Role Of Na Research Articles

Unravelling the strategies used by the wild tomato species Solanum pennellii to confront salt stress: From leaf anatomical adaptations to molecular responses

Despite the genus Solanum include wild species with a high level of tolerance to salinity, the adaptation processes used by these halotolerant species are still poorly understood. Here, the differential responses to salinity of cultivated tomato S. lycopersicum (Sl) and the salt tolerant species S. pennellii (Sp) were studied in order to advance in the understanding of the strategies used by the latter one to confront salt stress, and allow identifying key tolerance traits which could be further introduced in tomato. Several physiological traits, as well as the anatomical structure of leaf and the expression of genes involved in water and Na+ homeostasis, were analysed throughout the time period required by Sp to adapt to salinity (100mM NaCl for 7days). In this period, Sp was able to reduce water loss by regulating transpiration through reduced stomatal density and aperture. Other anatomical adaptations in Sp leaves, including larger and more turgid cells occupied by a giant vacuole, were associated to higher water and Na+ accumulation. Interestingly, a higher basal expression of PIP2;1 aquaporin was also found in leaf of Sp compared to Sl, which could be one of the causes of its better hydric status under salinity. With respect to the genes involved in Na+ homeostasis, our results suggest that SpHKT1;2 in combination with SpSOS1 play an important role in Na+-translocation from root to shoot, and therefore, in the determination of the includer behaviour in the wild species, which is in concordance with the higher transcript levels of Na+ vacuolar transporters SpNHX3 and SpNHX4 in Sp leaves. Overall, these results highlight the necessity of a coordinated regulation of anatomical, physiological and molecular mechanisms in the salt tolerance of the wild species Sp.

Intestinal response to salinity challenge in the Senegalese sole (Solea senegalensis)

Fish are continuously forced to actively absorb or expel water and ions through epithelia. Most studies have focused on the gill due to its role in Na+ and Cl− trafficking. However, comparatively few studies have focused on the changing function of the intestine in response to external salinity. Therefore, the present study investigated the main intestinal changes of long-term acclimation of the Senegalese sole (Solea senegalensis) to 5, 15, 38 and 55ppt. Through the measurement of short-circuit current (Isc) in Ussing chambers and biochemical approaches, we described a clear anterior/posterior functional regionalization of the intestine in response to salinity. The use of specific inhibitors in Ussing chamber experiments, revealed that the bumetanide-sensitive Na+/K+/Cl− co-transporters are the main effectors of Cl− uptake in both anterior intestine and rectum. Additionally, the use of the anion exchanger specific inhibitor, DIDS, showed a salinity/region dependency of anion exchanger function. Moreover, we also described ouabain-sensitive Na+/K+-ATPase (NKA) and Bafilomycin A1-sensitive H+-ATPase activities (HA), which displayed changes related to salinity and intestinal region. However, the most striking result of the present study is the description of an omeprazole-sensitive H+/K+-ATPase (HKA) in the rectum of Senegalese sole. Its activity was consistently measurable and increased at lower salinities, reaching rates even higher than those of the NKA. Together our results provide new insights into the changing role of the intestine in response to external salinity in teleost fish. The rectal activity of HKA offers an alternative/cooperative mechanism with the HA in the final processing of intestinal water absorption by apical titration of secreted bicarbonate.

Down-regulation of Na+/H+ exchanger 1 by Toll-like receptor stimulation in macrophages

The role of Na+/H+ exchanger 1 (NHE1) in various cell types, including inflammatory cells, has been extensively studied. However, regulation of NHE1 protein level in activated inflammatory cells is yet to be characterized. In this study, we investigated whether Toll-like receptor (TLR) ligands can regulate NHE1 protein level in the mouse macrophage-like RAW 264 cell line. We found that lipopolysaccharide (LPS), a TLR4 ligand, lowered NHE1 level and activity in RAW 264 cells and in primary murine macrophages. Other TLR ligands, such as zymosan A and poly(I:C), also displayed reduced NHE1 level. LPS promoted NHE1 ubiquitination and reduced the expression of calcineurin homologous protein 1 (CHP1), a regulator of NHE1 activity and stability. These responses were inhibited by c-Jun N-terminal kinase (JNK) inhibitor SP600125 and dexamethasone. A proteasome inhibitor, but not caspase-3 or lysosomal inhibitors, blocked the LPS-induced NHE1 down-regulation. These results suggested that LPS promotes the degranulation of NHE1 mediated by the ubiquitin-proteasome system and CHP1 downregulation resulting from activation of JNK.

