RESUMO
MAIN CONCLUSION: A systematic analysis of NaCl-dependent, plasma-membrane depolarization (∆∆Ψ) in rice roots calls into question the current leading model of rapid membrane cycling of Na+ under salt stress. To investigate the character and mechanisms of Na+ influx into roots, Na+-dependent changes in plasma-membrane electrical potentials (∆∆Ψ) were measured in root cells of intact rice (Oryza sativa L., cv. Pokkali) seedlings. As external sodium concentrations ([Na+]ext) were increased in a step gradient from 0 to 100 mM, membrane potentials depolarized in a saturable manner, fitting a Michaelis-Menten model and contradicting the linear (non-saturating) models developed from radiotracer studies. Clear differences in saturation patterns were found between plants grown under low- and high-nutrient (LN and HN) conditions, with LN plants showing greater depolarization and higher affinity for Na+ (i.e., higher Vmax and lower Km) than HN plants. In addition, counterion effects on ∆∆Ψ were pronounced in LN plants (with ∆∆Ψ decreasing in the order: Cl- > SO42- > HPO 4 2- ), but not seen in HN plants. When effects of osmotic strength, Cl- influx, K+ efflux, and H+-ATPase activity on ∆∆Ψ were accounted for, resultant Km and Vmax values suggested that a single, dominant Na+-transport mechanism was operating under each nutritional condition, with Km values of 1.2 and 16 mM for LN and HN plants, respectively. Comparing saturating patterns of depolarization to linear patterns of 24Na+ radiotracer influx leads to the conclusion that electrophysiological and tracer methods do not report the same phenomena and that the current model of rapid transmembrane sodium cycling may require revision.
Assuntos
Oryza/metabolismo , Sódio/metabolismo , Membrana Celular/metabolismo , Cloretos/metabolismo , Potenciais da Membrana , Pressão Osmótica , Radioisótopos de Potássio/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Radioisótopos de Sódio/metabolismoRESUMO
Provision of silicon (Si) to roots of rice (Oryza sativa L.) can alleviate salt stress by blocking apoplastic, transpirational bypass flow of Na+ from root to shoot. However, little is known about how Si affects Na+ fluxes across cell membranes. Here, we measured radiotracer fluxes of 24Na+, plasma membrane depolarization, tissue ion accumulation, and transpirational bypass flow, to examine the influence of Si on Na+ transport patterns in hydroponically grown, salt-sensitive (cv. IR29) and salt-tolerant (cv. Pokkali) rice. Si increased growth and lowered [Na+] in shoots of both cultivars, with minor effects in roots; neither root nor shoot [K+] were affected. In IR29, Si lowered shoot [Na+] via a large reduction in bypass flow, while in Pokkali, where bypass flow was small and not affected by Si, this was achieved mainly via a growth dilution of shoot Na+. Si had no effect on unidirectional 24Na+ fluxes (influx and efflux), or on Na+-stimulated plasma-membrane depolarization, in either IR29 or Pokkali. We conclude that, while Si can reduce Na+ translocation via bypass flow in some (but not all) rice cultivars, it does not affect unidirectional Na+ transport or Na+ cycling in roots, either across root cell membranes or within the bulk root apoplast.
Assuntos
Oryza/fisiologia , Transpiração Vegetal , Tolerância ao Sal , Silício/metabolismo , Radioisótopos de Sódio/metabolismo , Transporte Biológico , Membrana Celular/metabolismoRESUMO
PURPOSE OF REVIEW: This review focuses on the recent technological advances in quantitative sodium (Na) MRI to provide a noninvasive measure of tissue viability for use in clinical studies of patients with kidney disease. Na MRI is the only noninvasive imaging technique that allows for the absolute spatial quantification of tissue sodium concentration (TSC), providing assessment of the corticomedullary sodium gradient (CMSG) in the kidney, and allowing measures of TSC in the skin and muscle. RECENT FINDINGS: Na MRI of the kidney has demonstrated the sensitivity to measure the CMSG, providing the normal range in healthy individuals and demonstrating a reduction in CMSG in kidney disease and transplanted kidneys. Studies using Na and H MRI have shown that in humans, skeletal muscle and skin can store sodium without water retention, and that sodium concentrations in muscle and skin increase with advancing age. Recent studies have shown that TSC can be mobilised during haemodialysis, and that skin sodium content links closely to left ventricular mass in patients with chronic kidney disease. SUMMARY: Na MRI is currently a research technique, but with future advances, Na MRI has potential to become a noninvasive renal biomarker and a measure of tissue sodium storage for clinical studies.
