Extracellular NaCl Concentration Research Articles

Altered Neuronal Discharge in the Organum Vasculosum of the Lamina Terminalis Contributes to Dahl Salt-Sensitive Hypertension.

Salt-sensitive hypertension in humans and experimental models is associated with higher plasma and cerebrospinal fluid sodium chloride (NaCl) concentrations. Changes in extracellular NaCl concentrations are sensed by specialized neurons in the organum vasculosum of the lamina terminalis (OVLT). Stimulation of OVLT neurons increases sympathetic nerve activity (SNA) and arterial blood pressure (ABP), whereas chronic activation produces hypertension. Therefore, the present study tested whether OVLT neuronal activity was elevated and contributed to SNA and ABP in salt-sensitive hypertension. Male Dahl salt-sensitive (Dahl S) and Dahl salt-resistant (Dahl R) rats were fed 0.1% or 4.0% NaCl diets for 3 to 4 weeks and used for single-unit recordings of OVLT neurons or simultaneous recording of multiple sympathetic nerves during pharmacological inhibition of the OVLT. Plasma and cerebrospinal fluid Na+ and Cl- concentrations were higher in Dahl S rats fed 4% versus 0.1% or Dahl R rats fed either diet. In vivo single-unit recordings revealed a significantly higher discharge of NaCl-responsive OVLT neurons in Dahl S rats fed 4% versus 0.1% or Dahl R rats. Interestingly, intracarotid infusion of hypertonic NaCl evoked greater increases in OVLT neuronal discharge of Dahl S versus Dahl R rats regardless of NaCl diet. The activity of non-NaCl-responsive OVLT neurons was not different across strain or diets. Finally, inhibition of OVLT neurons by local injection of the gamma-aminobutyric acid agonist muscimol produced a greater decrease in renal SNA, splanchnic SNA, and ABP of Dahl S rats fed 4% versus 0.1% or Dahl R rats. A high salt diet activates NaCl-responsive OVLT neurons to increase SNA and ABP in salt-sensitive hypertension.

Post-Ganglionic Sympathetic Neurons can Directly Sense Raised Extracellular Na+ via SCN7a/Nax.

The relationship between dietary NaCl intake and high blood pressure is well-established, and occurs primarily through activation of the sympathetic nervous system. Nax, a Na+-sensitive Na+ channel, plays a pivotal role in driving sympathetic excitability, which is thought to originate from central regions controlling neural outflow. We investigated whether post-ganglionic sympathetic neurons from different ganglia innervating cardiac and vasculature tissue can also directly sense extracellular Na+. Using whole-cell patch clamp recordings we demonstrate that sympathetic neurons from three sympathetic ganglia (superior cervical, stellate and superior mesenteric/coeliac) respond to elevated extracellular NaCl concentration. In sympathetic stellate ganglia neurons, we established that the effect of NaCl was dose-dependent and independent of osmolarity, Cl− and membrane Ca2+ flux, and critically dependent on extracellular Na+ concentration. We show that Nax is expressed in sympathetic stellate ganglia neurons at a transcript and protein level using single-cell RNA-sequencing and immunohistochemistry respectively. Additionally, the response to NaCl was prevented by siRNA-mediated knockdown of Nax, but not by inhibition of other membrane Na+ pathways. Together, these results demonstrate that post-ganglionic sympathetic neurons are direct sensors of extracellular Na+ via Nax, which could contribute to sympathetic driven hypertension.

Integration of Hypernatremia and Angiotensin II by the Organum Vasculosum of the Lamina Terminalis Regulates Thirst.

