SIK1 is part of a cell sodium-sensing network that regulates active sodium transport through a calcium-dependent process

In mammalian cells, active sodium transport and its derived functions (e.g., plasma membrane potential) are dictated by the activity of the Na(+),K(+)-ATPase (NK), whose regulation is essential for maintaining cell volume and composition, as well as other vital cell functions. Here we report the existence of a salt-inducible kinase-1 (SIK1) that associates constitutively with the NK regulatory complex and is responsible for increases in its catalytic activity following small elevations in intracellular sodium concentrations. Increases in intracellular sodium are paralleled by elevations in intracellular calcium through the reversible Na(+)/Ca(2+) exchanger, leading to the activation of SIK1 (Thr-322 phosphorylation) by a calcium calmodulin-dependent kinase. Activation of SIK1 results in the dephosphorylation of the NK alpha-subunit and an increase in its catalytic activity. A protein phosphatase 2A/phosphatase methylesterase-1 (PME-1) complex, which constitutively associates with the NK alpha-subunit, is activated by SIK1 through phosphorylation of PME-1 and its dissociation from the complex. These observations illustrate the existence of a distinct intracellular signaling network, with SIK1 at its core, which is triggered by a monovalent cation (Na(+)) and links sodium permeability to its active transport.     

In Vitro Action of Insulin on Erythrocyte Sodium Transport Mechanisms: Its Possible Role in the Pathogenesis of Arterial Hypertension

The effects of insulin on sodium and potassium metabolism have been well known for many years; clinical observation and laboratory experience showed different results about the insulin effect on the sodium-potassium pump. Moreover, studies about the insulin effect on the sodium-potassium cotransport are not available. Therefore, the effects of insulin on Na,⁺K⁺ pump and Na⁺,K⁺ cotransport were evaluated. Results show that insulin inhibits Na⁺, K⁺ pump, while Na⁺, K⁺ cotransport is markedly activated. The possible link between pathogenesis of arterial hypertension in hyperinsulinemic subjects and present data is examined.     

Effect of phenylalanine and p-chlorophenylalanine on Na+, K+-ATPase activity in the synaptic plasma membrane from the cerebral cortex of rats

Na⁺, K⁺-ATPase activity was measured in synaptic plasma membrane from cerebral cortex of Wistar rats subjected to experimental phenylketonuria, i.e., chemical hyperphenylalaninemia induced by subcutaneous administration of 5.2 μmol phenylalanine /g body weight (twice a day) plus 0.9 μmol p-chlorophenylalanine /g body weight (once a day). The treatment was performed from the 6^th to the 14^th postpartum day and rats were killed 12 h after the last injection. Synaptic plasma membrane from cerebral cortex was prepared by a discontinuous density sucrose gradient for Na⁺, K⁺-ATPase activity determination. The results showed that the enzyme activity was decreased by 30% in animals subjected to experimental phenylketonuria when compared to control. Thein vitro effects of the drugs on Na⁺, K⁺-ATPase activity were also investigated. Phenylalanine and p-chlorophenylalanine inhibited the enzyme activity and this inhibition was reversed by alanine. In addition, competition between phenylalanine and p-chlorophenylalanine for binding to the enzyme was observed, suggesting a common binding site for these substances. Our results suggest that reduction of Na⁺, K⁺-ATPase activity may be one of the mechanisms related to the brain dysfunction observed in human PKU.     