Kinetic and thermodynamic analysis (with statistical optimization) of hydrogen production from crude glycerol using Clostridium pasteurianum

This study presents kinetic and thermodynamic analysis of crude glycerol fermentation for hydrogen production. Fermentation parameters have been optimized using central composite design (CCD) of experiments as: pH = 6.7, temperature = 36 °C, crude glycerol concentration = 7.4 g/L. Kinetic and thermodynamic analysis has been done using Haldane substrate inhibition model, Arrhenius plots and Eyring equation. Kinetic parameters Vmax and Km increase, while KI reduces for crude glycerol, as compared to pure glycerol. Moreover, activation energy (217 kJ/mol) and ΔG (103 kJ/mol) for crude glycerol are lesser than pure glycerol. Consequently, hydrogen yield in crude glycerol fermentation (0.627 mol H2/mol glycerol with 44% v/v H2 in product gas) is significantly higher than pure glycerol. Role of Na+ ions in improving crude glycerol fermentation has been experimentally confirmed. Kinetic and thermodynamic parameters for crude glycerol fermentation determined in this study have quantitatively substantiated its potential for biohydrogen production.

Association Between Polymorphisms of ADRBK1 Gene and Plasma Renin Activity in Hypertensive Patients: A Case-Control Study

BackgroundRenin is the first step of the RAS cascade, which is a major regulator of salt-volume homeostasis. Adrenergic beta receptor kinase 1 (ADRBK1) plays important roles in regulating blood pressure via the epithelial Na+ channel (ENaC), which plays an important role in Na+ reabsorption in the renal collecting duct. The present case-control study was designed to investigate the potential relationship between polymorphisms of ADRBK1 and plasma renin activity (PRA) in hypertension.Material/MethodsWe recruited 1831 hypertensive and 422 normotensive Han Chinese subjects. Sitting PRA (ng/mL/h) was measured using radioimmunoassay method. Hypertensive patients were classified into 4 renin categories via PRA quartile. Single-nucleotide polymorphisms (SNPs) of the ADRBK1 gene (rs1894111, rs4930416, rs7127431, rs12286664, and rs3730147) were identified via TaqMan polymerase chain reaction.ResultsComparison of the hypertensive group and the control group showed significant differences in distribution of genotypes and alleles of rs1894111 (P<0.05). Moreover, distribution of the dominant model (CC vs. CT+TT) in rs1894111 was lower in the hypertensive group than in the control group (P<0.05). Subjects were classified into 4 subgroups based on PRA quartile; the dominant model (CC vs. CT+TT) of rs1894111 was significantly lower in the quartile 1 group (the group with the lowest PRA) than in the control group (P<0.05). Logistic regression analysis demonstrated that the dominant model (CC vs. CT+TT) of rs1894111 was significantly different in the hypertensive group (OR=1.590, 95%CI=1.022–2.474, P<0.05), particularly in the quartile 1 group (OR=1.845, 95%CI=1.119–3.042, P<0.05), but not in the quartile 4 group.ConclusionsThe dominant model (CC vs. CT+TT) of rs1894111 polymorphism in the ADRBK1 gene might be associated with low-renin hypertension in Han Chinese.

Differential roles of caveolin-1 in ouabain-induced Na+/K+-ATPase cardiac signaling and contractility.

Binding of ouabain to cardiac Na+/K+-ATPase initiates cell signaling and causes contractility in cardiomyocytes. It is widely accepted that caveolins, structural proteins of caveolae, have been implicated in signal transduction. It is known that caveolae play a role in Na+/K+-ATPase functions. Regulation of caveolin-1 in ouabain-mediated cardiac signaling and contractility has never been reported. The aim of this study is to compare ouabain-induced cardiac signaling and contractility in wild-type (WT) and caveolin-1 knockout (cav-1 KO) mice. In contrast with WT cardiomyocytes, ouabain-induced signaling e.g., activation of phosphoinositide 3-kinase-α/Akt and extracellular signal-regulated kinases (ERK)1/2, and hypertrophic growth were significantly reduced in cav-1 KO cardiomyocytes. Interactions of the Na+/K+-ATPase α1-subunit with caveolin-3 and the Na+/K+-ATPase α1-subunit with PI3K-α were also decreased in cav-1 KO cardiomyocytes. The results from cav-1 KO mouse embryonic fibroblasts also proved that cav-1 significantly attenuated ouabain-induced ERK1/2 activation without alteration in protein and cholesterol distribution in caveolae/lipid rafts. Intriguingly, the effect of ouabain induced positive inotropy in vivo (via transient infusion of ouabain, 0.48 nmol/g body wt) was not attenuated in cav-1 KO mice. Furthermore, ouabain (1-100 μM) induced dose-dependent contractility in isolated working hearts from WT and cav-1 KO mice. The effects of ouabain on contractility between WT and cav-1 KO mice were not significantly different. These results demonstrated differential roles of cav-1 in the regulation of ouabain signaling and contractility. Signaling by ouabain, in contrast to contractility, may be a redundant property of Na+/K+-ATPase.