The organum vasculosum of the lamina terminalis (OVLT) contains NaCl-sensitive neurons to regulate thirst, neuroendocrine function, and autonomic outflow. The OVLT also expresses the angiotensin II (AngII) type1 receptor, and AngII increases Fos expression in OVLT neurons. The present study tested whether individual OVLT neurons sensed both NaCl and AngII to regulate thirst and body fluid homeostasis. A multifaceted approach, including in vitro whole-cell patch recordings, in vivo single-unit recordings, and optogenetic manipulation of OVLT neurons, was used in adult, male Sprague Dawley rats. First, acute intravenous infusion of hypertonic NaCl or AngII produced anatomically distinct patterns of Fos-positive nuclei in the OVLT largely restricted to the dorsal cap versus vascular core, respectively. However, in vitro patch-clamp recordings indicate 66% (23 of 35) of OVLT neurons were excited by bath application of both hypertonic NaCl and AngII. Similarly, in vivo single-unit recordings revealed that 52% (23 of 44) of OVLT neurons displayed an increased discharge to intracarotid injection of both hypertonic NaCl and AngII. In marked contrast to Fos immunoreactivity, neuroanatomical mapping of Neurobiotin-filled cells from both in vitro and in vivo recordings revealed that NaCl- and AngII-responsive neurons were distributed throughout the OVLT. Next, optogenetic excitation of OVLT neurons stimulated thirst but not salt appetite. Conversely, optogenetic inhibition of OVLT neurons attenuated thirst stimulated by hypernatremia or elevated AngII but not hypovolemia. Collectively, these findings provide the first identification of individual OVLT neurons that respond to both elevated NaCl and AngII concentrations to regulate thirst and body fluid homeostasis.SIGNIFICANCE STATEMENT Body fluid homeostasis requires the integration of neurohumoral signals to coordinate behavior, neuroendocrine function, and autonomic function. Extracellular NaCl concentrations and the peptide hormone angiotensin II (AngII) are two major neurohumoral signals that regulate body fluid homeostasis. Herein, we present the first compelling evidence that individual neurons located in the organum vasculosum of the lamina terminalis detect both NaCl and AngII. Furthermore, optogenetic interrogations demonstrate that these neurons play a pivotal role in the regulation of thirst stimulated by NaCl and AngII. These novel observations lay the foundation for future investigations for how such inputs as well as others converge onto unique organum vasculosum of the lamina terminalis neurons to coordinate body fluid homeostasis and contribute to disorders of fluid balance.

Abstract 107: Neurons in the Organum Vasculosum of the Lamina Terminalis Sense NaCl and Angiotensin II to Regulate Sympathetic Nerve Activity and Arterial Blood Pressure

The organum vasculosum of the lamina terminalis (OVLT) contains specialized neurons that sense changes in extracellular NaCl concentrations to regulate sympathetic nerve activity (SNA) and arterial blood pressure (ABP). However, OVLT neurons also abundantly express angiotensin II (AngII)-type I receptors. Therefore, we performed a series of in vitro and in vivo studies in adult male Sprague-Dawley rats to determine whether OVLT neurons sense both NaCl and AngII to impact SNA and ABP. First, in vitro whole-cell recordings revealed the majority of OVLT neurons (66%, 23/35) showed an increased discharge frequency to both +7.5mM NaCl (1.1±0.2 to 2.2±0.3Hz, P<0.001) and 100pM AngII (1.3±0.3 to 2.2±0.3Hz, P<0.001). Second, in vivo single-unit recordings demonstrate the majority of OVLT neurons (61%, 11/18) displayed an increase in cell discharge to intracarotid injection of 0.5M NaCl (3.6±0.6 to 9.0±1.6Hz, P<0.01) and 200ng AngII (3.2±0.7 to 9.2±1.9Hz, P<0.01). Third, optogenetic inhibition of OVLT neurons (rAAV2-CaMKII-eNpHR3.0-mCherry, 5x10 12 particles/mL, 561nm, 10mW) attenuated thirst responses to IV infusion of 2M NaCl (1.25mL/30min; Laser OFF: 6.1±0.5mL vs ON: 2.0±0.7mL, P<0.05) and AngII (Laser OFF 7.2±0.7mL vs ON: 2.2±1.2mL; n=5 per group, P<0.05). Fourth, OVLT injection (20nL) of 0.5M NaCl or 10pmol AngII increased SNA (121±6% or 117±5%, n=4/group; P<0.05 respectively) and mean ABP (7±1 or 9±2mmHg, n=4/group; P<0.05 respectively). To determine whether chronic activation of OVLT neurons produces hypertension, rats received an OVLT injection of rAAV2-hSyn-HM3D(Gq)-mCherry (3x10 12 particles/mL, 50nL) and given access to 0.1% NaCl chow and 0.9% NaCl drinking solution. After 4 weeks, daily administration of clozapine-N-oxide to the drinking solution (3mg/kg/day) significantly increased 24-h fluid intake (baseline: 60±10mL vs Day 7: 178±29mL; n=6, P<0.01) and 24-h mean ABP (baseline: 97±2mmHg vs Day 7: 108±1 mmHg; n=6, P<0.05). Ganglionic blockade with hexamethonium (30mg/kg, ip) produced a greater fall in mean ABP at Day 7 (baseline: -38±6 mmHg vs Day 7: -53±5 mmHg, P<0.05). These findings suggest OVLT neurons sense both NaCl and AngII to regulate body fluid homeostasis and SNA to produce a sympathetically-mediated hypertension.

