Activity Of Plasma Membrane Na Research Articles

Salt-Stress-Induced Ion Transport Contributes to K+/Na+ Homeostasis in Roots of Ping’ou Hybrid Hazelnut

Soil salinity is a worldwide problem that adversely affects plant growth and development. Soil salinization in Xinjiang of China is very serious. Ping’ou hybrid hazelnut, as an important ecological and economic tree species, as well as a salt-tolerant plant, has been grown in Xinjiang for over 20 years. Understanding the salt-tolerance mechanism of Ping’ou hybrid hazelnut is of great significance for the breeding of salt-tolerant varieties and the rational utilization of salinized land. In this study, ‘Liaozhen 7’, a fine variety of Ping’ou hybrid hazelnut, was selected as test material, and seedlings were treated with 0 (control), 50, 100 and 200 mM NaCl. Subsequently, the pattern of NaCl-induced fluxes of Na+, K+ and H+ in the root meristematic zone and their response to ion transport inhibitors were studied using non-invasive micro-test technology (NMT). Different concentrations of NaCl stress significantly increased the Na+ concentration in roots, while K+ concentration decreased first and then increased with the increase of NaCl concentration. Meanwhile, NaCl stress induced a significant decline in K+/Na+ ratio. Control and 200 mM NaCl-induced Na+ and K+ fluxes in roots exhibited an outward efflux, whereas an inward flux was observed for H+. Under 200 mM NaCl stress, the average rates of net Na+ and K+ efflux, as well as H+ influx in roots were significantly increased, which were 11.6, 6.7 and 2.3 times higher than that of control, respectively. Furthermore, pharmacological experiments showed that 200 mM NaCl-induced Na+ efflux; H+ influx was significantly suppressed by amiloride, an inhibitor of plasma membrane (PM) Na+/H+ antiporter, and sodium vanadate, an inhibitor of PM H+-ATPase. Net Na+ efflux and H+ influx induced by NaCl decreased by 89.9% and 135.0%, respectively. The NaCl-induced Na+ efflux was mediated by a Na+/H+ antiporter using energy provided by PM H+-ATPase. The NaCl-induced K+ efflux was significantly restricted by tetraethylamine chloride, a K+ channel inhibitor, and promoted by sodium vanadate, which decreased by 111.2% and increased by 80.8%, respectively, indicating that K+ efflux was regulated by depolarization-activated outward-rectifying K+ channels and non-selective cation channels (NSCCs). In conclusion, NMT data revealed that NaCl stress up regulated the root Na+/H+ antiporter and H+ pump (an activity of PM Na+/H+ antiport system) of ‘Liaozhen 7’, which compelled the Na+/H+ exchange across the PM and restricted K+ loss via depolarization-activated K+ channels and NSCCs simultaneously, thereby maintaining the K+/Na+ homeostasis and higher salt tolerance.

Assessing the role of root plasma membrane and tonoplast Na+/H+ exchangers in salinity tolerance in wheat: in planta quantification methods

This work investigates the role of cytosolic Na+ exclusion in roots as a means of salinity tolerance in wheat, and offers in planta methods for the functional assessment of major transporters contributing to this trait. An electrophysiological protocol was developed to quantify the activity of plasma membrane Na+ efflux systems in roots, using the microelectrode ion flux estimation (MIFE) technique. We show that active efflux of Na+ from wheat root epidermal cells is mediated by a SOS1-like homolog, energized by the plasma membrane H+-ATPase. SOS1-like efflux activity was highest in Kharchia 65, a salt-tolerant bread wheat cultivar. Kharchia 65 also had an enhanced ability to sequester large quantities of Na+ into the vacuoles of root cells, as revealed by confocal microscopy using Sodium Green. These findings were consistent with the highest level of expression of both SOS1 and NHX1 transcripts in plant roots in this variety. In the sensitive wheat varieties, a greater proportion of Na+ was located in the root cell cytosol. Overall, our findings suggest a critical role of cytosolic Na+ exclusion for salinity tolerance in wheat and offer convenient protocols to quantify the contribution of the major transporters conferring this trait, to screen plants for salinity tolerance.