Assuntos
Rim/diagnóstico por imagem , Rim/metabolismo , Imageamento por Ressonância Magnética/métodos , Radioisótopos de Sódio/metabolismo , Humanos , Músculo Esquelético/metabolismo , Nefrologia/métodos , Pele/metabolismo , Equilíbrio HidroeletrolíticoRESUMO
We investigate the potential of multiple quantum filtered (MQF) (23)Na NMR to probe intracellular [Na]i in the Langendorff perfused mouse heart. In the presence of Tm(DOTP) shift reagent the triple quantum filtered (TQF) signal originated largely from the intracellular sodium pool with a 32±6% contribution of the total TQF signal arising from extracellular sodium, whilst the rank 2 double-quantum filtered signal (DQF), acquired with a 54.7° flip-angle pulse, originated exclusively from the extracellular sodium pool. Given the different cellular origins of the (23)Na MQF signals we propose that the TQF/DQF ratio can be used as a semi-quantitative measure of [Na]i in the mouse heart. We demonstrate a good correlation of this ratio with [Na]i measured with shift reagent at baseline and under conditions of elevated [Na]i. We compare the measurements of [Na]i using both shift reagent and TQF/DQF ratio in a cohort of wild type mouse hearts and in a transgenic PLM(3SA) mouse expressing a non-phosphorylatable form of phospholemman, showing a modest but measurable elevation of baseline [Na]i. MQF filtered (23)Na NMR is a potentially useful tool for studying normal and pathophysiological changes in [Na]i, particularly in transgenic mouse models with altered Na regulation.
Assuntos
Coração/fisiopatologia , Preparação de Coração Isolado , Miocárdio/metabolismo , Animais , Coração/diagnóstico por imagem , Imageamento por Ressonância Magnética , Camundongos , Radiografia , Sódio/metabolismo , Radioisótopos de Sódio/administração & dosagem , Radioisótopos de Sódio/metabolismoRESUMO
BACKGROUND: Established imaging methods are still not confident in the determination of stroke onset. Sodium imaging in animal models and lately in humans implicates that the sodium signal intensity within the ischemic lesion increases in a time-dependent fashion. Sodium imaging usually requires a time-consuming change of resonators or magnetic resonance imaging systems. To avoid this, we used a double-tuned (1) H/(23) Na birdcage head coil in combination with a protocol minimizing T1 - and T2 *-weighting effects for measurement of sodium intensity in acute stroke patients. METHODS: Multinuclear (1) H/(23) Na data sets were obtained from 16 stroke patients [75 ± 9·9 (standard deviation) years old] 4-130 h after symptom onset. The protocol was acquired on a clinical 3T magnetic resonance imaging site using a double-tuned (1) H/(23) Na birdcage head coil. Sodium signal intensity within the lesion and homologous contralateral side was measured and compared. RESULTS: With an acquisition time of the complete magnetic resonance imaging protocol of 22 min, a nonlinear sodium signal intensity increase within the lesion over time after stroke onset was acknowledged. Onset time within six-hours showed an increase of only 8% or less, whereas onset time beyond 8·5 h demonstrated increases of 36% or more reaching a maximum of 170% > 120 h. In addition, some patients showed a difference in sodium signal intensity compared with diffusion weighted imaging lesion. CONCLUSIONS: The use of a double-tuned (1) H/(23) Na birdcage head coil in a clinical setting 'allowed sodium intensity measurements' in a justifiable time also for acute stroke patients, and heterogenous sodium signal intensity in the diffusion weighted imaging lesion might represent differences in tissue damage or repair.