Monomeric adiponectin modulates nitric oxide release and calcium movements in porcine aortic endothelial cells in normal/high glucose conditions

AimsPerivascular adipose tissue can be involved in the process of cardiovascular pathology through the release of adipokines, namely adiponectins. Monomeric adiponectin has been shown to increase coronary blood flow in anesthetized pigs through increased nitric oxide (NO) release and the involvement of adiponectin receptor 1 (AdipoR1). The present study was therefore planned to examine the effects of monomeric adiponectin on NO release and Ca2+ transients in porcine aortic endothelial cells (PAEs) in normal/high glucose conditions and the related mechanisms. Main methodsPAEs were treated with monomeric adiponectin alone or in the presence of intracellular kinases blocker, AdipoR1 and Ca2+-ATPase pump inhibitors. The role of Na+/Ca2+ exchanger was examined in experiments performed in zero Na+ medium. NO release and intracellular Ca2+ were measured through specific probes. Key findingsIn PAE cultured in normal glucose conditions, monomeric adiponectin elevated NO production and [Ca2+]c. Similar effects were observed in high glucose conditions, although the response was lower and not transient. The Ca2+ mobilized by monomeric adiponectin originated from an intracellular pool thapsigargin- and ATP-sensitive and from the extracellular space. Moreover, the effects of monomeric adiponectin were prevented by kinase blockers and AdipoR1 inhibitor. Finally, in normal glucose condition, a role for Na+/Ca2+ exchanger and Ca2+-ATPase pump in restoring Ca2+ was found. SignificanceOur results add new information about the control of endothelial function elicited by monomeric adiponectin, which would be achieved by modulation of NO release and Ca2+ transients. A signalling related to Akt, ERK1/2 and p38MAPK downstream AdipoR1 would be involved.

Structure and properties of Na2O-TiO2-SiO2 glasses: Role of Na and Ti on modifying the silica network

Changes in the structure and properties of xNa2O-yTiO2-(1-x-y)SiO2 glasses have been studied with regards to composition through Raman spectroscopy, Brillouin light scattering, and dilatometry. Twelve glasses with compositions x=10, 15, 20, and 25, and y=4, 7, and 10 were prepared through a traditional melt quench method. Na2O was found to increase the density, refractive index, coefficient of thermal expansion (CTE), bulk modulus, and Poisson's ratio of glasses, and to decrease Young's modulus and shear modulus. Density, refractive index, Young's modulus, shear modulus, bulk modulus and Poisson's ratio increase with the increase of TiO2 content. CTE was found to increase with the initial addition of 4mol% TiO2 and to remain constant with further additions, regardless of the Na2O content. Changes in the Raman spectra with composition have been discussed and related to the changes in resulting properties.

Glioma-mediated microglial activation promotes glioma proliferation and migration: roles of Na+/H+ exchanger isoform 1.