Estrogen receptor modulators and estrogen receptor beta immunolabelling in human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC) exposed to the female sex hormone estradiol show different kinds of effects including increased elasticity, activation of plasma membrane Na+/H+ exchange, prostacyclin production, prevention of apoptosis and many others. The aim of this study was the systematic analysis of the immunolabelling of estrogen receptors (ERs), ERalpha and ERbeta, in HUVEC after stimulation with different commercially available ER modulators and ER agonists or antagonists. HUVEC response to these substances was shown to be regulated via ERbeta. ERalpha immunolabelling or up-regulation was abrogated after application of estrogen derivatives, selective estrogen receptor modulators (SERM) and ER agonists or antagonists. Immunolabelling of ERbeta was significantly increased by estradiol, estrone, ethinylestradiol and tumour necrosis factor alpha (TNFalpha). SERM, such as Tamoxifen, and pure antagonists, such as ICI 182.780, stimulated ERbeta in HUVEC at low concentrations, whereas higher concentrations inhibited ERbeta immunolabelling. The pure estrogen receptor agonist 2,3-bis (4-hydroxyphenyl) proprionitrile (DPN) exhibited its activating potential at low concentrations. In contrast, higher concentrations resulted in a down-regulation of ERbeta. Estrogenic effects in HUVEC, independent of stimulation or inhibition, are mediated via the ERbeta. SERM such as Tamoxifen and ER antagonists such as ICI 182.780 act as ER activators in low concentrations, whereas higher concentrations lead to inhibitory effects.

Human umbilical vascular endothelial cells express estrogen receptor beta (ERβ) and progesterone receptor A (PR-A), but not ERα and PR-B

Several reports deal with possible effects of female sex hormones on human umbilical vein endothelial cells (HUVEC) including elasticity, activation of plasma membrane Na+/H+ exchange, VEGF receptor Flk-1/KDR and many others. In contrast to those findings, some publications pointed out that HUVEC lack expression of both the estrogen receptor (ER) and/or the progesterone receptor (PR). Because the majority of these investigations were carried out at a time period, when only one ER and one PR was known, the aim of this study was the systematic analysis of ERalpha and ERbeta as well as PR-A and PR-B expression in HUVEC with specific monoclonal antibodies by immunocytochemistry and quantitative RT-PCR (TaqMan). As a result, we could show that HUVEC lack ERalpha but express ERbeta. The expression of ERbeta could be significantly upregulated with 17beta-estradiol on mRNA and protein level. In addition, HUVEC express PR-A but not PR-B. PR-A expression could be significantly upregulated with progesterone, again on mRNA and protein level. We conclude that estrogenic effects on HUVEC are mediated via the ERbeta and gestagens act via the PR-A pathway.

Functional comparison of plasma-membrane Na+/H+ antiporters from two pathogenic Candida species

BackgroundThe virulence of Candida species depends on many environmental conditions. Extracellular pH and concentration of alkali metal cations belong among important factors. Nevertheless, the contribution of transporters mediating the exchange of alkali metal cations for protons across the plasma membrane to the cell salt tolerance and other physiological properties of various Candida species has not been studied so far.ResultsThe tolerance/sensitivity of four pathogenic Candida species to alkali metal cations was tested and the role of one of the cation transporters in that tolerance (presumed to be the plasma-membrane Na+/H+ antiporter) was studied. The genes encoding these antiporters in the most and least salt sensitive species, C. dubliniensis and C. parapsilosis respectively, were identified, cloned and functionally expressed in the plasma membranes of Saccharomyces cerevisiae cells lacking their own cation exporters. Both CpCnh1 and CdCnh1 antiporters had broad substrate specificity and transported Na+, K+, Li+, and Rb+. Their activity in S. cerevisiae cells differed; CpCnh1p provided cells with a much higher salt tolerance than the CdCnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters.ConclusionWe have cloned two genes encoding putative Na+/H+ antiporters in C. parapsilosis and C. dubliniensis, and characterized the transport properties of encoded proteins. Our results show that the activity of plasma-membrane Na+/H+ antiporters is one of the factors determining the tolerance of pathogenic Candida species to high external concentrations of alkali metal cations.

