Activity Of Na Research Articles

Cyclic AMP-dependent protein kinase A and EPAC mediate VIP and secretin stimulation of PAK4 and activation of Na+,K+-ATPase in pancreatic acinar cells.

Rat pancreatic acinar cells possess only the p21-activated kinase (PAKs), PAK4 of the group II PAK, and it is activated by gastrointestinal hormones/neurotransmitters stimulating PLC and by a number of growth factors. However, little is known generally of cAMP agents causing PAK4 activation, and there are no studies with gastrointestinal hormones/neurotransmitters activating cAMP cascades. In the present study, we examined the ability of VIP and secretin, which stimulate cAMP generation in pancreatic acini, to stimulate PAK4 activation, the signaling cascades involved, and their possible role in activating sodium-potassium adenosine triphosphatase (Na+,K+-ATPase). PAK4 activation was compared with activation of the well-established cAMP target, cyclic AMP response element binding protein (CREB). Secretin-stimulated PAK4 activation was inhibited by KT-5720 and PKA Type II inhibitor (PKI), protein kinase A (PKA) inhibitors, whereas VIP activation was inhibited by ESI-09 and HJC0197, exchange protein directly activated by cAMP (EPAC) inhibitors. In contrast, both VIP/secretin-stimulated phosphorylation of CREB (pCREB) via EPAC activation; however, it was inhibited by the p44/42 inhibitor PD98059 and the p38 inhibitor SB202190. The specific EPAC agonist 8-CPT-2- O-Me-cAMP as well 8-Br-cAMP and forskolin stimulated PAK4 activation. Secretin/VIP activation of Na+,K+-ATPase, was inhibited by PAK4 inhibitors (PF-3758309, LCH-7749944). These results demonstrate PAK4 is activated in pancreatic acini by stimulation of both VIP-/secretin-preferring receptors, as is CREB. However, they differ in their signaling cascades. Furthermore, PAK4 activation is needed for Na+,K+ATPase activation, which mediates pancreatic fluid secretion. These results, coupled with recent studies reporting PAKs are involved in both pancreatitis/pancreatic cancer growth/enzyme secretion, show that PAK4, similar to PAK2, likely plays an important role in both pancreatic physiological/pathological responses. NEW & NOTEWORTHY Pancreatic acini possess only the group II p21-activated kinase, PAK4, which is activated by PLC-stimulating agents/growth factors and is important in enzyme-secretion/growth/pancreatitis. Little information exists on cAMP-activating agents stimulating group II PAKs. We studied ability/effect of cyclic AMP-stimulating agents [vasoactive intestinal polypeptide (VIP), secretin] on PAK4 activity in rat pancreatic-acini. Both VIP/secretin activated PAK4/CREB, but the cAMP signaling cascades differed for EPAC, MAPK, and PKA pathways. Both hormones require PAK4 activation to stimulate sodium-potassium adenosine triphosphatase activity. This study shows PAK4 plays an important role in VIP-/secretin-stimulated pancreatic fluid secretion and suggests it plays important roles in pancreatic acinar physiological/pathophysiological responses mediated by cAMP-activating agents.

Protein kinase C mediates juvenile hormone–dependent phosphorylation of Na+/K+-ATPase to induce ovarian follicular patency for yolk protein uptake

In oviparous animals, vitellogenesis is prerequisite to egg production and embryonic growth after oviposition. For successful insect vitellogenesis and oogenesis, vitellogenin (Vg) synthesized in the fat body (homologue to vertebrate liver and adipose tissue) must pass through the intercellular channels, a condition known as patency in the follicular epithelium, to reach the surface of oocytes. This process is controlled by juvenile hormone (JH) in many insect species, but the underlying mechanisms remain elusive. Previous work has suggested the possible involvement of Na+/K+-ATPase in patency initiation, but again, the regulatory cascade of Na+/K+-ATPase for patency initiation has been lacking. Using the migratory locust Locusta migratoria as a model system, we report here that RNAi-mediated knockdown of gene coding for Na+/K+-ATPase, inhibition of its phosphorylation, or suppression of its activity causes loss of patency, resulting in blocked Vg uptake, arrested oocyte maturation, and impaired ovarian growth. JH triggers G protein-coupled receptor (GPCR), receptor tyrosine kinase (RTK), phospholipase C (PLC), inositol trisphosphate receptor (IP3R), and protein kinase C (PKC) to phosphorylate Na+/K+-ATPase α-subunit at amino acid residue Ser8, consequently activating Na+/K+-ATPase for the induction of patency in vitellogenic follicular epithelium. Our results thus point to a previously unidentified mechanism by which JH induces the phosphorylation and activation of Na+/K+-ATPase via a signaling cascade of GPCR, RTK, PLC, IP3R, and PKC. The findings advance our understanding of JH regulation in insect vitellogenesis and oogenesis.