Microglia play important roles in extracellular matrix remodeling, tumor invasion, angiogenesis, and suppression of adaptive immunity in glioma. Na(+)/H(+) exchanger isoform 1 (NHE1) regulates microglial activation and migration. However, little is known about the roles of NHE1 in intratumoral microglial activation and microglia-glioma interactions. Our study revealed up-regulation of NHE1 protein expression in both glioma cells and tumor-associated Iba1(+) microglia in glioma xenografts and glioblastoma multiforme microarrays. Moreover, we observed positive correlation of NHE1 expression with Iba1 intensity in microglia/macrophages. Glioma cells, via conditioned medium or non-contact glioma-microglia co-cultures, concurrently upregulated microglial expression of NHE1 protein and other microglial activation markers (iNOS, arginase-1, TGF-β, IL-6, IL-10 and the matrix metalloproteinases MT1-MMP and MMP9). Interestingly, glioma-stimulated microglia reciprocally enhanced glioma proliferation and migration. Most importantly, inhibition of microglial NHE1 activity via small interfering RNA (siRNA) knockdown or the potent NHE1-specific inhibitor HOE642 significantly attenuated microglial activation and abolished microglia-stimulated glioma migration and proliferation. Taken together, our findings provide the first evidence that NHE1 function plays an important role in glioma-microglia interactions, enhancing glioma proliferation and invasion by stimulating microglial release of soluble factors. NHE1 upregulation is a novel marker of the glioma-associated microglial activation phenotype. Inhibition of NHE1 represents a novel glioma therapeutic strategy by targeting tumor-induced microglial activation.

COMMD10: A Novel Regulator Of ENaC Trafficking?

The Epithelial Sodium channel (ENaC) is a heterotrimeric (consisting of one α, one β and one γ subunit) Na+ channel and is located at the apical membrane of polarised epithelia, such as the distal kidney, lung and colon, where it has an important role in Na+ reabsorption for maintenance of total body Na+, blood pressure and blood volume. ENaC's trafficking to and from the plasma membrane is a highly regulated but not completely understood process. One protein that has been identified as playing a role in ENaC trafficking is COMMD1, a member of the COMMD (Copper Metabolism Murr1 domain containing) family of proteins. The other family members however have not been as extensively studied, including COMMD10, the focus of our study. Preliminary data has suggested an interaction between COMMD10 and Arf1 (a small GTP binding protein with various functions throughout the trafficking pathway) which suggests a role for COMMD10 in protein trafficking. Our work aims to investigate whether COMMD10 is a novel regulator of the protein trafficking pathway, and thus, whether it is involved in ENaC trafficking to and/or from the plasma membrane. A stable COMMD10 knockdown reduces amiloride‐sensitive short circuit current in αβγ‐ENaC transfected FRT epithelia. A COMMD10 knockdown reduces protein expression of some proteins (Vps35 and COMMD1) involved in the retromer complex (a complex implicated in the recycling of ENaC back to the plasma membrane). These results suggest that when COMMD10 is knocked down ENaC trafficking is disrupted. We have also identified that COMMD10 and Arf1, both in its wild‐type form as well as constitutively active and constitutively inactive forms, co‐localise. However, no co‐localisation was observed between COMMD10 and other Arf family members (Arf4 and Arf5) suggesting an Arf1 specific function for COMMD10. Using a cell‐line in which COMMD10 is stabily knocked down we have identified a change in localisation of the active form of Arf1, that when taken with the other results suggests a role for COMMD10 in the protein trafficking pathway.Support or Funding InformationUniversity of Otago PhD Scholarship

Hydronephrosis and Urinary Bladder Hypertrophy in ROMK Bartter's Mouse

The renal outer medullary potassium channel (ROMK; Kir1.1) plays an important role in Na+ and K+ homeostasis. ROMK knockout mice (KO) show a similar phenotype to Bartter's syndrome of salt wasting and dehydration due to reduced Na‐2Cl‐K‐cotransporter activity. ROMK KO mice also show hydronephrosis; however, the mechanism of this phenotype has not been understood. We investigated ROMK expression and regulation by high and low K intake in mouse bladder, by Western blot and immunofluorescence (IF) staining. We also compared the bladder weight, bladder capacity, and thickness of urothelium and smooth muscle layer between WT and ROMK KO mice. Western blotting detected ROMK expression at 45~48 kDA, and IF staining showed that the luminal membrane of umbrella cells were strongly stained in WT compared to KO mice. The expression of ROMK in the luminal membrane disappeared and signal intensity of the detrusor muscle was decreased by low potassium diet (LK) in WT mice. In contrast, there was no significant change in ROMK intensity, but the thickness of urothelium was increased after mice were treated with HK diet. The thickness of urothelium was 168.1±25.8 μm in normal K (NK) and was 300.7±83.6 μm (p&lt;0.001) in the HK treated group compared with NK in WT mice at the same age. The bladder weight, bladder capacity and the thickness of urothelium in ROMK KO showed average increases 2~4 fold greater than WT at both 2 months and 12 months of age. The thickness of the urothelium was 648.8±33.2 μm vs 302.7±16.5 μm (p&lt;0.001) and the detrusor muscle 1940.7±98.9 μm vs 1308.2±102.1 μm (p=0.013) respectively in 12‐month ROMK KO mice. In conclusion, ROMK expressed in the urinary bladder can be regulated by K+ intake; significant enlargement and hypertrophy of the bladder may contribute to hydronephrosis in ROMK KO mice.Support or Funding InformationNIH RO1 DK099284 and PO1 DK 17433