Assuntos
Isquemia Encefálica/complicações , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/etiologia , Idoso , Idoso de 80 Anos ou mais , Mapeamento Encefálico , Imagem de Difusão por Ressonância Magnética , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Cintilografia , Radioisótopos de Sódio/metabolismo , Acidente Vascular Cerebral/diagnóstico por imagem , Trítio/metabolismoRESUMO
In plant research, radioisotope imaging provides useful information about physiological activities in various tissues and elemental transport between plant organs. To expand the usage of imaging techniques, a new system was developed to visualize beta particles, x-rays and gamma-rays emitted from plant bodies. This real-time radioisotope imaging system (RRIS) visualizes radioactivity after conversion into light with a CsI(Tl) scintillator plate. Herein, the RRIS detection properties of the gamma-ray emitters (22)Na, (65)Zn, (86)Rb, (109)Cd and (137)Cs were evaluated in comparison with those of radioluminography (RLG) using an imaging plate. The lower quantitative detection limit (Bq mm(-2)) during a 15 min period ranged from 0.1 to 4, depending on the nuclide, similar to that of RLG. When the quantitative ability to detect radiation from various Arabidopsis tissues was analyzed, the quantitative capability in silique and the thick internode tended to be low. In an EGS5 simulation, beta particles were the greatest contributors to RRIS imaging of (22)Na, (86)Rb and (137)Cs, and low-energy x-rays contributed significantly to (65)Zn and (109)Cd detection. Thus, both self-absorption and air space between the sample and scintillator surface could impair quantitative RRIS imaging. Despite these issues, RRIS is suggested for quantitative time-course measurements of radionuclide motion within plants.
Assuntos
Arabidopsis/metabolismo , Radioisótopos/metabolismo , Cintilografia/métodos , Radioisótopos de Cádmio/química , Radioisótopos de Cádmio/metabolismo , Radioisótopos de Césio/química , Radioisótopos de Césio/metabolismo , Radioquímica , Radioisótopos/química , Radioisótopos de Rubídio/química , Radioisótopos de Rubídio/metabolismo , Radioisótopos de Sódio/química , Radioisótopos de Sódio/metabolismo , Fatores de Tempo , Radioisótopos de Zinco/química , Radioisótopos de Zinco/metabolismoRESUMO
Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Animais , Azidas/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Transporte de Cátions/isolamento & purificação , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Magnésio/metabolismo , Marcadores de Fotoafinidade/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteólise , Células Sf9 , Radioisótopos de Sódio/metabolismo , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Transfecção , Raios UltravioletaRESUMO
There is a growing body of evidence implicating the involvement of crustacean hyperglycaemic hormone (CHH) in ionic homeostasis in decapod crustaceans. However, little is known regarding hormonally influenced osmoregulatory processes in terrestrial decapods. As many terrestrial decapods experience opposing seasonal demands upon ionoregulatory physiologies, we reasoned that these would make interesting models in which to study the effect of CHH upon these phenomena. In particular, those (tropical) species that also undergo seasonal migrations might be especially informative, as we know relatively little regarding the nature of CHHs in terrestrial decapods, and hormonally mediated responses to seasonal changes in metabolic demands might also be superimposed or otherwise integrated with those associated with ionic homeostasis. Using Discoplax celeste as a model crab that experiences seasonal extremes in water availability, and exhibits diurnal and migratory activity patterns, we identified two CHHs in the sinus gland. We biochemically characterised (cDNA cloning) one CHH and functionally characterised (in terms of dose-dependent hyperglycaemic responses and glucose-dependent negative feedback loops) both CHHs. Whole-animal in situ branchial chamber (22)NaCl perfusion experiments showed that injection of both CHHs increased gill Na(+) uptake in a seasonally dependent manner, and (51)Cr-EDTA clearance experiments demonstrated that CHH increased urine production by the antennal gland. Seasonal and salinity-dependent differences in haemolymph CHH titre further implicated CHH in osmoregulatory processes. Intriguingly, CHH appeared to have no effect on gill Na(+)/K(+)-ATPase or V-ATPase activity, suggesting unknown mechanisms of this hormone's action on Na(+) transport across gill epithelia.