Ni0 NPs anchored on acid-activated montmorillonite (Ni0-Mont) as a highly efficient and reusable nanocatalyst for synthesis of biscoumarins and bisdimedones

Ni0 nanoparticles were immobilized on acid-activated montmorillonite (Ni0-Mont) by anchoring Ni(OAc)2·4H2O on modified micro/mesoporous montmorillonite followed by reduction with ethylene glycol. The prepared clay composite system was then characterized using Fourier-transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, X-ray diffraction (XRD) analysis, and Brunauer–Emmett–Teller (BET) analysis. SEM analysis revealed perfect dispersion of metallic nickel nanoparticles with size distribution of 6‒41 nm on the clay matrix. Micro/mesoporous montmorillonite was prepared by activation of Na+-montmorillonite with HCl (4 M) under controlled conditions. The catalytic activity of the prepared Ni0-Mont clay was studied in one-pot microwave-assisted synthesis of biscoumarins and bisdimedones via Knoevenagel reaction of aromatic aldehydes with 4-hydroxycoumarin or dimedone in solvent-free conditions. All reactions were carried out at room temperature within 5‒15 min (for biscoumarins) and 5–20 min (for bisdimedones) to afford the products in high to excellent yield. The reusability of the Ni0-Mont catalyst was also investigated in seven consecutive cycles, without significant loss of catalytic activity.

Effects of Neostigmine and Physostigmine on Activity of Na+,K+-ATPase in Various Subdivisions of Rat Brain.

The effects of intramuscular administration of neostigmine and physostigmine on Na+,K+-ATPase activity in various cerebral subdivisions were examined in rats. In CNS and peripheral tissues, both agents rapidly and significantly reduced activity of cholinesterases by 30-50%. The development of intoxication did not change the marker indices of stress reaction. In the cerebral cortex, physostigmine increased Na+,K+-ATPase activity, whereas neostigmine suppressed it. In addition, neostigmine decreased activity of this enzyme in the cerebellum. In contrast, both agents produced no effects on Na+,K+-ATPase activity in the striatum. The data corroborate the view on functional interaction between Na+,K+-ATPase and nicotinic cholinoreceptors in rat cerebral cortex.

234 - Lung epithelial PDIA3 plays a critical role in influenza infection

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and that this interaction is required for efficient oxidative folding of HA in vitro. However it is unknown whether these host-viral protein interactions occur during active infection and whether this interaction represent a putative target for the treatment of influenza infection. Current therapies targeting viral proteins are often limited in their effectiveness due to rapid development of viral resistance; targeting host proteins may avoid these limitations. Here we show that PDIA3 is specifically upregulated in IAV infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Among PDI inhibitors tested, we found LOC14 inhibits PDIA3 at an IC50 of 5μM. Treatment with LOC14 inhibits PDIA3 activity in lung epithelial cells and subsequently decreases disulfide bonds, and oligomerization (maturation) of HA in both pandemic H1N1 and seasonal H3N2 infected lung epithelial cells. Disulfide labeling analysis indicated that inhibition of PDIA3 activity also decreased disulfide bonds in IAV-neuraminidase (NA) and these differences in oxidative folding correspond to a subsequent decrease in NA activity. Furthermore, these decreases in disulfide bonds significantly decrease viral load, and pro-inflammatory response from primary lung epithelial cells. Lung epithelial specific deletion of PDIA3 in mice results in a significant decreases in viral burden and levels of inflammatory-immune markers in mouse lung, as well as significantly improved airway mechanics (function). Taken together, these results suggest PDIA3 is required for effective influenza pathogenesis in vivo, and inhibition of PDIA3 may aid future development anti-influenza therapies based on inhibition of host PDIs to circumvent antigenic drifts and shifts of influenza and may lend critically needed help during pandemic and severe influenza seasons.