Zinc Deficiency Stimulates Sodium Chloride Cotransporter (NCC) Upregulation via Modulation of Calcineurin Activity in Mouse Distal Convoluted Tubular Cells

BackgroundZn2+ deficiency (ZnD) has been implicated in the development of hypertension. However, the mechanisms continue to be defined. The Sodium Chloride Cotransporter (NCC) plays a critical role in Na+ handling by the distal convoluted tubule and subsequent blood pressure homeostasis. Furthermore, experimental studies support a direct role for the Zn2+‐dependent enzyme calcineurin in NCC regulation.HypothesisThe objective of this study is to investigate the effects of ZnD on NCC. We hypothesize that Zn2+ regulates NCC via modulation of calcineurin activity.MethodsTo test this hypothesis, mouse distal convoluted tubular (mDCT) cells were exposed to the Zn2+ chelator N,N,N′,N′‐Tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN) or vehicle (DMSO) for 24 hours. To investigate the role of calcineurin in NCC regulation, mDCT cells were treated with the calcineurin inhibitor tacrolimus for 24 hours. NCC expression (qRT‐PCR, Western blot and immunocytochemistry) and activity (thiazide‐sensitive Na+ uptake) were examined as well as calcineurin activity (phosphatase assay).ResultsThe data show that NCC expression is upregulated in mDCT cells following intracellular Zn2+‐depletion with TPEN compared to vehicle‐treated cells. Additionally, calcineurin phosphatase activity is blocked in Zn2+ depleted mDCT cells. Consistent with TPEN findings, inhibition of calcineurin with tacrolimus stimulates NCC expression and activity.ConclusionsTaken together, data suggest that ZnD induces NCC upregulation by inhibiting calcineurin activity.SignificanceThese novel findings identify NCC as a possible mediator of ZnD‐induced hypertension. Understanding the mechanisms by which ZnD contributes to blood pressure dysregulation may have an important impact on the treatment of hypertension.Support or Funding InformationT32‐DK00756 (CRW), R25‐DK101390 (MSL), K08‐DK081728 (BK), VA‐MERIT (JLG), R01‐DK085097 (RSH)

134 Essential Role of Na+/CA2+ Exchanger 1 in the Mechanosensitivity of Human Esophageal Motor Neurons

134 Essential Role of Na+/CA2+ Exchanger 1 in the Mechanosensitivity of Human Esophageal Motor Neurons

Distinguishing subgroups among μ-opioid receptor agonists using Na+,K+-ATPase as an effector mechanism

We evaluated the effects of intracerebroventricular administration of ouabain on the antinociception induced by five μ-opioid receptor agonists in a tail flick test on female CD-1 mice and the effects of these μ-opioid receptor agonists on mice forebrain synaptosomal ouabain-sensitive Na+,K+-ATPase activity. The subcutaneous administration of the μ-opioid receptor agonists tested produced a dose-dependent antinociceptive effect. The antinociception induced by morphine (1–32mg/kg), levorphanol (0.4–6.4mg/kg), and buprenorphine (0.02–0.64mg/kg) was antagonised in a dose-dependent manner by ouabain (0.001–10 ng, i.c.v.), whilst the antinociception produced by fentanyl (0.02–0.16mg/kg) and methadone (2–10mg/kg) was not influenced significantly by ouabain (1–100 ng, i.c.v.). Incubation in vitro of forebrain synaptosomes with morphine (10−9–10−4 M), levorphanol (10−10–10−4 M), buprenorphine (10−10–10−5 M), or fentanyl (10−10–10−5 M) stimulated significantly ouabain-sensitive Na+,K+-ATPase activity in a concentration-dependent way. The order of efficacy (using the Emax as a measure of intrinsic efficacy) was: morphine (29.83±0.56%)>levorphanol (18.61±1.26%)>buprenorphine (14.91±0.74%)>fentanyl (10.10±1.73%). On the other hand, methadone (10−10–10−5 M) did not significantly modify the ouabain-sensitive Na+,K+-ATPase activity (Emax=5.11±0.92%). These results suggest that Na+,K+-ATPase activity is involved in the antinociceptive effects of morphine, levorphanol and buprenorphine, but not in that produced by fentanyl and methadone. Thus, we can conclude that at least two subgroups can be distinguished among the μ-opioid receptor agonists taking into consideration the role of Na+,K+-ATPase in their antinociceptive effects.