Assuntos
Proteínas de Artrópodes/metabolismo , Braquiúros/fisiologia , Homeostase/fisiologia , Hormônios de Invertebrado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Análise de Variância , Animais , Austrália , Glicemia/metabolismo , Braquiúros/metabolismo , Cromatografia Líquida de Alta Pressão , Radioisótopos de Cromo/metabolismo , Retroalimentação Fisiológica/fisiologia , Fluorimunoensaio , Hemolinfa/metabolismo , Ácido Láctico/metabolismo , Estações do Ano , Radioisótopos de Sódio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Radioisotopes (particularly (22)Na, (24)Na, (42)K, and (86)Rb) have been used for many decades to trace the fluxes and accumulation of sodium and potassium ions in plant tissues. In this article, standard procedures for the tracing of ion fluxes are described, with emphasis on special problems encountered when examining K(+) and Na(+) transport under salinity conditions. We focus in particular on unidirectional influx measurements, while also providing a brief introduction to compartmental analysis by tracer efflux.
Assuntos
Plantas/metabolismo , Potássio/metabolismo , Salinidade , Sódio/metabolismo , Transporte de Íons , Cinética , Potássio/química , Radioisótopos de Potássio/metabolismo , Traçadores Radioativos , Sódio/química , Radioisótopos de Sódio/metabolismoRESUMO
Soil sodium, while toxic to most plants at high concentrations, can be beneficial at low concentrations, particularly when potassium is limiting. However, little is known about Na(+) uptake in this 'high-affinity' range. New information is provided here with an insight into the transport characteristics, mechanism, and ecological significance of this phenomenon. High-affinity Na(+) and K(+) fluxes were investigated using the short-lived radiotracers (24)Na and (42)K, under an extensive range of measuring conditions (variations in external sodium, and in nutritional and pharmacological agents). This work was supported by electrophysiological, compartmental, and growth analyses. Na(+) uptake was extremely sensitive to all treatments, displaying properties of high-affinity K(+) transporters, K(+) channels, animal Na(+) channels, and non-selective cation channels. K(+), NH(4)(+), and Ca(2+) suppressed Na(+) transport biphasically, yielding IC(50) values of 30, 10, and <5 µM, respectively. Reciprocal experiments showed that K(+) influx is neither inhibited nor stimulated by Na(+). Sodium efflux constituted 65% of influx, indicating a futile cycle. The thermodynamic feasibility of passive channel mediation is supported by compartmentation and electrophysiological data. Our study complements recent advances in the molecular biology of high-affinity Na(+) transport by uncovering new physiological foundations for this transport phenomenon, while questioning its ecological relevance.
Assuntos
Hordeum/metabolismo , Radioisótopos de Potássio/farmacocinética , Radioisótopos de Sódio/farmacocinética , Transporte Biológico , Hordeum/química , Hordeum/genética , Cinética , Radioisótopos de Potássio/metabolismo , Radioisótopos de Sódio/metabolismoRESUMO
This study examined the role of cell volume modulation in plasma membrane rupture and death documented in ouabain-treated renal epithelial cells. Long-term exposure to ouabain caused massive death of C11-MDCK (Madin-Darby canine kidney) epithelial cells, documented by their detachment, chromatin cleavage and complete loss of lactate dehydrogenase (LDH), but did not affect the survival of vascular smooth muscle cells (VSMCs) from the rat aorta. Unlike the distinct impact on cell survival, 2-h exposure to ouabain led to sharp elevation of the [Naâº](i)/[Kâº](i) ratio in both cell types. A similar increment of Naâº(i) content was evoked by sustained inhibition of Naâº,Kâº-ATPase in Kâº-free medium. However, in contrast to ouabain, C11-MDCK cells survived perfectly during 24-h exposure to Kâº-free medium. At 3 h, the volume of ouabain-treated C11-MDCK cells and VSMCs, measured by the recently developed dual-image surface reconstruction technique, was increased by 16 and 12%, respectively, whereas 5-10 min before the detachment of ouabain-treated C11-MDCK cells, their volume was augmented by ~30-40%. To examine the role of modest swelling in the plasma membrane rupture of ouabain-treated cells, we compared actions of hypotonic medium on volume and LDH release. We observed that LDH release from hypoosmotically swollen C11-MDCK cells was triggered when their volume was increased by approximately fivefold. Thus, our results showed that the rupture of plasma membranes in ouabain-treated C11-MDCK cells was not directly caused by cell volume modulation evoked by Naâº,Kâº-ATPase inhibition and inversion of the [Naâº](i)/[Kâº](i) ratio.