Abstract 083: Neurons in the Organum Vasculosum of the Lamina Terminalis Contribute to Salt-sensitive Hypertension

Excess dietary salt intake raises plasma and cerebrospinal fluid NaCl concentrations to elevate sympathetic nerve activity (SNA) and arterial blood pressure (ABP). Changes in extracellular NaCl concentrations are sensed by neurons in the organum vasculosum of the lamina terminalis (OVLT) - a circumventricular organ that lacks a complete blood-brain barrier. The purpose of the present study was to investigate the hypothesis that salt-sensitive hypertension was mediated, in part, by an elevated activity of OVLT neurons. Dahl-Salt-Sensitive or Sprague-Dawley rats (8-10 weeks) were fed 0.5% or 4.0% NaCl diets for 3-4 weeks. First, in vivo single-unit recordings demonstrate the discharge of OVLT neurons in Dahl-Salt-Sensitive rats was higher after a 4.0% versus 0.5% NaCl diet (4.1±0.4 Hz vs 1.9±0.3 Hz, n=6 per group, P<0.05). OVLT neuronal discharge of Sprague-Dawley rats was not different after a 4.0% or 0.5% NaCl diet (2.1±0.4 Hz vs 1.7±0.3 Hz, n=6-9 per group, P>0.5). In a second set of experiments, injection of hypertonic NaCl (1.0M NaCl, 20nL) into the OVLT produced significantly greater increases in lumbar SNA (131±6% vs 116±3%, n=4 per group, P<0.05) and mean ABP (14±2 vs 8±2 mmHg, n=4 per group, P<0.05) of Dahl-Salt-Sensitive rats fed 4.0% versus 0.5% NaCl respectively. Sprague-Dawley rats fed 4.0% versus 0.5% NaCl exhibited responses of smaller magnitude for both lumbar SNA (115±4 vs 108±3%, n=4 per group, P<0.05) and mean ABP (9±2 vs 6±2 mmHg, n=4 per group, P<0.05). Interestingly, the duration of the response was much longer in Dahl-Salt-Sensitive versus Sprague-Dawley rats (data not shown). Finally, inhibition of neuronal activity by injection of the GABA agonist muscimol (5mM, 20nL) into the OVLT produced a significantly greater fall in lumbar SNA (-25±4% vs -11±3%, n=4 per group, P<0.05) and mean ABP (-19±4 vs -6±2 mmHg, n=4 per group, P<0.05) of Dahl-Salt-Sensitive rats fed 4.0% versus 0.5% NaCl, respectively. Injection of muscimol into the OVLT of Sprague-Dawley rats did not significantly affect SNA or mean ABP. Collectively, these findings suggest a high salt diet increases the activity of OVLT neurons to elevate SNA and ABP in salt-sensitive hypertension.

Hypertonic NaCl versus osmotic stimuli: distinct OVLT neurones can sense the difference to control sympathetic outflow and blood pressure.

