partially dependent on TRPV4 channels. (AHA 0665100Y and 0230353N)

Distinct P2Y Receptor Subtypes Regulate Calcium Signaling in Human Retinal Pigment Epithelial Cells

Nucleotide signaling plays a role in retinal pigment epithelial (RPE) function, and receptors for nucleotides are potential therapeutic targets for various ocular diseases. The purpose of this study was to investigate the expression of P2Y receptor subtypes in native and cultured human RPE cells. Intracellular Ca(2+) levels were monitored using real-time fluorescence imaging in cultured human RPE cells loaded with Fura-2. Expression of P2Y receptors in native and cultured RPE cells was determined by quantitative RT-PCR and Western blot analysis. Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), 2-methylthio ATP (2MeSATP), and uridine diphosphate (UDP) produced concentration-related increases in [Ca(2+)](i) in cultured RPE cells. However, differences between the magnitude and shape of agonist responses were observed. ATP and UTP showed similar response characteristics, including a distinct Ca(2+) influx component. ATP and UTP were equipotent (EC(50), 6 muM) and maximum responses were equivalent, suggesting activation of a P2Y(2) receptor. Maximal responses to ADP and 2MeSATP were equivalent with EC(50)s of 1 muM and 0.3 muM. The P2Y(1) antagonist MRS 2179 (10 muM) inhibited these responses, confirming functional expression of P2Y(1) receptors. The presence of a response to UDP suggested P2Y(6) expression. There was no influx component to P2Y(1)- and P2Y(6)-mediated responses. mRNA for P2Y(1), P2Y(2,) P2Y(4), and P2Y(6) receptor subtypes was found in cultured RPE cells, and for P2Y(1), P2Y(2,) P2Y(4,) P2Y(6), and P2Y(12) it was found in native RPE cells. Expression of P2Y(1), P2Y(2), and P2Y(6) protein was found in native and cultured RPE cells. These data define the expression profile of P2Y receptors in human RPE and show that different P2Y subtypes control distinct calcium responses in these cells.

Short and long-term uptake of Hg in white lupin plants:Kinetics and stress indicators

The intrinsic characteristics of white lupin regarding biomass production and tolerance to abiotic stresses could make it a good candidate to be used in degraded mine soils containing mercury (Hg), but white lupin behaviour in response to Hg has to be previously evaluated. With this aim, kinetic parameters of Hg uptake in short and long-term experiments, and Hg resistance of white lupin plants using several stress indicators were studied. The plants were grown with increasing Hg doses in nutrient solutions (0, 5 and 10 μM). Hg uptake showed an active component in Hg influx, suggesting the existence of a low affinity root transporter that can be used for Hg uptake into white lupin root cells. K m and V max values obtained for the saturable component were 217.7 ± 27.6 μM and 3.78 ± 0.18 μmol Hg g FW −1 h −1. Hg accumulation was concentration–time-dependent, showing a saturable behaviour for the lower doses but a linear behaviour for the highest ones. A high ability of Hg absorption by white lupin was observed both in short and long-term uptake experiments. The highest Hg dose reduced biomass production especially in the shoots. Moreover, increases in chlorophylls, malondialdehyde, total thiols and phenols were observed in Hg-stressed plants. The enhancement of total thiols and phenols levels in roots reduced oxidative stress for the 5 μM dose, but not for higher Hg levels. The deleterious effect of Hg was less marked in root tissues, in spite of their accumulation of very high Hg amounts (99%) because of, at least in part, a combined increase in total thiols and phenolics able to minimize oxidative stress. Our results suggested that phenolic content in roots could be used as a new and easy-to-use indicator of Hg presence. On the whole, white lupin showed a certain ability to survive in Hg-contaminated media and it would be possible to include it in combined decontamination strategies.