Effect of low potassium concentration on cadmium induced epileptiform activity of leech retzius neurons

Epilepsies have a large significance and require detailed investigation of cellular mechanisms that lead to this disorder. Environmental, especially industrial, toxins are having increasingly more prominent role in these investigations. The aim of our research was to investigate the significance of Cd2+ in generation of epileptiform electrical activity of neurons, and the role of Na+/K+ pump in mechanisms that lead to cessation of this activity. Experiments were performed on Retzius nerve cells of the leech Haemopis sanguisuga. Intracellularly placed microelectrodes were used to measure membrane potential changes upon administration of Cd2+ (100 µmol/l), and the same concentration of Cd2+ in low K+ (1 mmol/l) solution. In our experiments Cd2+ led to generation of rhythmic repetitive oscillatory activity. This activity closely resembles paroxysmal depolarizing shifts (PDS) which represent the cellular basis of epilepsy. Cd2+ induced epileptiform activity had the following characteristics: frequency of 3.9±0.8 PDS/minute, PDS duration of 4.0±0.3 s, and PDS amplitude of 8.1±0.7 mV. Cd2+ induces effects similar to those of Ni2+ and Co2+, but in 30 times smaller concentration. Application of Cd2+ in low K+ solution led to a significant reduction of PDS frequency (by 2.34±0.55 PDS/minute, p<0.05, Student's t-test), highly significant increase in PDS duration (by 2.84±0.23 s, p<0.01, Student's t-test) and highly significant reduction in PDS amplitude (by 1.91±0.33 mV, p=0.01, Student's t-test). Our results show that Cd2+ is a potent initiator of epileptiform activity, and that Na+/K+ pump significantly affects this activity and has a potentially important role in mechanisms that lead to its cessation.

Role of Na+ and Ca2+ currents in computational model of in-vitro sigh generation

Eupneic breathing in mammals is periodically interrupted by spontaneous augmented breaths (sighs) that are characterized by a biphasic larger-amplitude inspiratory burst followed by post-sigh apnea. Previous in vitro studies in newborn rodents have demonstrated that the respiratory oscillator of the pre-Botzinger complex (preBotC) can generate the distinct inspiratory-related motor patterns for both eupnea- and sigh-like activity [1,2]. However it remains debated whether these two types of inspiratory activities are produced by the same neuronal population or by distinct sub-networks. Based on recent in vitro data obtained in the mouse embryo [3], we have built a computational model consisting of two compartments, one dedicated to sigh generation and the other generating eupneic bursts, interconnected through appropriate synapses (Figure ​(Figure11 A). Figure 1 Sigh and eupnea activity pattern in silico. (A) Diagrams of sigh (left) and eupnea (right) network models. Except for the ER capacity, INaP and Ih, the sigh and eupnea models are identical. (B) Intracellular Ca2+ (top) and voltage (bottom) obtained for ... The model reproduces basic features of simultaneous sigh and eupnea generation: two types of bursts differing in terms of shape, amplitude and frequency of occurrence and mimics the effect of glycinergic synapses blockade. We designed a two-compartment computational model for sigh and eupnea subpopulations of neurons with several different parameters reflecting distinct burst generating mechanisms. The sigh subpopulation generates a low frequency rhythm based on slow intracellular Ca2+ oscillations and the eupnea subnetwork generates fast oscillations mainly driven by activation/inactivation of the persistent Na+ current (Fig 1 B,C).Furthermore, we used this model to make predictions that were subsequently tested on the isolated preBotC in brainstem slice preparations. Through a combination of our in vitro and in silico approaches we found that 1), sigh events are less sensitive to network excitability than eupneic activity, 2)The combination of voltage-gated calcium current and persistent sodium current control the sigh period of, and 3), specific parameters of Ih activation set the low sensitivity to excitability in the sigh neuronal subset. Altogether, our results strongly support the hypothesis that distinct subpopulations within the preBotC network are responsible for sigh and eupnea rhythmogenesis.