Role of the Na~+/K~+/2Cl~- cotransporter NKCC1 in cell cycle progression in human esophageal squamous cell carcinoma

AIM:To investigate the role of Na+/K+/2Cl-cotransporter 1(NKCC1)in the regulation of genes involved in cell cycle progression and the clinicopathological significance of its expression in esophageal squamous cell carcinoma(ESCC).METHODS:An immunohistochemical analysis was performed on 68 primary tumor samples obtained from ESCC patients that underwent esophagectomy.NKCC1expression in human ESCC cell lines was analyzed by Western blotting.Knockdown experiments were conducted using NKCC1 small interfering RNA,and the effects on cell cycle progression were analyzed.The gene expression profiles of cells were analyzed by microarray analysis.RESULTS:Immunohistochemical staining showed that NKCC1 was primarily found in the cytoplasm of carcinoma cells and that its expression was related to the histological degree of differentiation of SCC.NKCC1 was highly expressed in KYSE170 cells.Depletion of NKCC1in these cells inhibited cell proliferation via G2/M phase arrest.Microarray analysis identified 2527 genes with altered expression levels in NKCC1depleted KYSE170.Pathway analysis showed that the top-ranked canonical pathway was the G2/M DNA damage checkpoint regulation pathway,which involves MAD2L1,DTL,BLM,CDC20,BRCA1,and E2F5.CONCLUSION:These results suggest that the expression of NKCC1 in ESCC may affect the G2/M checkpoint and may be related to the degree of histological differentiation of SCCs.We have provided a deeper understanding of the role of NKCC1 as a mediator and/or a biomarker in ESCC.     

Platelet Na+,K+-ATPase activity as a possible peripheral marker for the neurotoxic effects of phenylalanine in phenylketonuria

Thein vitro effects of phenylalanine or alanine alone or combined on Na⁺,K⁺-ATPase activity in membranes from human platelets were investigated. The enzyme activity was assayed in membranes prepared from platelet-rich plasma of healthy donors. Phenylalanine or alanine were added to the assay to final concentrations of 0.3 to 1.2 mM, similar to those found in plasma of phenylketonuric patients. Phenylalanine inhibited Na⁺,K⁺-ATPase activity by 20–50% [F(4,25)=11.47; p<0.001]. Alanine had no effect on Na⁺,K⁺-ATPase activity but when combined with phenylalanine prevented the enzyme inhibition. These results, allied to others previously reported on brain Na⁺,K⁺-ATPase activity, may reflect a general inhibitory effect of phenylalanine on this important enzyme activity. Therefore, it is possible that measurement of Na⁺,K⁺-ATPase activity in platelets from PKU patients may be a useful peripheral marker for the neurotoxic effects of phenylalanine.     

Establishment of a neuroepithelial barrier by Claudin5a is essential for zebrafish brain ventricular lumen expansion

Lumen expansion driven by hydrostatic pressure occurs during many morphogenetic processes. Although it is well established that members of the Claudin family of transmembrane tight junction proteins determine paracellular tightness within epithelial/endothelial barrier systems, functional evidence for their role in the morphogenesis of lumenized organs has been scarce. Here, we identify Claudin5a as a core component of an early cerebral-ventricular barrier system that is required for ventricular lumen expansion in the zebrafish embryonic brain before the establishment of the embryonic blood–brain barrier. Loss of Claudin5a or expression of a tight junction-opening Claudin5a mutant reduces brain ventricular volume expansion without disrupting the polarized organization of the neuroepithelium. Perfusion experiments with the electron-dense small molecule lanthanum nitrate reveal that paracellular tightness of the cerebral-ventricular barrier decreases upon loss of Claudin5a. Genetic analyses show that the apical neuroepithelial localization of Claudin5a depends on epithelial cell polarity and provide evidence for concerted activities between Claudin5a and Na⁺,K⁺-ATPase during luminal expansion of brain ventricles. These data establish an essential role of a barrier-forming Claudin in ventricular lumen expansion, thereby contributing to brain morphogenesis.     