Assuntos
Membrana Celular/metabolismo , Tamanho Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Transporte de Íons/efeitos dos fármacos , Rim/metabolismo , Músculo Liso Vascular/metabolismo , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio , Animais , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatina/metabolismo , Cães , Células Epiteliais/citologia , Rim/citologia , L-Lactato Desidrogenase/metabolismo , Músculo Liso Vascular/citologia , Osmose/efeitos dos fármacos , Pressão Osmótica/efeitos dos fármacos , Potássio/metabolismo , Ratos , Rubídio/metabolismo , Isótopos de Sódio/metabolismo , Radioisótopos de Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Many terrestrial arthropods display tight osmotic and ionic regulation of the hemolymph during dehydration. In this study, we sought to quantify the level of regulation of the major hemolymph cations in the terrestrial isopod Armadillidium vulgare (Isopoda, Oniscidea). Inulin space measurements showed that the hemolymph comprises 52 ± 2.2% of the hydrated water content but contributes 71 ± 9.8% of water losses during desiccation. Hemolymph concentrations of Na+, K+ and Ca²+ were measured in variably dehydrated animals using ion-selective microelectrodes and compared with predicted concentrations assuming no regulation. Na+ and Ca²+ are quite tightly regulated, showing respective concentration increases of 20.8% and 7.1% following a 50% reduction in hemolymph volume, but K+ showed no measurable regulation. The excreted cation fraction during desiccation is negligible. Sites of ion sequestration were examined by injecting ²²Na and 45Ca into the hemolymph of hydrated animals and assaying tissue-specific activities following dehydration. Na+ is apparently sequestered non-specifically by an unknown mechanism. Ca²+ accumulates in the dorsal somatic tissues, possibly in the calcium pool of the cuticle. How A. vulgare avoids significant disruptions of E(m) and neuromuscular function in the absence of K+ regulation, and how it sequesters Na+, both pose intriguing challenges for future work.
Assuntos
Cálcio/sangue , Desidratação/metabolismo , Isópodes/fisiologia , Potássio/sangue , Sódio/sangue , Animais , Radioisótopos de Cálcio/metabolismo , Sistema Digestório/metabolismo , Cabeça/fisiologia , Hemolinfa/química , Hemolinfa/metabolismo , Especificidade de Órgãos , Radioisótopos de Sódio/metabolismoRESUMO
Malignant gliomas are the most common primary brain tumors associated with significant morbidity and mortality. How to target the tumor in situ, and inhibit tumor cell proliferation and invasion is the key for therapy. Gliomas express a glioma-specific chloride ion channel that is sensitive to toxins including BmKCT. In the current study, the inhibitory effect of BmKCT on glioma growth was observed in vivo using the glioma/SD rat model. Furthermore, BmKCT prevented the metastasis of glioma cells in vivo. Moreover, biodistribution experiments with (l3l)I-labeled or Cy5.5-conjugated BmKCT revealed that BmKCT selectively targeted the glioma in situ. Our data suggest that BmKCT could be exploited as a potential therapeutic for glioma diagnosis and therapy.