17β-estradiol increases volume, apical surface and elasticity of human endothelium mediated by Na+/H+ exchange

17beta-estradiol is known to delay the onset of atherosclerosis in women but cellular mechanisms are still unclear. Estrogens bind to specific receptors and initiate a signaling cascade that involves the activation of plasma membrane Na(+)/H(+) exchange. We hypothesized that estrogens interfere with ion transport across the plasma membrane and thus control endothelial structure and function. Therefore, we investigated the effects of the sex steroids 17beta-estradiol, progesterone, and testosterone on volume, apical surface and elasticity in human endothelium. The atomic force microscope was used as an imaging tool and as an elasticity sensor. We applied the antiestrogen tamoxifen, the Na(+)/H(+) exchange blocker cariporide and the epithelial Na(+)channel blocker amiloride to elucidate the role of transmembrane ion transport in hormone-treated human umbilical vein endothelial cells (HUVEC). Incubation with 17beta-estradiol for 72 h led to a dose-dependent increase of endothelial cell volume (41%), apical cell surface (22%), and cell elasticity (53%) as compared to non-17beta-estradiol treated controls. Block of the 17beta-estradiol receptor by tamoxifen and of plasma membrane Na(+)/H(+) exchange by cariporide prevented the hormone-induced changes. Progesterone and testosterone were ineffective. 17beta-estradiol increases HUVEC water content and HUVEC elasticity mediated by activated estrogen receptors. The estrogen response depends on the activation of plasma membrane Na(+)/H(+) exchange. The increase in endothelial cell elasticity could be one of the vasoprotective mechanisms postulated for 17beta-estradiol.

Involvement of Na+/H+ exchanger in desmopressin-induced platelet procoagulant response.

Desmopressin (DDAVP) action on platelets is associated with the development of procoagulant response but the underlying mechanism of this phenomenon is not known. We investigated whether this effect of DDAVP might be due to activation of plasma membrane Na+/H+ exchanger. The DDAVP-induced platelet procoagulant response, measured as phospholipid-dependent thrombin generation, was dose dependent and significantly weaker than that produced by collagen or monensin (mimics Na+/H+ antiport). Both the DDAVP- and collagen-produced procoagulant responses were less pronounced in the presence of EIPA, an Na+/H+ exchanger inhibitor. Flow cytometry studies revealed that in vitro treatment of platelets with DDAVP or collagen was associated with the appearance of both degranulated (and fragmented) and swollen cells. The DDAVP-evoked rise in size and granularity heterogeneity was similar to that produced by collagen or monensin and was not observed in the presence of EIPA. Using flow cytometry and annexin V-FITC as a probe for phosphatidylserine (PS) we demonstrated increased and uniform binding of this marker to all subsets of DDAVP-treated platelet population. The DDAVP-evoked PS expression was dose dependent, strongly reduced by EIPA and weaker than that caused by monensin or collagen. As judged by optical swelling assay, DDAVP in a dose dependent manner produced a rise in platelet volume. The swelling was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Electronic cell-sizing measurements showed an increase in mean platelet volume and a decrease in platelet count and platelet crit upon treatment with DDAVP. DDAVP elicited a slow (much slower than collagen) alkalinization of platelet cytosol. Altogether the data indicate an involvement of Na+/H+ exchanger in the generation of procoagulant activity in DDAVP-treated platelets.

Involvement of Na+/H+ exchanger in desmopressin-induced platelet procoagulant response.

Desmopressin (DDAVP) action on platelets is associated with the development of procoagulant response but the underlying mechanism of this phenomenon is not known. We investigated whether this effect of DDAVP might be due to activation of plasma membrane Na+/H+ exchanger. The DDAVP-induced platelet procoagulant response, measured as phospholipid-dependent thrombin generation, was dose dependent and significantly weaker than that produced by collagen or monensin (mimics Na+/H+ antiport). Both the DDAVP- and collagen-produced procoagulant responses were less pronounced in the presence of EIPA, an Na+/H+ exchanger inhibitor. Flow cytometry studies revealed that in vitro treatment of platelets with DDAVP or collagen was associated with the appearance of both degranulated (and fragmented) and swollen cells. The DDAVP-evoked rise in size and granularity heterogeneity was similar to that produced by collagen or monensin and was not observed in the presence of EIPA. Using flow cytometry and annexin V-FITC as a probe for phosphatidylserine (PS) we demonstrated increased and uniform binding of this marker to all subsets of DDAVP-treated platelet population. The DDAVP-evoked PS expression was dose dependent, strongly reduced by EIPA and weaker than that caused by monensin or collagen. As judged by optical swelling assay, DDAVP in a dose dependent manner produced a rise in platelet volume. The swelling was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Electronic cell-sizing measurements showed an increase in mean platelet volume and a decrease in platelet count and platelet crit upon treatment with DDAVP. DDAVP elicited a slow (much slower than collagen) alkalinization of platelet cytosol. Altogether the data indicate an involvement of Na+/H+ exchanger in the generation of procoagulant activity in DDAVP-treated platelets.