Acidic pH and short-chain fatty acids activate Na+ transport but differentially modulate expression of Na+/H+ exchanger isoforms 1, 2, and 3 in omasal epithelium

Low sodium content in feed and large amounts of salivary sodium secretion are essential requirements to efficient sodium reabsorption in the dairy cow. It is already known that Na+/H+ exchange (NHE) of the ruminal epithelium plays a key role in Na+ absorption, and its function is influenced by the presence of short-chain fatty acids (SCFA) and mucosal pH. By contrast, the functional role and regulation of NHE in omasal epithelium have not been completely understood. In the present study, we used model studies in small ruminants (sheep and goats) to investigate NHE-mediated Na+ transport and the effects of pH and SCFA on NHE activity in omasal epithelium and on the expression of NHE isoform in omasal epithelial cells. Conventional Ussing chamber technique, primary cell culture, quantitative PCR, and Western blot were used. In native omasal epithelium of sheep, the Na+ transport was electroneutral, and it was inhibited by the specific NHE3 inhibitor 3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride, which decreased mucosal-to-serosal, serosal-to-mucosal, and net flux rates of Na+ by 80% each. The application of low mucosal pH (6.4 or 5.8) in the presence of SCFA activated the Na+ transport across omasal epithelium of sheep compared with that at pH 7.4. In cultured omasal epithelial cells of goats, mRNA and protein of NHE1, NHE2, and NHE3 were detected. The application of SCFA increased NHE1 mRNA and protein expression, which was most prominent when the culture medium pH decreased from 7.4 to 6.8. At variance, the mRNA and protein expression of NHE2 and NHE3 were decreased with low pH and SCFA, which was contrary to the published data from ruminal epithelial studies. In conclusion, this paper shows that (1) NHE1, NHE2, and NHE3 are expressed in omasal epithelium; (2) NHE3 mediates the major portion of transepithelial Na+ transport in omasal epithelium; and (3) SCFA and acidic pH acutely activate Na+ transport but suppress the expression of NHE2 and NHE3 in the longer term. By contrast, the expression of NHE1 is increased by SCFA and acidic pH, indicating a prominent role for NHE1 in the regulation of intracellular pH of omasal epithelium. Our results suggest a regulatable Na+ absorption in ruminal and omasal epithelium. It is of benefit for intracellular pH homeostasis and highly relevant to dairy cows fed on high-concentrate diets.

Role of the Na+/H+exchanger 3 in angiotensin II-induced hypertension in NHE3-deficient mice with transgenic rescue of NHE3 in small intestines

The role of Na+/H+ exchanger 3 (NHE3) in the kidney in angiotensin II (ANG II)-induced hypertension remains unknown. The present study used global NHE3-deficient mice with transgenic rescue of the Nhe3 gene in small intestines (tgNhe3−/−) to test the hypothesis that genetic deletion of NHE3 selectively in the kidney attenuates ANG II-induced hypertension. Six groups of wild-type (tgNhe3+/+) and tgNhe3−/− mice were infused with either vehicle or ANG II (1.5 mg/kg/day, i.p., 2 weeks, or 10 nmol/min, i.v., 30 min), treated with or without losartan (20 mg/kg/day, p.o.) for 2 weeks. Basal systolic blood pressure (SBP) and mean intra-arterial blood pressure (MAP) were significantly lower in tgNhe3−/− mice (P < 0.01). Basal glomerular filtration rate, 24 h urine excretion, urinary Na+ excretion, urinary K+ excretion, and urinary Cl− excretion were significantly lower in tgNhe3−/− mice (P < 0.01). These responses were associated with significantly elevated plasma ANG II and aldosterone levels, and marked upregulation in aquaporin 1, the Na+/HCO3 cotransporter, the α1 subunit isoform of Na+/K+-ATPase, protein kinase Cα, MAP kinases ERK1/2, and glycogen synthase kinase 3 α/β in the renal cortex of tgNhe3−/− mice (P < 0.01). ANG II infusion markedly increased SBP and MAP and renal cortical transporter and signaling proteins in tgNhe3+/+, as expected, but all of these responses to ANG II were attenuated in tgNhe3−/− mice (P < 0.01). These results suggest that NHE3 in the kidney is necessary for maintaining normal blood pressure and fully developing ANG II-dependent hypertension.