The Role of Sodium in Diabetic Cardiomyopathy.

Cardiovascular complications are the major cause of mortality and morbidity in diabetic patients. The changes in myocardial structure and function associated with diabetes are collectively called diabetic cardiomyopathy. Numerous molecular mechanisms have been proposed that could contribute to the development of diabetic cardiomyopathy and have been studied in various animal models of type 1 or type 2 diabetes. The current review focuses on the role of sodium (Na+) in diabetic cardiomyopathy and provides unique data on the linkage between Na+ flux and energy metabolism, studied with non-invasive 23Na, and 31P-NMR spectroscopy, polarography, and mass spectroscopy. 23Na NMR studies allow determination of the intracellular and extracellular Na+ pools by splitting the total Na+ peak into two resonances after the addition of a shift reagent to the perfusate. Using this technology, we found that intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control possibly due to combined changes in the activity of Na+–K+ pump, Na+/H+ exchanger 1 (NHE1) and Na+-glucose cotransporter. We hypothesized that the increase in Na+ activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to a loss of intramitochondrial Ca2+, with a subsequent alteration in mitochondrial bioenergetics and function. Using isolated mitochondria, we showed that the addition of Na+ (1–10 mM) led to a dose-dependent decrease in oxidative phosphorylation and that this effect was reversed by providing extramitochondrial Ca2+ or by inhibiting the mitochondrial Na+/Ca2+ exchanger with diltiazem. Similar experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3–30 mM) led to significantly decreased ATP levels and that this effect was stronger in diabetic rats. These data suggest that in diabetic cardiomyocytes, increased Na+ leads to abnormalities in oxidative phosphorylation and a subsequent decrease in ATP levels. In support of these data, using 31P-NMR, we showed that the baseline β-ATP and phosphocreatine (PCr) were lower in diabetic cardiomyocytes than in control, suggesting that diabetic cardiomyocytes have depressed bioenergetic function. Thus, both altered intracellular Na+ levels and bioenergetics and their interactions may significantly contribute to the pathology of diabetic cardiomyopathy.

Regulation of mitochondrial biosynthesis and function by dietary carbohydrate levels and lipid sources in juvenile blunt snout bream Megalobrama amblycephala

This study aimed to investigate the effects of dietary non-protein energy adjustments on the mitochondrial biosynthesis and function of juvenile Megalobrama amblycephala. Fish (average weight: 37.98 ± 0.07 g) were fed eight diets containing two dietary carbohydrate levels (30% and 43%) and four lipid sources (fish oil, soybean oil, palm oil and the mixed oil) for 11 weeks. Liver mitochondrial respiratory chain complex V activity and ATP (adenosine triphosphate) content both increased significantly with increasing dietary carbohydrate levels, whereas the opposite was true for the AMP (adenosine 5′-monophosphate)/ATP ratio, hepatic transcripts of AMP-activated protein kinase α1 (AMPKα1), AMPKα2, peroxisome proliferators γ-activated receptor coativator-1α (PGC-1α), NADH dehydrogenase 1 and cytochrome c oxidase 1 (COX1) as well as the activities of Na+-K+-ATPase, succinate dehydrogenase (SDH), citrate synthase (CS) and mitochondrial respiratory chain complex I, III and IV. Additionally, hepatic ATP content, the transcripts of AMPKα, COX1 and ATP6 and the activities of Na+-K+-ATPase, SDH, CS and mitochondrial respiratory chain complex III were all significantly affected by lipid sources. Furthermore, an interaction between dietary carbohydrate levels and lipid sources was also observed in the activities of liver mitochondrial Na+-K+-ATPase and respiratory chain complex III as well as the transcripts of ATP6 and PGC-1α. Overall, these findings suggested that dietary carbohydrate levels and lipid sources remarkably affected the mitochondrial biosynthesis and function of M. amblycephala. A diet containing 30% carbohydrate and FO could boost its mitochondrial biosynthesis, while that of 30% carbohydrate and SO could enhance the mitochondrial function.

Recovery from hypoxia-induced internalization of cardiac Na+ /H + exchanger 1 requires an adequate intracellular store of antioxidants.