Assuntos
Glioma/patologia , Metástase Neoplásica/patologia , Venenos de Escorpião/uso terapêutico , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Clonagem Molecular , Feminino , Amplificação de Genes , Glioma/tratamento farmacológico , Glutationa Transferase/metabolismo , Metástase Neoplásica/prevenção & controle , Ratos , Ratos Sprague-Dawley , Venenos de Escorpião/genética , Venenos de Escorpião/toxicidade , Escorpiões , Radioisótopos de Sódio/metabolismo , Distribuição TecidualRESUMO
The interaction of sodium and potassium ions in the context of the primary entry of Na(+) into plant cells, and the subsequent development of sodium toxicity, has been the subject of much recent attention. In the present study, the technique of compartmental analysis with the radiotracers (42)K(+) and (24)Na(+) was applied in intact seedlings of barley (Hordeum vulgare L.) to test the hypothesis that elevated levels of K(+) in the growth medium will reduce both rapid, futile Na(+) cycling at the plasma membrane, and Na(+) build-up in the cytosol of root cells, under saline conditions (100 mM NaCl). We reject this hypothesis, showing that, over a wide (400-fold) range of K(+) supply, K(+) neither reduces the primary fluxes of Na(+) at the root plasma membrane nor suppresses Na(+) accumulation in the cytosol. By contrast, 100 mM NaCl suppressed the cytosolic K(+) pool by 47-73%, and also substantially decreased low-affinity K(+) transport across the plasma membrane. We confirm that the cytosolic [K(+)]:[Na(+)] ratio is a poor predictor of growth performance under saline conditions, while a good correlation is seen between growth and the tissue ratios of the two ions. The data provide insight into the mechanisms that mediate the toxic influx of sodium across the root plasma membrane under salinity stress, demonstrating that, in the glycophyte barley, K(+) and Na(+) are unlikely to share a common low-affinity pathway for entry into the plant cell.
Assuntos
Hordeum/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Transporte Biológico , Hordeum/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Radioisótopos de Potássio/metabolismo , Radioisótopos de Sódio/metabolismoRESUMO
Plant salinity tolerance is a polygenic trait with contributions from genetic, developmental, and physiological interactions, in addition to interactions between the plant and its environment. In this study, we show that in salt-tolerant genotypes of barley (Hordeum vulgare), multiple mechanisms are well combined to withstand saline conditions. These mechanisms include: (1) better control of membrane voltage so retaining a more negative membrane potential; (2) intrinsically higher H(+) pump activity; (3) better ability of root cells to pump Na(+) from the cytosol to the external medium; and (4) higher sensitivity to supplemental Ca(2+). At the same time, no significant difference was found between contrasting cultivars in their unidirectional (22)Na(+) influx or in the density and voltage dependence of depolarization-activated outward-rectifying K(+) channels. Overall, our results are consistent with the idea of the cytosolic K(+)-to-Na(+) ratio being a key determinant of plant salinity tolerance, and suggest multiple pathways of controlling that important feature in salt-tolerant plants.
Assuntos
Membrana Celular/metabolismo , Hordeum/metabolismo , Raízes de Plantas/metabolismo , Potássio/metabolismo , Cloreto de Sódio/metabolismo , Adaptação Fisiológica , Genótipo , Homeostase/fisiologia , Hordeum/genética , Hordeum/fisiologia , Potenciais da Membrana , Técnicas de Patch-Clamp , Epiderme Vegetal/metabolismo , Raízes de Plantas/fisiologia , Canais de Potássio/metabolismo , Bombas de Próton/metabolismo , Protoplastos/metabolismo , Salinidade , Sódio/metabolismo , Radioisótopos de Sódio/metabolismo , TetraetilamônioRESUMO
The general phosphate need in mammalian cells is accommodated by members of the P(i) transport (PiT) family (SLC20), which use either Na(+) or H(+) to mediate inorganic phosphate (P(i)) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na(+)-dependent P(i) (NaP(i)) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with (32)P(i) as a traceable P(i) source. For PiT1, the Michaelis-Menten constant for P(i) was determined as 322.5 +/- 124.5 microM. PiT2 was analyzed for the first time and showed positive cooperativity in P(i) uptake with a half-maximal activity constant for P(i) of 163.5 +/- 39.8 microM. PiT1- and PiT2-mediated Na(+)-dependent P(i) uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na(+) dependency patterns. However, only PiT2 was capable of Na(+)-independent P(i) transport at acidic pH. Study of the impact of divalent cations Ca(2+) and Mg(2+) revealed that Ca(2+) was important, but not critical, for NaP(i) transport function of PiT proteins. To gain insight into the NaP(i) cotransport function, we analyzed PiT2 and a PiT2 P(i) transport knockout mutant using (22)Na(+) as a traceable Na(+) source. Na(+) was transported by PiT2 even without P(i) in the uptake medium and also when P(i) transport function was knocked out. This is the first time decoupling of P(i) from Na(+) transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E(55) and E(575) are responsible for linking P(i) import to Na(+) transport in PiT2.