Shape and volume of living aldosterone-sensitive cells imaged with the atomic force microscope.

The steroid hormone aldosterone, which is synthesized in the suprarenal glands and secreted in response to a reduction in circulating blood volume, increases water and sodium reabsorption in the kidney (,). Although kidney is the major target organ, various other cell types, including different epithelia, smooth muscle, and endothelium, respond to the hormone (, , , ). The acute aldosterone-induced responses of target cells are an intracellular calcium change and an intracellular pH increase (,,) along with activation of plasma membrane Na+/H+ exchange and plasma membrane proton conductance (,). Therefore, it is not surprising that researchers have postulated that these acute transmembrane shifts of electrolytes are accompanied by cell swelling (,). Cell swelling or shrinkage plays a critical role in endothelial cell (EC) function. ECs tightly coat the luminal surface of blood vessels, playing an important role in the regulation of vascular tone, in vascular remodeling, in the pathogenesis of arteriosclerosis, and in arterial hypertension of humans (). It has been shown that swelling of EC may disturb cell-to-cell interactions, resulting in an increase of transendothelial permeability, a precursor mechanism in the development of arteriosclerosis (,). Moreover, environmental stress (e.g., mechanical forces or hyperosmolarity) induces changes in cell volume and stimulates tissue plasminogen activator synthesis (, , , ). Direct evidence for the importance of cell-volume regulation of endothelial cells is the existence of a volume-sensitive protein kinase ().

Functional coupling of Na+/H+ and Na+/Ca2+ exchangers in the alpha 1-adrenoreceptor-mediated activation of hepatic metabolism.

The purpose of this study was to characterize the role of ions other than Ca2+ in hepatic responses to alpha 1-adrenergic stimulation. We report that the alpha 1-adrenoreceptor activation of hepatic functions is accompanied by extracellular acidification and an increase in intracellular pH. These effects are dependent on extracellular Na+ concentration and are inhibited by the Na+/H+ antiporter blocker 5-(N-ethyl-N-isopropyl) amiloride under conditions that preclude antagonistic effects on agonist binding. Thus, the activation of plasma membrane Na+/H+ exchange is an essential feature of the hepatic alpha-adrenoreceptor-coupled signaling pathway. The following observations indicate that the sustained hepatic alpha 1-adrenergic actions rely on a functional coupling between the plasma membrane Na+/H+ and Na+/Ca2+ exchangers, resulting in the stimulation of Ca2+ influx. 1) Inhibition of the Na+/K(+)-ATPase does not prevent the alpha 1-adrenergic effects. However, alpha 1-adrenoreceptor stimulation fails to induce intracellular alkalinization and to acidify the extracellular medium in the absence of extracellular Ca2+. 2) A non-receptor-induced increase in intracellular Na+ concentration, caused by the ionophore monensin, stimulates Ca2+ influx and increases vascular resistance. 3) Inhibition of Na+/Ca2+ exchange prevents, in a concentration-dependent manner, most of the alpha 1-agonist-induced responses. 4) The actions of Ca(2+)-mobilizing vasoactive peptide receptors or alpha 2-adrenoreceptors, which produce neither sustained extracellular acidification nor release of Ca2+, are insensitive to Na+/H+ exchange blockers.

Isolation and characterization of a polarized isolated hepatocyte preparation in the skate Raja erinacea.