Proline Administration Decreases Na+,K+-ATPase Activity in the Synaptic Plasma Membrane from Cerebral Cortex of Rats

Buffered proline was injected subcutaneously into rats twice a day at 8 h intervals from the 6^th to the 28^th day of age. Control rats received saline in the same volumes. The animals were weighed and killed by decapitation 12 h after the last injection. Cerebral cortex was used for the determination of Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities. Body, whole brain and cortical weights were similar in the two groups. Na⁺,K⁺-ATPase activity was significantly reduced (by 20%) in membranes from the proline-treated group compared to the controls, whereas Mg²⁺-ATPase activity was not affected by proline. In another set of experiments, synaptic plasma membranes were prepared from cerebral cortex of 29-day-old rats and incubated with proline at final concentrations ranging from 0.1 to 2.0 mM. Na⁺,K⁺-ATPase activity, but not Mg²⁺-ATPase activity, was inhibited by 20-30%. Since proline concentrations in plasma of chronically treated rats and of type ll hyperprolinemic children are of the same order of magnitude as those tested in vitro, the results suggest that reduction of Na⁺,K⁺-ATPase activity may contribute to the neurological dysfunction found in some patients affected by type ll hyperprolinemia.     

Effect of hypermethioninemia on some parameters of oxidative stress and on Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity in hippocampus of rats

In the present study we investigated the effect of chronic administration of methionine, a metabolite accumulated in many inherited pathological conditions such as methionine adenosyltransferase deficiency and homocystinuria, on some parameters of oxidative stress, namely thiobarbituric acid reactive substances (TBARS), catalase activity and total thiol content, as well as on Na⁺,K⁺-ATPase activity in rat hippocampus. For chronic treatment, rats received subcutaneous injections of methionine (1.34–2.68 μmol/g of body weight), twice a day, from the 6th to the 28th day of age and controls received saline. Animals were killed 12 h after the last injection. Results showed that chronic hypermethioninemia significantly increased TBARS, decreased Na⁺,K⁺-ATPase activity but did not alter catalase and total thiol content. Since chronic hypermethioninemia altered TBARS and Na⁺,K⁺-ATPase activity at 12 h after methionine administration, we also investigated the effect of acute administration of this amino acid on the same parameters studied after chronic methionine administration. For acute treatment,29-day-old rats received one single injection of methionine (2.68 μmol/g of body weight) or saline and were killed 1, 3 or 12 h later. Results showed that rats subjected to acute hypermethioninemia presented a reduction of Na⁺,K⁺-ATPase activity and an increase in TBARS when the animals were killed at 3 and 12 h, but not at 1 h, after methionine administration. These data indicate that hypermethioninemia increases lipid peroxidation which may, at least partially, explain the effect of methionine on the reduction in Na⁺,K⁺-ATPase activity. If confirmed in human beings, our findings could suggest that the induction of oxidative stress and the inhibition of Na⁺,K⁺-ATPase activity caused by methionine might contribute to the neurophysiopathology observed in patients with severe hypermethioninemia.     

Effects of short-term acid and aluminum exposure on the parr-smolt transformation in Atlantic salmon (Salmo salar): disruption of seawater tolerance and endocrine status

Episodic acidification resulting in increased acidity and inorganic aluminum (Al_i) is known to interfere with the parr-smolt transformation of Atlantic salmon (Salmo salar), and has been implicated as a possible cause of population decline. To determine the extent and mechanism(s) by which short-term acid/Al exposure compromises smolt development, Atlantic salmon smolts were exposed to either control (pH 6.7–6.9) or acid/Al (pH 5.4–6.3, 28–64 μg l⁻¹ Al_i) conditions for 2 and 5 days, and impacts on freshwater (FW) ion regulation, seawater (SW) tolerance, plasma hormone levels and stress response were examined. Gill Al concentrations were elevated in all smolts exposed to acid/Al relative to controls confirming exposure to increased Al_i. There was no effect of acid/Al on plasma ion concentrations in FW however, smolts exposed to acid/Al followed by a 24 h SW challenge exhibited greater plasma Cl⁻ levels than controls, indicating reduced SW tolerance. Loss of SW tolerance was accompanied by reductions in gill Na⁺,K⁺-ATPase (NKA) activity and Na⁺,K⁺,2Cl⁻ (NKCC) cotransporter protein abundance. Acid/Al exposure resulted in decreased plasma insulin-like growth factor (IGF-I) and 3,3′,5′-triiodo-l-thyronine (T₃) levels, whereas no effect of treatment was seen on plasma cortisol, growth hormone (GH), or thyroxine (T₄) levels. Acid/Al exposure resulted in increased hematocrit and plasma glucose levels in FW, but both returned to control levels after 24 h in SW. The results indicate that smolt development and SW tolerance are compromised by short-term exposure to acid/Al in the absence of detectable impacts on FW ion regulation. Loss of SW tolerance during short-term acid/Al exposure likely results from reductions in gill NKA and NKCC, possibly mediated by decreases in plasma IGF-I and T₃.     