Assuntos
Fosfatos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico , Cálcio/metabolismo , Enzimas de Restrição do DNA/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Magnésio/metabolismo , Oócitos/citologia , Oócitos/fisiologia , Fosfatos/química , Radioisótopos de Fósforo/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Complementar/metabolismo , Sódio/química , Radioisótopos de Sódio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Xenopus laevisRESUMO
Atrial natriuretic peptide (ANP) decreases plasma Na+ concentration and promtes seawater (SW) adaptation in eels. The hyponatremia may most probably be caused by increased branchial extrusion of Na+, but the mechanism has not been determined yet. The present study examined initially the effects of ANP on branchial Na+ efflux in vivo using isotopic 22Na. However, the efflux rate was not altered by infusion of a hyponatremic dose of ANP (5 pmol.kg(-1).min(-1)). Therefore, we sought to examine whether the ANP-mediated hyponatremia is caused by a decrease in the uptake of Na+ from the environment. Since a decrease in drinking was highly correlated with a degree of hyponatremia, conscious SW eels were infused with dilute SW into the stomach at a normal drinking rate to offset the antidipsogenic effect of ANP. Under this regimen, the hyponatremic effect of ANP was abolished. Then, we examined the site of Na+ absorption in the alimentary tract by measuring the changes in ion composition of intraluminal fluid along the tract. Since Na+ was absorbed at the esophagus and anterior/middle intestine, a sac was prepared at each site and the effects of ANP were examined in situ in conscious SW eels. ANP infusion did not alter Na+ absorption at the esophagus, but it profoundly reduced the absorption at the intestine. Together with our previous finding that ANP does not alter renal Na+ excretion, we propose that ANP reduces plasma Na+ concentration in SW eels by inhibiting drinking and subsequent absorption of Na+ by the intestine.
Assuntos
Anguilla/metabolismo , Fator Natriurético Atrial/metabolismo , Ingestão de Líquidos/fisiologia , Hiponatremia/metabolismo , Mucosa Intestinal/metabolismo , Radioisótopos de Sódio/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Análise de Variância , Anguilla/fisiologia , Animais , Transporte Biológico Ativo , Transporte de Íons , Japão , Água do Mar , Radioisótopos de Sódio/sangueRESUMO
RATIONALE AND OBJECTIVES: The aim of the study was to investigate the feasibility of using sodium magnetic resonance imaging (MRI) as a noninvasive quantitative technique for measuring proteoglycan (PG) content in an in vivo porcine model of osteoarthritis (OA). MATERIALS AND METHODS: Biochemical conditions similar to those of OA were created by an intra-articular injection of recombinant porcine interleukin-1beta (IL-1beta) into the knee joint of pigs (n = 6) before performing MRI. The contralateral knee joint was given a saline injection to serve as an internal control. Sodium MRI data were acquired on a 4-T clinical MR scanner and used to compute quantitative sodium and fixed charge density (FCD) maps based on a previously established methodology. In vivo FCD maps were compared with FCD maps obtained using ex vivo patellae harvested from the specimens. The tissue and joint fluid were subjected to histologic and immunohistochemical analyses as independent measurements of IL-1beta activity and PG loss. RESULTS: The average FCD of IL-1beta-treated patellae was measured to be 49% lower than that of saline-treated patellae, indicating a loss of PG content. These results were supported by histologic and immunochemical findings, most notably a reduction in staining for PG and an increase in matrix metalloproteinases in the synovial fluid. CONCLUSION: Sodium MRI can serve as a quantitative method to measure in vivo changes in PG content in an animal model of OA. The use of a noninvasive quantitative in vivo PG measurement technique such as sodium MRI on an animal model would aid greatly in efforts to monitor the efficacy of treatments for OA. Furthermore, these results indicate that early degenerative events could be detected noninvasively in vivo in humans with PG-depleting diseases such as OA.