Despite large fluctuations in salt and water intake, mammals are able to maintain electrolyte concentrations, particularly sodium (Na+) as the major determinant of osmolality of the extracellular fluid (ECF), within narrow physiological limits (∼300 mOsm kg−1). The two distinct sensory systems monitoring Na+ concentration and osmolality in the hypothalamic area of the brain are located outside the blood–brain barrier and are therefore known as circumventricular organs (CVOs). The osmosensitive neurones of CVOs are located in the subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT). Direct projections from these areas to hypothalamic nuclei containing neurosecretory magnocellular neurones promote the release of water-conserving hormones such as arginine vasopressin (McKinley et al. 2001). Interestingly, these neurones also project to smaller parvocellular neurones located within other brain structures, such as the paraventricular nucleus of the hypothalamus (PVN) that modulate sympathetic nerve activity (SNA), an important mechanism in blood pressure (BP) homeostasis. The distinct physiological responses to maintain fluid balance triggered by infusion of hypertonic sodium chloride (NaCl) and equi-osmolar solutions have been debated for decades. McKinley and colleagues (1978) have shown that a peripheral osmoreceptor mechanism was most likely to be responsible for the thirst and antidiuresis induced by intracarotid infusion of hypertonic NaCl or sucrose in sheep. However, they also found support for a periventricular Na+ receptor mechanism because of differential drinking responses to intracerebroventricular (i.c.v.) administration of hypertonic NaCl or sucrose. The SFO seems to be the primary locus of NaCl sensing for the control of salt-intake behaviour at the central level, identified as a sensory mechanism that relies on activation of a Na+ channel (Nax); it is not osmosensing (Hyiama et al. 2004). The SFO is also involved in mediating the central Na+-induced pressor response, since administration of NaCl-rich artificial cerebrospinal fluid (aCSF) into the SFO caused an increase in BP, which was blunted by an angiotensin II type 1 (AT1) receptor antagonist at the same site. However, equi-osmotic infusion of manitol did not change BP, indicating that the pressor response is Na+ specific and not due to osmolality or volume effects (Tiruneh et al. 2013). Not only SFO, but also OVLT neurones are capable of detecting extracellular NaCl concentration and altering the sympathetic nerve discharge in order increase BP (Kinsman et al. 2017b). However, to date, no study has been able to show whether distinct cellular processes of osmosensitive neurones can differentiate between hyperosmolality versus hypertonic NaCl stimulus to selectively control SNA and BP. In this issue of The Journal of Physiology, Kinsman and co-workers (2017a) addressed this important issue by performing an elegant in vivo neurophysiological study. They determined how subsets of OVLT neurones respond differentially to hypertonic NaCl versus osmolality and subsequently regulate the sympathetic nervous system. More precisely, they showed that local injection of hypertonic saline into the OVLT produced an increase in SNA and BP, whereas an equi-osmotic stimulus of mannitol/sorbitol solutions had no effect. Moreover, these authors combined in vitro whole-cell recordings to demonstrate that stimulation with hypertonic saline caused the discharge frequency of OVLT neurones to increase, unlike stimulation with the equi-osmotic mannitol solution. These findings by Kinsman and colleagues (2017a) open several interesting perspectives for further investigation regarding the functional significance of distinct cellular responses of Na+ and osmosensitive OVLT neurones: (i) What are the physiological mechanisms, phenotype and neurotransmitters released by the functionally distinct OVLT neurones? (ii) How do these differences translate to hypertonic NaCl and osmotically driven sympathetic responses and BP control? (iii) Do salt-sensitive rats (e.g. Dahl-Salt) show alterations in OVLT neuronal populations that control hydroelectrolytic balance, sympathetic hyperactivity and hypertension in response to high salt diet? (iv) It would be interesting to determine the contribution of the SFO compared to the OVLT to the sympathetic responses to hypertonic saline and osmolality challenges. Understanding the characteristics of the CVO neurones relating to control of body fluid and cardiovascular homeostasis in response to distinct osmotic stimuli will require a range of techniques from anatomical, molecular, biochemical and pharmaco/opto-genetic tools, and this paper by Kinsman and co-workers (2017a) is a great kick-off in elucidating the complex hierarchy and mechanisms of the osmoregulatory signalling network at the CNS level. None declared. V.R.A. is supported by The National Council for Scientific and Technological Development (CNPq) Research Fellow, and The State of Sao Paulo Research Funding Agency (FAPESP) no. 2016-21991-3.

Organum Vasculosum of the Lamina Terminalis Detects NaCl to Elevate Sympathetic Nerve Activity and Blood Pressure.

High-salt diet elevates NaCl concentrations in the cerebrospinal fluid to increase sympathetic nerve activity (SNA) in salt-sensitive hypertension. The organum vasculosum of the lamina terminalis (OVLT) resides along the rostral wall of the third ventricle, lacks a complete blood-brain barrier, and plays a pivotal role in body fluid homeostasis. Therefore, the present study used a multifaceted approach to examine whether OVLT neurons of Sprague-Dawley rats are intrinsically sensitive to changes in extracellular NaCl concentrations and mediate the sympathoexcitatory responses to central NaCl loading. Using in vitro whole-cell recordings, step-wise increases in extracellular NaCl concentrations (2.5-10 mmol/L) produced concentration-dependent excitation of OVLT neurons. Additionally, these excitatory responses were intrinsic to OVLT neurons because hypertonic NaCl evoked inward currents, despite pharmacological synaptic blockade. In vivo single-unit recordings demonstrate that the majority of OVLT neurons (72%, 13/19) display concentration-dependent increases in neuronal discharge to intracarotid (50 μL/15 s) or intracerebroventricular infusion (5 μL/10 minutes) of hypertonic NaCl. Microinjection of hypertonic NaCl (30 nL/60 s) into the OVLT, but not adjacent areas, increased lumbar SNA, adrenal SNA, and arterial blood pressure in a concentration-dependent manner. Renal SNA decreased and splanchnic SNA remained unaffected. Finally, local inhibition of OVLT neurons with the GABAA receptor agonist muscimol (24 nL/10 s) significantly attenuated the sympathoexcitatory and pressor responses to intracerebroventricular infusion of 0.5 mol/L or 1.0 mol/L NaCl. Collectively, these findings indicate that OVLT neurons detect changes in extracellular NaCl concentrations to selectively alter SNA and raise arterial blood pressure.