TMIC-06GLIOMA-MEDIATED MICROGLIAL ACTIVATION PROMOTES GLIOMA PROLIFERATION AND MIGRATION: ROLES OF NA + /H+ EXCHANGER 1

Glioma-associated microglia and macrophages (GAMs) comprise up to 30% of the brain tumor mass, making them substantial contributors to the tumor environment. Once within the tumor niche, infiltrating GAMs are quickly converted to pro-tumorigenic phenotypes that enhance glioma growth and migration. Here we have characterized the role of Na + /H+ exchanger isoform-1 (NHE1) in the bidirectional communication between glioma cells and microglia. NHE1 protein expression was increased in tumor xenographs in both microglia and glioma cells. We found that microglia cells exposed to glioma-conditioned medium increased their NHE1 expression, as well as expression of the M1 marker iNOS and the M2 marker Arg1. This is in accordance with the mixed activation profile observed with GAMs. The levels of a panel of other candidates were also evaluated, revealing a large increase in expression of the cytokines IL6, IL10, and TGFβ as well as membrane type 1-matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase 9. Regulation of all these proteins was abolished when NHE1 protein in microglia was inhibited with the selective NHE1 inhibitor HOE642. We next looked at the communication from glioma-stimulated microglia back to glioma cells. Glioma cells were exposed to conditioned medium from glioma-stimulated microglia grown with or without the NHE1 inhibitor HOE642. Glioma cells treated with glioma/microglia conditioned medium showed increased levels of phosphylated-Akt, increased proliferation, and enhanced migration, but not if microglial NHE1 function had been blocked by HOE 642. Our results show that NHE1 plays important roles in both the activation of microglia in response to tumor-secreted signals as well as microglial production of secreted signals which stimulate tumor progression. Thus, NHE1 is a promising therapeutic target for the treatment of glioma. Funding sources include NIH grants R01NS075995 and R01NS048216, the HEADRUSH Brain Tumor Research Professorship, the Roger Loff Memorial GBM Research Fund, and Department of Neurological Surgery.

Abstract 141: G-Protein Coupled Receptors GPR37 and GPR37L1 Regulate Sodium Reabsorption in Renal Proximal Tubule Cells

GPR37 and GPR37L1 are closely related G-protein coupled receptors that are expressed mainly in brain glial cells and muscle-myenteric nerve layers in the GI tract. GPR37L1 transgenic mice have decreased systolic blood pressure (SBP) whereas GPR37L1 KO mice have increased SBP. However, there are no studies reporting kidney expression of GPR37 or GPR37L1 or their role in renal blood pressure regulation. Immunostaining and immunoblotting showed that GPR37 and GPR37L1 are expressed in the apical membrane of proximal tubules of the mouse kidney; RT-PCR on proximal tubule and collecting duct cells obtained by laser capture micro-dissection of mouse kidney sections, confirmed these findings. In addition, chronic high salt diet increased the renal expression of prosaposin, a precursor for saposin C, a natural ligand for GPR37 and GPR37L1. Infusion of prosaptide, a synthetic ligand for GPR37 and GPR37L1, decreased SBP in mice by 10 mm Hg. To determine the roles of GPR37 and GPR37L1 in renal Na+ transport, we over-expressed separately the two proteins in human renal proximal tubule (RPT) cells (n=3). Intracellular Na+ was increased in GPR37- or GPR37L1-transfected RPT cells (3.1 ± 0.8 and 3.2 ± 0.6 fold respectively, P&lt;0.001) compared with mock-transfected cells. Immunoblot analyses showed increased phosphorylation of Erk1/2 (1.33 ± 0.03 and 1.52 ± 0.06 fold, P&lt;0.05), and ribosomal S6 protein (1.29 ± 0.03.5 and 1.39 ± 0.08 fold, P&lt;0.01) in RPT cells over-expressing GPR37 or GPR37L1, respectively. Na+,K+-ATPase expression was decreased by 29% ± 3.5 (P&lt;0.05) in GPR37L1 transfected RPT cells, but was unaltered in GPR37 transfected RPT cells. Taken together, these results show that both GPR37 and GPR37L1 are expressed in RPT cells and signal via the Erk1/2 pathway. GPR37L1 increases intracellular Na+ in RPT cells by decreasing the exit of Na+ due to a decrease in Na+,K+-ATPase expression and activity at the basolateral membrane while GPR37 increases intracellular Na+, by a mechanism independent of Na+,K+-ATPase. These results indicate that GPR37 and GPR37L1 may play a role in Na+ reabsorption in RPT cells and may be novel targets to designing drugs to treat patients with hypertension.