The heart is highly active metabolically but relatively underperfused and, therefore, vulnerable to ischemia. In addition to acidosis, a key component of ischemia is hypoxia that can modulate gene expression and protein function as part of an adaptive or even maladaptive response. Here, using cardiac-derived HL-1 cells, we investigate the effect of various hypoxic stimuli on the expression and activity of Na+ /H + exchanger 1 (NHE1), a principal regulator of intracellular pH. Acute (10 min) anoxia produced a reversible decrease in the sarcolemmal NHE1 activity attributable to NHE1 internalization. Treatment with either 1% O 2 or dimethyloxaloylglycine (DMOG; 1 mM) for 48-hr stabilized hypoxia-inducible factor 1 and reduced the sarcolemmal NHE1 activity by internalization, but without a change in total NHE1 immunoreactivity or message levels of the coding gene ( SLC9A1) determined in whole-cell lysates. Unlike the effect of DMOG, which was rapidly reversed on washout, reoxygenation after a prolonged period of hypoxia did not reverse the effects on NHE1, unless media were also supplemented with a membrane-permeant derivative of glutathione (GSH). Without a prior hypoxic episode, GSH supplementation had no effect on the NHE1 activity. Thus, posthypoxic NHE1 reinsertion can only take place if cells have a sufficient reservoir of a reducing agent. We propose that oxidative stress under prolonged hypoxia depletes intracellular GSH to an extent that curtails NHE1 reinsertion once the hypoxic stimulus is withdrawn. This effect may be cardioprotective, as rapid postischaemic restoration of the NHE1 activity is known to trigger reperfusion injury by producing an intracellular Na + -overload, which is proarrhythmogenic.

Tolerance of roach for modified water mineralization levels and temperatures expressed by biochemical indicators

Abstract Changes in the activity of Na+/K+-ATPase, lactate dehydrogenase, and succinate dehydrogenase in the white muscles and gill petals of roach, Rutilus rutilus (L), were studied for the combined actions of water mineralization and water temperature. It was established that decreased mineralization to 260.0 mg dm-3 and increased water temperature to 32°C increased aerobic processes in roach tissues. A sharp increase in water temperature and changes in water mineralization significantly affected the tolerance and physiological state of fish and could impact their abundance in waterbodies.

KADMİYUMA MARUZ BIRAKILAN DİYABETİK RATLARA ALFA LİPOİK ASİT VE İNSÜLİN UYGULANMASININ OLASI DÜZENLEYİCİ ETKİLERİ

OBJECTIVE: Environmental exposure to the cadmium (Cd), is associated with hyperglycemia and reduced serum insulin. This investigation was planned to assess the effects of Lipoic Acid (LA) and insulin on glycolytic enzymes, liver marker enzymes and lipids in Cd exposed diabetic rats. MATERIAL AND METHODS: Male Wistar rats were separated into 7 groups (n=8 in each group). Groups were designed as control, diabetic control, diabetic + CdCl2, diabetic + insulin, diabetic + CdCl2 + insulin, diabetic + CdCl2 + LA, anddiabetic + CdCl2 + insulin + LA groups. Type 1 diabetes was established by intraperitoneal (i.p.) injection of streptozotocin (STZ) (65 mg/kg) into 6 groups. Insulin (4 IU/kg/day) was given subcutaneously (s.c.) to insulin treated groups. CdCl2 (1,2 mg/kg/day) was given s.c. to CdCl2 treated groups. LA (100 mg/kg/day) was given i.p. to LA treated groups. CdCl2, LA, and insulin treatment were started 2 days after intraperitoneal STZ injection and continued for 3 weeks. Serum glucose, AST, ALT, BUN, LDL, HDL, and TG levels and liver hexokinase (HK), pyruvate kinase (PK), whole blood HbA1c level, and Na+/K+ATPase activity were evaluated. RESULTS: In diabetic group, serum glucose, HbA1c, TG, LDL, AST, ALT, ALP, and BUN levels were higher than cont- rol, but HDL was lower. In liver tissue, activities of Na+/ K+ATPase, HK and PK activities were decreased in dia- betic control group. PK, HK and Na+/K+ATPase activities were increased in liver in diabetic+CdCl2 and Diabeti- c+Insulin+CdCl2 groups. An increase was determined in activities of HK, PK, and Na+/K+ATPase in insulin and LA treated groups compared with diabetic control group. CONCLUSIONS: These results suggest that application of insulin and LA could be an effective therapeutic intervention against liver injury caused by Cd and STZ.