Hypernatremia-induced vasopressin secretion is not altered in TRPV1-/- rats.

Changes in osmolality or extracellular NaCl concentrations are detected by specialized neurons in the hypothalamus to increase vasopressin (VP) and stimulate thirst. Recent in vitro evidence suggests this process is mediated by an NH2-terminal variant of the transient receptor potential vanilloid type 1 (TRPV1) channel expressed by osmosensitive neurons of the lamina terminalis and vasopressinergic neurons of the supraoptic nucleus. The present study tested this hypothesis in vivo by analysis of plasma VP levels during acute hypernatremia in awake control and TRPV1(-/-) rats. TRPV1(-/-) rats were produced by a Zinc-finger-nuclease 2-bp deletion in exon 13. Intravenous injection of the TRPV1 agonist capsaicin produced hypotension and bradycardia in control rats, but this response was absent in TRPV1(-/-) rats. Infusion of 2 M NaCl (1 ml/h iv) increased plasma osmolality, electrolytes, and VP levels in both control and TRPV1(-/-) rats. However, plasma VP levels did not differ between strains at any time. Furthermore, a linear regression between plasma VP versus osmolality revealed a significant correlation in both control and TRPV1(-/-) rats, but the slope of the regression lines was not attenuated in TRPV1(-/-) versus control rats. Hypotension produced by intravenous injection of minoxidil decreased blood pressure and increased plasma VP levels similarly in both groups. Finally, both treatments stimulated thirst; however, cumulative water intakes in response to hypernatremia or hypotension were not different between control and TRPV1(-/-) rats. These findings suggest that TRPV1 channels are not necessary for VP secretion and thirst stimulated by hypernatremia.

Regulation of hamster sperm hyperactivation by extracellular Na+

Mammalian sperm motility has to be hyperactivated to be fertilization-competent. Hyperactivation is regulated by extracellular environment. Osmolality of mammalian semen is higher than that in female reproductive tract; however, the effect of them on hyperactivation has not been investigated. So we investigated the effect of osmotic environment on hyperactivation using hamster spermatozoa at first. Increase in the osmolality of the media (∼370 mOsm) by increasing the concentration of NaCl (∼150 mmol/L) caused the delay of the expression of hyperactivation. When NaCl concentration varied in the same range (75-150 mmol/L) whereas the osmolality was fixed at 370 mOsm by adding mannitol, the delay of hyperactivation occurred dependent on NaCl concentration. Increase in NaCl concentration also caused suppression of curvilinear velocity, bend angle, and sliding velocity of the flagellum at the onset of incubation, suggesting that NaCl concentration affect both activation and hyperactivation in hamster spermatozoa. Hamster sperm intracellular Ca(2+) concentration decreased as extracellular NaCl concentration increased, whereas membrane potential and intracellular pH were unaffected by extracellular NaCl concentration. SN-6 and SEA0400, inhibitors of Na(+)-Ca(2+) exchanger (NCX), increased intracellular Ca(2+) and accelerated hyperactivation in the presence of 150 mmol/L NaCl. Tyrosine phosphorylation on fibrous sheath proteins was unaffected by extracellular NaCl concentration. These results suggest that extracellular Na(+) suppresses hamster sperm hyperactivation by reducing intracellular Ca(2+) via an action of NCX in a tyrosine phosphorylation-independent manner. It seems that the removal of suppression by extracellular Na(+) leads to the expression of hyperactivated motility.