Hepatocytes of the small skate (Raja erinacea) were isolated by collagenase perfusion and evaluated by a variety of functional and morphologic criteria. Cell yield was 1.45 X 10(8) +/- 1.3 X 10(7) cells per isolation, and as long as 8 h after isolation 98% of the hepatocytes excluded Trypan blue and no leakage of lactate dehydrogenase (LDH) or cell associated potassium could be detected. Oxygen consumption averaged 1.6 +/- 0.5 nmol/min/mg cell protein, was not stimulated by 1 mM succinate, and also remained stable for up to 8 h following isolation. However, 2,4,-dinitrophenol (5 X 10(-5) M) produced a 55% increase in oxygen utilization while ouabain, (1 mM) or sodium removal decreased oxygen consumption by 31 +/- 6 and 33 +/- 7%, respectively, indicating that a significant portion of the cells energy utilization is coupled to the activity of plasma membrane Na+, K+-ATPase. Light microscopic studies showed that the individual hepatocytes had diameters of 28 +/- 5 microns and contained large lipid droplets. Electron microscopy revealed groups of three to five cells with normal ultrastructure and tight junctions and desmosomes surrounding a single bile canaliculus. These studies indicate that skate hepatocytes can be isolated in high yield that retain their structural polarity in the form of clusters of cells formed around a single bile canaliculus. These hepatocytes remain morphologically intact and metabolically stable for a prolonged period of time.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane anomalies in Huntington's disease fibroblasts.

Plasma membranes, microsomes, and mitochondria were isolated from paired, passage number matched, cultured human fibroblasts. The cells were obtained from skin biopsies of Huntington's disease (HD) subjects and from sex and age matched controls. All fibroblasts were cultured in identical media for three to seven passages. Enrichment of surface marker enzymes such as Na+,K+-ATPase indicated a 10-fold purification of the isolated plasma membrane. The specific activity of Na+,K+-ATPase was 62 and 82% greater in the crude homogenate and isolated plasma membrane, respectively, of HD fibroblasts than in control fibroblasts. The specific activity of plasma membrane Na+,K+-ATPase was correlated with lipid composition and with membrane structure as determined by measurement of the rotational relaxation time and limiting anisotropy of fluorescence probe molecules. Major alterations in the structure of the plasma membranes in HD fibroblasts were not noted. The rotational relaxation time and limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene and of trans-parinaric acid were not significantly different between the plasma membrane, microsomes, or mitochondria of HD versus those of control fibroblasts. trans-Parinaric acid demonstrated the coexistence of fluid and solid domains in all three subcellular membrane fractions of the normal and HD skin fibroblasts. Lastly, both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene displayed characteristic breakpoints in Arrhenius plots of absorbance corrected fluorescence in plasma membranes, microsomes, and mitochondria. In all cases, similar breakpoint temperatures, indicative of phase alterations, were noted near 20 degrees and 30 degrees C. These breakpoints were unaltered in HD. In summary, the data do not support the concept of major membrane structural defects in HD.

The effects of nitrogen mustard (HN2) on activities of the plasma membrane of PC6A mouse plasmacytoma cells

Nitrogen mustard, HN2 (10 −5 M), inhibited the transport of the potassium congener 86rubidium into PC6A mouse plasmacytoma cells by 45% after a 4 hr incubation at 37° in vitro. HN2 (10 −3 M) had a rapid effect on the profile of 86rubidium transport into PC6A cells when added simultaneously with the 86rubidium whereas a monofunctional analogue of HN2((2-chloroethyl)dimethylamine) had no effect at 10 −3 M. The transport of the amino acid analogues α-aminoisobutyric acid and cycloleucine into PC6A cells was inhibited by 19% and 5% respectively after a 4 hr incubation with 10 −5 M HN2. The results suggest that the activity of plasma membrane Na +K +-ATPase may be affected by HN2. This enzyme may play a pivotal role in controlling cell growth and division. Crude cell membrane preparations from PC6A cells had variable Na +K +-ATPase activity which was possibly due to contamination with mitochondrial Mg 2+-ATPase. Incubation of a crude cell membrane preparation in the presence of 40 nM dicyclohexylcarbodiimide gave constant Na +K +-ATPase activity which was inhibited by 44% on incubation with HN2 (10 −3 M) for 0.5 hr. The monofunctional analogue of HN2 inhibited this preparation by only 7% under the same conditions. It is suggested that inhibition of Na +K +-ATPase by HN2 may be an important facet of its cytotoxic activity.

Hormonal control in vitro of plasma membrane-bound [formula omitted] of rat liver

Epinephrine, glucagon and cyclic AMP reduced in vitro the activity of (Na +−K +)-ATPase of plasma membranes isolated from rat liver. Insulin did not significantly modify the activity, but partially counteracted the effect of both hormones. Propranolol, a β-blocking agent, prevented the action of epinephrine. These data suggest that epinephrine, glucagon and insulin may modulate the activity of plasma membrane Na +−K +)-ATPase , by an action probably mediated by cyclic AMP.