Tuning NaO2 Cube Sizes by Controlling Na+ and Solvent Activity in Na–O2 Batteries

Understanding the kinetics of electrochemical oxygen reduction reaction (ORR) and controlling the chemistry, morphology, and size of discharge products are critical to realize reversible operation of metal–air batteries. Here we show that increasing Na+ activity and free DME (not coordinated to Na+) activity in the solution increases the solubility of NaO2 and size of NaO2 cubes in Na–O2 cells. With increasing Na salt concentration, Raman spectroscopy revealed that Na+ activity increased while free DME activity decreased. NaO2 solubility and NaO2 cube size were found to exhibit a maximum at a medium concentration of Na+, which was accompanied by the highest full discharge capacity. This trend was attributed to two competing effects that stabilize NaO2 in solution; both higher Na+ activity and higher free DME activity can enhance NaO2 solubility. These results highlight immense opportunities in the design of discharge/charge characteristics such as reaction product sizes and discharge capacity through the ...

Gill Morphology and Na+/K+-ATPase Activity of Gobionellus oceanicus (Teleostei: Gobiidae) in an Estuarine System.

Gobionellus oceanicus is a species widely distributed on the Atlantic coast and may be susceptible to anthropic effects. This study evaluated the morphology and the Na+/K+-ATPase activity of G. oceanicus gills considering the concentration of metals in the fish and Subaé River estuary. Although the metal concentrations detected in the water and sediment did not exceed certain limits, CONAMA (Brazilian Environment Council), TEL (Threshold Effect Level), and PEL (Probable Effect Level), the metals levels in gills plus muscle and skin of G. oceanicus were above the permitted setting of the Ministry of Health, Brazil. The pavement epithelial cells (PVC) of the gill filament was observed that there were long microridges either in the apical surface or in the lamella; especially, microridges degeneration was shown in some PVC from filament epithelium. The number of ionocyte and the volume density were (0.02 ± 0.001)/μm and (0.38 ± 0.27)%, respectively. The activity of Na+/K+-ATPase was 1.13 ± 0.76μMPimgprotein-1h-1. We describe the volume density and number of ionocytes and Na+/K+-ATPase enzymatic activity in G. oceanicus for the first time, which is useful for basic and comparative future studies to support aquatic biomonitoring.

Characterization and expression of Na+/K+-ATPase in gills and kidneys of the Teleost fish Oreochromis mossambicus, Oreochromis urolepis hornorum and their hybrids in response to salinity challenge

Tilapia (Oreochromis mossambicus, O. urolepis hornorum, their hybrids O. mossambicus♀ × O. hornorum♂ and O. hornorum♀ × O. mossambicus♂) were exposed to a high salinity environment to evaluate their osmoregulatory responses. The plasma osmolality of all the tilapia species were elevated with the salinity challenge. The activities of Na+/K+-ATPase (NKA) in both the gill and kidney showed a similar increased change tendency compared with the control. The distribution of NKA α1 mRNA in all the examined tissues suggested that NKA α1 has a possible housekeeping role for this isoform. The amount of NKA α1 mRNA in the gill and kidney was elevated in the four fishes with similar expression patterns after transfer from freshwater to seawater. The NKAα1 mRNA expression levels in the gill reached their peak level at 24 h after transfer (P < 0.01) compared to the freshwater group, following decreases in the pretreatment level at 48 h (P > 0.05). However, the NKAα1 mRNA expression levels in the kidney were not significantly affected with increasing environmental salinity (P > 0.05). The differences in the responses to saltwater challenge may be associated with differences in saltwater tolerance between the four tilapia. The drastic increase in the plasma osmolality, NKA activities and mRNA expression suggested that the hybrids (O. mossambicus♀ × O. hornorum♂) possess heterosis in salinity responsiveness compared to that of both the parents, indicating a maternal effect on the salinity tolerance of the tilapia hybrids. This study provides a theoretical basis to further study the mechanism of fish osmoregulation response to salinity challenge.

Nitric oxide augments single persistent Na+ channel currents via the cGMP/PKG signaling pathway in Kenyon cells isolated from cricket mushroom bodies.