Endogenous 5-HT outflow from chicken aorta by 5-HT uptake inhibitors and amphetamine derivatives

Chemoreceptor cells aggregating in clusters in the chicken thoracic aorta contain 5-hydroxytryptamine (5-HT) and have voltage-dependent ion channels and nicotinic acetylcholine receptors, which are characteristics typically associated with neurons. The aim of the present study was to investigate the effects of 5-HT uptake inhibitors, fluvoxamine, fluoxetine and clomipramine (CLM), and amphetamine derivatives, p-chloroamphetamine (PCA) and methamphetamine (MET), on endogenous 5-HT outflow from the isolated chick thoracic aorta in vitro. 5-HT was measured by using a HPLC system with electrochemical detection. The amphetamine derivatives and 5-HT uptake inhibitors caused concentration-dependent increases in endogenous 5-HT outflow. PCA was about ten times more effective in eliciting 5-HT outflow than MET. The 5-HT uptake inhibitors examined had similar potency for 5-HT outflow. PCA and CLM increased 5-HT outflow in a temperature-dependent manner. The outflow of 5-HT induced by PCA or 5-HT uptake inhibitors was independent of extracellular Ca2+ concentration. The 5-HT outflow induced by CLM, but not that by PCA, was dependent on the extracellular NaCl concentration. These results suggest that the 5-HT uptake system of 5-HT-containing chemoreceptor cells in the chicken thoracic aorta has characteristics similar to those of 5-HT-containing neurons in the mammalian central nervous system (CNS).

C-2005: Effect of extracellular concentration profile on the precision of the experimentally determined cell membrane permeabilities through osmotic shift tests using a microperfusion chamber

The transportation of water and cryoprotective agents across the cell membrane plays an important role in cell survival during freezing and thawing of cells. A microperfusion chamber was developed for studying cellular response to osmotic change. The cells are immobilized in the microperfusion chamber and exposed to the solution which is osmotic solution shift to hypertonic solution. The change of extracellular concentration has a certain impact on fitting cell membrane permeability parameters. The microperfusion chamber can be modeled through the mechanism of convection diffusion, and thus the Finite Element Analysis (FEA) was applied for the simulation of extracellular concentration. The validity of the concentration shift model was confirmed by comparing the cell-free experimental results used by the optical image processing with the results of simulation model. The CuCl 2 solution was used to do the cell-free experiment and NaCl solution was used to do the determined cell membrane permeabilities experiment, thus the dynamic viscosity and diffusion coefficient of the solution at the certain concentration difference were measured to ensure the accuracy of the stimulant results. The simulation results have shown that the T-pipe and Y-pipe make no difference on the concentration change, but the size of T-pipe has a significant impact on extracellular NaCl concentration. The equilibration time of the low concentration shift to high concentration is about 100s, and the simulation shows the equilibration time of concentration function affects the measurement of hydraulic conductivity. Thus using FEA to analysis the extracellular concentration profile of microperfusion chamber has a great prospect to service for the cryobiology research by improving the precision of the experimentally determined cell membrane permeabilities.

Salt Tolerance in Astragalus cicer Microsymbionts: The Role of Glycine Betaine in Osmoprotection

In this study, we have investigated intrinsic salt tolerance of Astragalus cicer microsymbionts (USDA3350, ACMP18) and the role of exogenous glycine betaine in osmoprotection in these bacteria. Salt stress was imposed by NaCl concentrations ranging from 0.5 to 2%. A. cicer mesorhizobia were capable of tolerating up to 2% sodium chloride with a population count that was inversely proportional to the salt content. When the extracellular concentration of NaCl was raised to 2%, the generation time of the UDSA3350 strain in the mid-exponential phase of growth was 3.9-times greater than that in the no-salt control medium, whereas the ACMP18 strain survived under the same conditions but did not multiply. Application of 1mM glycine betaine into the salt-stressed rhizobium cultures increased the number of culturable bacteria, pointing out that this molecule was involved in restoration of osmotic balance. The decline in A. cicer symbiont viability in the medium with sodium chloride and the osmoprotective role of glycine betaine for these bacteria was confirmed in the experiment using the live/dead Bac Light Bacterial Vibility Kit. Data presented in this study showed the presence of proU-like genes in the genomes of A. cicer rhizobia with high sequence similarity to the genes of the ProU-like system in Sinorhizobium meliloti and the proU operon of Escherichia coli.