Assuntos
Imageamento por Ressonância Magnética , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/metabolismo , Proteoglicanas/efeitos dos fármacos , Proteoglicanas/metabolismo , Animais , Biomarcadores/análise , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Colagenases/metabolismo , Modelos Animais de Doenças , Imunoquímica , Interleucina-1/administração & dosagem , Metaloproteinase 13 da Matriz , Metaloproteinase 3 da Matriz/metabolismo , Patela/diagnóstico por imagem , Patela/metabolismo , Patela/patologia , Intensificação de Imagem Radiográfica , Radioisótopos de Sódio/metabolismo , SuínosRESUMO
BACKGROUND & OBJECTIVES: The biochemical mechanisms underlying the development of sensitization-induced airway hyperresponsiveness (AHR) in asthma are poorly defined. Alterations in the regulation of intracellular calcium may play an important role in its pathogenesis. We carried out this study to see the effect of sensitization with ovalbumin on membrane ion fluxes and intracellular calcium in a guinea pig model. METHODS: Airway reactivity to inhaled histamine was measured initially and after sensitization with ovalbumin in 28 guineapigs. Intracellular calcium [Ca(2+)]i was measured in tracheal smooth muscle cells and peripheral leukocytes using fluorescent dye FURA 2AM. Calcium and sodium ion influx across the cell membrane was measured in leukocytes. Ouabain-sensitive Rubidium ((86)Rb) influx was measured in tracheal smooth muscles cells. The activities of Na(+), K(+) ATPase and Ca(2+) ATPase were measured in tracheal smooth muscle cells. Lipid peroxides were measured in plasma. RESULTS: Airway responsiveness was significantly (P<0.001) increased after sensitization along with an increase in [Ca2+]i levels in leukocytes and tracheal smooth muscle cells, higher rates of (45)Ca and (22)Na influx in leukocytes and higher (86)Rb influx rates in tracheal smooth muscle cells, and increased levels of lipid peroxides in plasma. INTERPRETATION & CONCLUSION: In guineapig model of asthma sensitization to allergen increased the membrane permeability to calcium and sodium, and intracellular calcium levels. These alterations may play a role in the pathogenesis of airway hyper-responsiveness following sensitization.
Assuntos
Hiper-Reatividade Brônquica/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Fura-2/análogos & derivados , Íons/metabolismo , Animais , Cálcio/química , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Cobaias , Histamina/metabolismo , Leucócitos/citologia , Leucócitos/metabolismo , Peroxidação de Lipídeos , Masculino , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Ovalbumina/metabolismo , Radioisótopos de Rubídio/metabolismo , Radioisótopos de Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Traqueia/citologiaRESUMO
In the proximal tubule, the apical Na(+)/H(+) exchanger identified as NHE3 mediates most NaCl and NaHCO(3) absorption. The purpose of this study was to analyze the long-term regulation of NHE3 during alkalosis induced by dietary NaHCO(3) loading and changes in NaCl intake. Sprague-Dawley rats exposed to a low-NaCl, high-NaCl, or NaHCO(3) diet for 6 days were studied. Renal cortical apical membrane vesicles (AMV) were prepared from treated and normal rats. Na(+)/H(+) exchange was assayed as the initial rate of (22)Na(+) uptake in the presence of an outward H(+) gradient. (22)Na(+) uptake measured in the presence of high-dose 5-(N-ethyl-N-isopropyl) amiloride was not different among models. Changes in NaCl intake did not affect NHE3 activity, whereas NaHCO(3) loading inhibited (22)Na(+) uptake by 30%. AMV NHE3 protein abundance assessed by Western blot analysis was unaffected during changes in NaCl intake. During NaHCO(3) loading, NHE3 protein abundance was decreased by 65%. We conclude that proximal NHE3 adapts to chronic metabolic acid-base disorders but not to changes in dietary NaCl intake.