The nitric oxide (NO)/cyclic GMP signaling pathway has been suggested to be important in the formation of olfactory memory in insects. However, the molecular targets of the NO signaling cascade in the central neurons associated with olfactory learning and memory have not been fully analyzed. In this study, we investigated the effects of NO donors on single voltage-dependent Na+ channels in intrinsic neurons, called Kenyon cells, in the mushroom bodies in the brain of the cricket. Step depolarization on cell-attached patch membranes induces single-channel currents with fast-activating and -inactivating brief openings at the beginning of the voltage steps followed by more persistently recurring brief openings all along the 150-ms pulses. Application of the NO donor S-nitrosoglutathione (GSNO) increased the number of channel openings of both types of single Na+ channels. This excitatory effect of GSNO on the activity of these Na+ channels was diminished by KT5823, an inhibitor of protein kinase G (PKG), indicating an involvement of PKG in the downstream pathway of NO. Application of KT5823 alone decreased the activity of the persistent Na+ channels without significant effects on the fast-inactivating Na+ channels. The membrane-permeable cGMP analog 8Br-cGMP increased the number of channel openings of both types of single Na+ channels, similar to the action of NO. Taken together, these results indicate that NO acts as a critical modulator of both fast-inactivating and persistent Na+ channels and that persistent Na+ channels are constantly upregulated by the endogenous cGMP/PKG signaling cascade. NEW & NOTEWORTHY This study clarified that nitric oxide (NO) increases the activity of both fast-inactivating and persistent Na+ channels via the cGMP/PKG signaling cascade in cricket Kenyon cells. The persistent Na+ channels are also found to be upregulated constantly by endogenous cGMP/PKG signaling. On the basis of the present results and the results of previous studies, we propose a hypothetical model explaining NO production and NO-dependent memory formation in cricket large Kenyon cells.

DRm217 attenuates myocardial ischemia-reperfusion injury via stabilizing plasma membrane Na+-K+-ATPase, inhibiting Na+-K+-ATPase/ROS pathway and activating PI3K/Akt and ERK1/2

Na+-K+-ATPase has close relationship with myocardial ischemia/reperfusion (IR) injury. Activation of Na+-K+-ATPase with its DR region specific antibody produces cardioprotective effect. In this study, we aimed to explore whether DRm217, a proved DR region specific antibody, could protect myocardial cells against IR injury and uncover the mechanisms under it. By employing H9c2 myocardial cell and SD rat, we found that DRm217 protected cardiac cells against IR-induced cell injury and apoptosis. DRm217 produced protective effect via stabilizing Na+-K+-ATPase membrane expression and inhibiting Na+-K+-ATPase/Src/NADPH oxidase dependent ROS accumulation. PI3K/Akt and ERK1/2 participated in DRm217-induced cardiomyocyte survival, but not in DRm217-related ROS reduction. Therefore, DRm217 can be used as a potential cardioprotective adjuvant in myocardial IR therapy and interference of Na+-K+-ATPase/ROS pathway will be a promising modality for clinical myocardial IR therapy.

The Role of Ca2+ and BK Channels of Locus Coeruleus (LC) Neurons as a Brake to the CO2 Chemosensitivity Response of Rats

The cellular mechanisms by which LC neurons respond to hypercapnia are usually attributed to an “accelerator” whereby hypercapnic acidosis causes an inhibition of K+ channels or activation of Na+ and Ca+2 channels to depolarize CO2-sensitive neurons. Nevertheless, it is still unknown if this “accelerator” mechanism could be controlled by a brake phenomenon. Whole-cell patch clamping, fluorescence imaging microscopy and plethysmography were used to study the chemosensitive response of the LC neurons. Hypercapnic acidosis activates L-type Ca2+ channels and large conductance Ca-activated K+ (BK) channels, which function as a “brake” on the chemosensitive response of LC neurons. Our findings indicate that both Ca2+ and BK currents develop over the first 2 weeks of postnatal life in rat LC slices and that this brake pathway may cause the developmental decrease in the chemosensitive firing rate response of LC neurons to hypercapnic acidosis. Inhibition of this brake by paxilline (BK channel inhibitor) returns the magnitude of the chemosensitive firing rate response from LC neurons in rats older than P10 to high values similar to those in LC neurons from younger rats. Inhibition of BK channels in LC neurons by bilateral injections of paxilline into the LC results in a significant increase in the hypercapnic ventilatory response of adult rats. Our findings indicate that a BK channel-based braking system helps to determine the chemosensitive respiratory drive of LC neurons and contributes to the hypercapnic ventilatory response. Perhaps, abnormalities of this braking system could result in hypercapnia-induced respiratory disorders and panic responses.