NaCl Potentiates Human Fibrocyte Differentiation

Excessive NaCl intake is associated with a variety of fibrosing diseases such as renal and cardiac fibrosis. This association has been attributed to increased blood pressure as the result of high NaCl intake. However, studies in patients with high NaCl intake and fibrosis reveal a connection between NaCl intake and fibrosis that is independent of blood pressure. We find that increasing the extracellular concentration of NaCl to levels that may occur in human blood after high-salt intake can potentiate, in serum-free culture conditions, the differentiation of freshly-isolated human monocytes into fibroblast-like cells called fibrocytes. NaCl affects the monocytes directly during their adhesion. Potassium chloride and sodium nitrate also potentiate fibrocyte differentiation. The plasma protein Serum Amyloid P (SAP) inhibits fibrocyte differentiation. High levels of extracellular NaCl change the SAP Hill coefficient from 1.7 to 0.8, and cause a four-fold increase in the concentration of SAP needed to inhibit fibrocyte differentiation by 95%. Together, our data suggest that NaCl potentiates fibrocyte differentiation. NaCl-increased fibrocyte differentiation may thus contribute to NaCl-increased renal and cardiac fibrosis.

Rapid Response of the Yeast Plasma Membrane Proteome to Salt Stress

The plasma membrane separates the cell from the external environment and plays an important role in the stress response of the cell. In this study, we compared plasma membrane proteome modifications of yeast cells exposed to mild (0.4 m NaCl) or high (1 m NaCl) salt stress for 10, 30, or 90 min. Plasma membrane-enriched fractions were isolated, purified, and subjected to iTRAQ labeling for quantitative analysis. In total, 88-109 plasma membrane proteins were identified and quantified. The quantitative analysis revealed significant changes in the abundance of several plasma membrane proteins. Mild salt stress caused an increase in abundance of 12 plasma membrane proteins, including known salt-responsive proteins, as well as new targets. Interestingly, 20 plasma membrane proteins, including the P-type H(+)-ATPase Pma1, ABC transporters, glucose and amino acid transporters, t-SNAREs, and proteins involved in cell wall biogenesis showed a significant and rapid decrease in abundance in response to both 0.4 m and 1 m NaCl. We propose that rapid protein internalization occurs as a response to hyper-osmotic and/or ionic shock, which might affect plasma membrane morphology and ionic homeostasis. This rapid response might help the cell to survive until the transcriptional response takes place.

Relationship between extracellular osmolarity, NaCl concentration and cell volume in rat glioma cells

The cell volume, which controls numerous cellular functions, is theoretically linearly related with the inverse osmolarity. However, deviations from this law have often been observed. In order to clarify the origin of these deviations we electronically measured the mean cell volume of rat glioma cells under three different experimental conditions, namely: at different osmolarities and constant NaCl concentration; at different NaCl concentrations and constant osmolarity and at different osmolarities caused by changes in NaCl concentration. In each condition, the osmolarity was maintained constant or changed with NaCl or mannitol. We showed that the cell volume was dependent on both the extracellular osmolarity and the NaCl concentration. The relationship between cell volume, osmolarity and NaCl concentration could be described by a new equation that is the product of the Boyle-van't Hoff law and the Michaelis-Menten equation at a power of 4. Together, these results suggest that in hyponatriemia, the cell volume deviates from the Boyle-van't Hoff law because either the activity of aquaporin 1, expressed in glioma cells, is decreased or the reduced NaCl influx decreases the osmotically obliged influx of water.

Characterization of duplicated Dunaliella viridis SPT1 genes provides insights into early gene divergence after duplication

The sodium-dependent phosphate transporter gene from unicellular green algae Dunaliella viridis, DvSPT1, shares similarity with members of Pi transporter family. Sequencing analysis of D. viridis BAC clone containing the DvSPT1 gene revealed two inverted duplicated copies of this gene ( DvSPT1 and DvSPT1-2 respectively). The duplication covered most of both genes except for their 3′ downstream region. The duplicated genomic sequences exhibited 97.9% identity with a synonymous divergence of Ks = 0.0126 in the coding region. This data indicated very recent gene duplication in D. viridis genome, providing an excellent opportunity to investigate sequence and expression divergence of duplicated genes at an early stage. Scatted point mutations and length polymorphism of simple sequence repeats (SSRs) were predominant among the sequence divergence soon after gene duplication. Due to sequence divergence in the 5′ regulatory regions and a swap of the entire 3′ downstream regions (3′-UTR), DvSPT1 and DvSPT1–2 showed expression divergence in response to extra-cellular NaCl concentration changes. According to their expression patterns, the two diverged gene copies would provide better adaptation to a broader range of extra-cellular NaCl concentration. Furthermore, Southern blot analysis indicated that there might be a large phosphate transporter gene family in D. viridis.