Rice OsHKT2;1 transporter mediates large Na+ influx component into K+-starved roots for growth

Excessive accumulation of sodium in plants causes toxicity. No mutation that greatly diminishes sodium (Na+) influx into plant roots has been isolated. The OsHKT2;1 (previously named OsHKT1) transporter from rice functions as a relatively Na+-selective transporter in heterologous expression systems, but the in vivo function of OsHKT2;1 remains unknown. Here, we analyzed transposon-insertion rice lines disrupted in OsHKT2;1. Interestingly, three independent oshkt2;1-null alleles exhibited significantly reduced growth compared with wild-type plants under low Na+ and K+ starvation conditions. The mutant alleles accumulated less Na+, but not less K+, in roots and shoots. OsHKT2;1 was mainly expressed in the cortex and endodermis of roots. (22)Na+ tracer influx experiments revealed that Na+ influx into oshkt2;1-null roots was dramatically reduced compared with wild-type plants. A rapid repression of OsHKT2;1-mediated Na+ influx and mRNA reduction were found when wild-type plants were exposed to 30 mM NaCl. These analyses demonstrate that Na+ can enhance growth of rice under K+ starvation conditions, and that OsHKT2;1 is the central transporter for nutritional Na+ uptake into K+-starved rice roots.

Water transport by GLUT2 expressed in Xenopus laevis oocytes

The glucose transporter GLUT2 has been shown to also transport water. In the present paper we investigated the relation between sugar and water transport in human GLUT2 expressed in Xenopus oocytes. Sugar transport was determined from uptakes of non-metabolizable glucose analogues, primarily 3-O-methyl-D-glucopyranoside; key experimental results were confirmed using D(+)-glucose. Water transport was derived from changes in oocyte volume monitored at a high resolution (20 pl, 1 s). Expression of GLUT2 induced a sugar permeability, P(S), of about 5 x 10(-6) cm s(-1) and a passive water permeability, L(p), of 5.5 x 10(-5) cm s(-1). Accordingly, the passive water permeability of a GLUT2 protein is about 10 times higher than its sugar permeability. Both permeabilities were abolished by phloretin. Isosmotic addition of sugar to the bathing solution (replacing mannitol) induced two parallel components of water influx in GLUT2, one by osmosis and one by cotransport. The osmotic driving force arose from sugar accumulation at the intracellular side of the membrane and was given by an intracellular diffusion coefficient for sugar of 10(-6) cm(2) s(-1), one-fifth of the free solution value. The diffusion coefficient was determined in oocytes coexpressing GLUT2 and the water channel AQP1 where water transport was predominantly osmotic. By the cotransport mechanism about 35 water molecules were transported for each sugar molecule by a mechanism within the GLUT2. These water molecules could be transported uphill, against an osmotic gradient, energized by the flux of sugar. This capacity for cotransport is 10 times smaller than that of the Na(+)-coupled glucose transporters (SGLT1). The physiological role of GLUT2 for intestinal transport under conditions of high luminal sugar concentrations is discussed.

Polar auxin transport and asymmetric auxin distribution.

Polar auxin transport and asymmetric auxin distribution.

Isolation of ripening rice ( Oryza sativa L.) aleurone protoplasts for uptake studies

The aleurone layer of the developing rice ( Oryza sativa L.) caryopsis is the first filial tissue in a position to take up sucrose released into the endosperm apoplast by the maternal nucellus. Adhesion of the aleurone layer to the nucellus during ripening makes it difficult to characterize sucrose transport by the aleurone layer. As an alternative, aleurone protoplasts were isolated enzymatically in a two-step process in which the cells were released from the caryopsis before the protoplasts were isolated. The procedure prevented the formation of maternal and giant (fused) aleurone protoplasts. Low protoplast yield (10% of the number of cells) was correlated with the low viability of the isolated cells. To test the utility of the protoplast system, sucrose uptake by the protoplasts was investigated using the silicone oil centrifugation technique. The concentration-dependence of the influx was biphasic and could be fitted by a function that depended on three parameters to describe the saturable and non-saturable components of the influx. These results demonstrate that it is possible to isolate protoplasts rapidly from the aleurone layer in high enough yield for protoplast uptake studies and are consistent with the aleurone layer having a saturable and a non-saturable component of sucrose influx.