Inhibition of Na+/K+‐ATPase and KIR channels abolishes hypoxic hyperaemia in resting but not contracting skeletal muscle of humans

Increasing blood flow (hyperaemia) to exercising muscle helps match oxygen delivery and metabolic demand. During exercise in hypoxia, there is a compensatory increase in muscle hyperaemia that maintains oxygen delivery and tissue oxygen consumption. Nitric oxide (NO) and prostaglandins (PGs) contribute to around half of the augmented hyperaemia during hypoxic exercise, although the contributors to the remaining response are unknown. In the present study, inhibiting NO, PGs, Na+ /K+ -ATPase and inwardly rectifying potassium (KIR ) channels did not blunt augmented hyperaemia during hypoxic exercise beyond previous observations with NO/PG block alone. Furthermore, although inhibition of only Na+ /K+ -ATPase and KIR channels abolished hyperaemia during hypoxia at rest, it had no effect on augmented hyperaemia during hypoxic exercise. This is the first study in humans to demonstrate that Na+ /K+ -ATPase and KIR channel activation is required for augmented muscle hyperaemia during hypoxia at rest but not during hypoxic exercise, thus providing new insight into vascular control. Exercise hyperaemia in hypoxia is augmented relative to the same exercise intensity in normoxia. During moderate-intensity handgrip exercise, endothelium-derived nitric oxide (NO) and vasodilating prostaglandins (PGs) contribute to ∼50% of the augmented forearm blood flow (FBF) response to hypoxic exercise (HypEx), although the mechanism(s) underlying the remaining response are unclear. We hypothesized that combined inhibition of NO, PGs, Na+ /K+ -ATPase and inwardly rectifying potassium (KIR ) channels would abolish the augmented hyperaemic response in HypEx. In healthy young adults, FBF responses were measured (Doppler ultrasound) and forearm vascular conductance was calculated during 5min of rhythmic handgrip exercise at 20% maximum voluntary contraction under regional sympathoadrenal inhibition in normoxia and isocapnic HypEx (O2 saturation ∼80%). Compared to control, combined inhibition of NO, PGs, Na+ /K+ -ATPase and KIR channels (l-NMMA+ketorolac+ouabain+BaCl2; Protocol 1; n=10) blunted the compensatory increase in FBF during HypEx by ∼50% (29±6mLmin-1 vs. 62±8mLmin-1 , respectively, P<0.05). By contrast, ouabain+BaCl2 alone (Protocol 2; n=10) did not affect this augmented hyperaemic response (50±11mLmin-1 vs. 60±13mLmin-1 , respectively, P>0.05). However, the blocked condition in both protocols abolished the hyperaemic response to hypoxia at rest (P<0.05). We conclude that activation of Na+ /K+ -ATPase and KIR channels is involved in the hyperaemic response to hypoxia at rest, although it does not contribute to the augmented exercise hyperaemia during hypoxia in humans.

NCX-DB: a unified resource for integrative analysis of the sodium calcium exchanger super-family

Na+/Ca2+ exchangers are low-affinity high-capacity transporters that mediate Ca2+ extrusion by coupling Ca2+ efflux to the influx of Na+ ions. The Na+/Ca2+ exchangers form a super-family comprised of three branches each differing in ion-substrate selectivity: Na+/Ca2+ exchangers (NCX), Na+/Ca2+/K+ exchangers, and Ca2+/cation exchangers. Their primary function is to maintain Ca2+ homeostasis and play a particularly important role in excitable cells that experience transient Ca2+ fluxes. Research into the role and activity of Na+/Ca2+ exchangers has focused extensively on the cardio-vascular system, however, growing evidence suggests that Na+/Ca2+ exchangers play a key role in neuronal processes such as memory formation, learning, oligodendrocyte differentiation, neuroprotection during brain ischemia and axon guidance. They have also been implicated in pathologies such as Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis and Epilepsy, however, a clear understanding of their mechanism during disease is lacking. To date, there has never been a central resource or database for Na+/Ca2+ exchangers. With clear disease relevance and ever-increasing research on Na+/Ca2+ exchangers from both model and non-model species, a database that unifies the data on Na+/Ca2+ exchangers is needed for future research. NCX-DB is a publicly available database with a web interface that enables users to explore various Na+/Ca2+ exchangers, perform cross-species sequence comparison, identify new exchangers, and stay-up to date with recent literature. NCX-DB is available on the web via an interactive user interface with an intuitive design, which is applicable for the identification and comparison of Na+/Ca2+ exchanger proteins across diverse species.