The P-113 Fragment of Histatin 5 Requires a Specific Peptide Sequence for Intracellular Translocation inCandida albicans, Which Is Independent of Cell Wall Binding

The activity of histatin 5 (Hst 5) against Candida albicans is initiated through cell wall binding, followed by translocation and intracellular targeting. The C. albicans cell wall protein Ssa2 is involved in the transport of Hst 5 into cells as part of cell killing. P-113 (a 12-amino-acid candidacidal active fragment of Hst 5) and P-113Q2.10 (which is inactivated by a glutamine substitution of the Lys residues at positions 2 and 10) were compared for their levels of cell wall binding and intracellular translocation in Candida wild-type (wt) and ssa2Delta strains. Both P-113 and P-113Q2.10 bound to the walls of C. albicans wt and ssa2Delta cells, although the quantity of P-113Q2.10 in cell wall extracts was higher than that of P-113 in both strains. Increasing the extracellular NaCl concentration to 100 mM completely inhibited the cell wall association of both peptides, suggesting that these interactions are primarily ionic. The accumulation of P-113 in the cytosol of wt cells reached maximal levels within 15 min (0.26 microg/10(7) cells), while ssa2Delta mutant cells had maximal cytosolic levels of less than 0.2 microg/10(7) cells even after 30 min of incubation. Furthermore, P-113 but not P-113Q2.10 showed specific binding with a peptide array of C. albicans Ssa2p. P-113Q2.10 was not transported into the cytosol of either C. albicans wt or ssa2Delta cells, despite the high levels of cell wall binding, showing that the two cationic lysine residues at positions 2 and 10 in the P-113 peptide are important for transport into the cytosol and that binding and transport are independent functional events.

Mutation of the 4F2 heavy-chain carboxy terminus causes y+LAT2 light-chain dysfunction

Heteromeric amino acid transporters are composed of two subunits--a multipass membrane protein called the 'light chain'--and a single pass glycoprotein called the 'heavy chain'. The light chain contains the transport pore, while the heavy chain appears to be necessary for trafficking the light chain to the plasma membrane. In this study, the role of the 4F2hc heavy chain in the function of the y+ LAT2 light chain was investigated. Carboxy terminal truncations and site specific mutants of 4F2hc were co-expressed in Xenopus laevis oocytes with the y+ LAT2 light chain, and the oocytes were analysed for transport activity and surface expression. Truncations of the 4F2hc carboxy terminus ranging between 15 and 404 residues caused a complete loss of light chain function, although all heterodimers were expressed at the cell surface. This indicated that the 15 carboxy-terminal residues of 4F2hc are required for the transport function of the heterodimer. Mutation of the conserved residue leucine 523 to glutamine in the carboxy terminus reduced the Vmax of arginine and leucine uptake. The affinity of the transporter for both arginine and leucine remained unaltered, but the Km-value of Na+, being cotransported with leucine, increased about three-fold. The change of the Na+ Km caused a specific defect of leucine efflux, whereas uptake of leucine at high extracellular NaCl concentration was unaffected.

TMAO and other organic osmolytes in the muscles of amphipods (Crustacea) from shallow and deep water of Lake Baikal

Concentrations of trimethylamine oxide (TMAO) and other ‘compatible’ osmolytes were analyzed in the muscle tissue of Lake Baikal amphipods (Crustacea) in relation to water depth of the freshwater Lake Baikal. Using HPLC and mass spectrometry, glycerophosphoryl choline (GPC), betaine, S-methyl-cysteine, sarcosine, and taurine were detected for the first time in freshwater amphipods. These osmolytes were frequently found in the five species studied but mixtures were too complex to be quantified. The pattern of these osmolytes did not change with respect to water depth. The TMAO concentration, however, was significantly higher in the muscle tissue of amphipods living in deep water than of those living in shallow water, which supports the hypothesis that TMAO acts as a protective osmolyte at increased hydrostatic pressure. We propose that eurybathic amphipods, exposed to raised hydrostatic pressure in the extremely deep freshwater Lake Baikal, have elevated TMAO levels to counteract the adverse effect of high pressure on protein structure. The elevated intracellular osmotic pressure is balanced by upregulating the extracellular hemolymph NaCl concentration.

Proteomic Analysis of Cellular Response to Osmotic Stress in Thick Ascending Limb of Henle’s Loop (TALH) Cells

Epithelial cells of the thick ascending limb of Henle's loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins alpha-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.