Perinatal α-tocopherol overload programs alterations in kidney development and renal angiotensin II signaling pathways at birth and at juvenile age: Mechanisms underlying the development of elevated blood pressure

α-Tocopherol (α-Toc) overload increases the risk of dying in humans (E.R. Miller III et al. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality Ann Int Med. 142 (2005) 37–46), and overload during early development leads to elevation of blood pressure at adult life, but the mechanism(s) remains unknown. We hypothesized that α-Toc overload during organogenesis affects the renal renin angiotensin system (RAS) components and renal Na+ handling, culminating with late elevated blood pressure. Pregnant Wistar rats received α-Toc or the superoxide dismutase mimetic tempol throughout pregnancy. We evaluated components of the intrarenal renin angiotensin system in neonate and juvenile offspring: Ang II-positive cells, Ang II receptors (AT1 and AT2), linked protein kinases, O2− production, NADPH oxidase abundance, lipid peroxidation and activity of Na+-transporting ATPases. In juvenile offspring we followed the evolution of arterial blood pressure. Neonates from α-Toc and tempol mothers presented with accentuated retardment in tubular development, pronounced decrease in glomerular Ang II-positive cells and AT1/AT2 ratio, intense production of O2− and upregulation of the α, ε and λ PKC isoforms. α-Toc decreased or augmented the abundance of renal (Na++K+)ATPase depending on the age and α-Toc dose. In juvenile rats the number of Ang II-positive cells returned to control values as well as PKCα, but co-existing with marked upregulation in the activity of (Na++K+) and Na+-ATPase and elevated arterial pressure at 30 days. We conclude that the mechanisms of these alterations rely on selective targeting of renal RAS components through genic and pro-oxidant effects of the vitamin.

FTY720P Increases, at 2 hrs, the Activity of the Na+/K+ ATPase in Caco‐2 cells via PKC and PI3K

Fingolimod phosphate (FTY720P), an analogue of sphingosine‐1‐phosphate (S1P), was shown previously to modulate, in a time‐dependent manner, the activity of hepatic Na+/K+ ATPase with an inhibitory effect appearing at 15 min and a stimulatory one at 2hrs. Because in the colon, the ATPase plays a pivotal role in the absorption of sodium and water, a time response study was conducted in human epithelial colorectal adenocarcinoma (Caco‐2) cells to examine if FTY720P regulates the activity of the ATPase in a similar fashion. An inhibitory effect was already demonstrated at 15 min. This work investigates the effect of FTY720P at 2 hrs and attempts to determine the major signaling molecules involved. The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate released in presence and absence of ouabain, a specific inhibitor of the enzyme. FTY720P exerted a significant and prominent stimulatory effect on the ATPase at 2 hours. This effect disappeared completely in presence of JTE‐013, a blocker of S1P receptor 2 (S1PR2), as well as in presence of each of calphostin and wortmannin, respective inhibitors of protein kinase C (PKC) and phosphoinositide 3‐kinase (PI3K). Moreover, Phorbol‐12‐Myristate‐13‐Acetate (PMA), an activator of PKC, simulated the stimulatory effect of FTY720P on Na+/K+ ATPase further confirming the involvement of PKC. BAPT/AM, a chelator of calcium ions, cancelled the effect of FTY720P on the ATPase, indicating thus an involvement of calcium ions in the signaling pathway. Since only conventional PKC isomers require the presence of calcium ions for their activation, FTY720P probably acts via a conventional PKC isomer. The stimulatory effect of PMA disappeared in presence of wortmannin, suggesting that PKC is upstream of PI3K. It was concluded that at 2 hours, FTY720‐P binds to S1PR2 leading to the activation of conventional PKC which, in turn, activates directly or indirectly PI3K, resulting in Na+/K+ ATPase stimulation.Support or Funding InformationUniversity Research BoardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.