Ouabain-Like and Non-Ouabain-Like Factors in Plasma of Patients with Essential Hypertension

Circulating inhibitor of Na⁺, K⁺-ATPase and ouabain-like immuno-reactivity were studied in patients with essential hypertension. In the plasma of patients, two types of Na⁺, K⁺-ATPase inhibitors (ouabain-like and non-ouabain-like inhibitors) and ouabain-like immunoreactivity were detected. Ouabain-like inhibitor was clearly detected at a low KC1 concentration (0.1 mM) in the assay buffer, and non-ouabain-like inhibitor was detected at a high KC1 concentration (10 mM). The plasma level of ouabain-like inhibitor correlated significantly with that of ouabain-like immunoreactivity (p<0.001) and with a mean blood pressure (p<0.01). The plasma level of non-ouabain-like inhibitor was not correlated with the levels of either ouabain-like immunoreactivity or mean blood pressure. The level of plasma ouabain-like inhibitor did not correlate with that of plasma non-ouabain like inhibitor. Both ouabain-like inhibitor and ouabain-like immunoreactivity in the plasma of patients with essential hypertension were significantly higher than those in normotensive subjects, but the plasma level of non-ouabain-like inhibitor in patients with essential hypertension was not higher than that in normotensive subjects. These results suggest that the plasma from patients with essential hypertension contains ouabain-like factor(s) which is important to mantain the high blood pressure.     

Assay of a Circulating Sodium Pump Inhibitor in Patients with Essential Hypertension and Normotensive Subjects

The plasma levels of a sodium pump inhibitor (Na⁺PI) were measured by a modified method of Hamlyn et al, using dog kidney Na⁺, K⁺-ATPase. When the level of Na⁺PI was expressed as the % inhibition of the enzyme and compared with that of a control solution, it was found to be 9.0 ± 0.7% in 43 untreated patients with essential hypertension. This was significantly higher than 5.0 ± 0.4% for 56 normotensive subjects (p < 0.01). Male patients with essential hypertension showed the highest mean value of 10.5 ± 1.1%, disclosing an apparent sex difference in the patient group (p< 0.01). Only in female patients was there a significant positive correlation between the inhibitor's level and the mean blood pressure (r = 0.649, p < 0.01). These results provided additional evidence for increased Na⁺ PI in the plasma of patients with essential hypertension, which might bear an important role in the pathogenesis of the disease.     

Kinetics of Red Cell Na+ and K+ Transport in Prague Hypertensive Rats

Kinetics of ouabain-sensitive, furosemide-sensitive (FS), bumetanide-sensitive (BS) and -resistant Na⁺ and K⁺ transport were studied in erythrocytes of Prague hypertensive rats (PHR) and Prague normotensive rats (PNR). Maximal transport rates (V_max) and apparent affinities for either intracellular Na⁺ or extracellular K⁺ (replaced by Rb⁺) were determined in red cells in which Na⁺ content varied around the physiological range and that were incubated in Na⁺ media. No major differences between PHR and PNR were disclosed in the kinetics of ion transport mediated by the Na⁺-K⁺ pump or BS inward Na⁺-K⁺ cotransport. FS Rb⁺ uptake was higher (due to a greater V_max) in red cells of PHR as compared to PNR. In cells with a lowered Na⁺ content this elevation of FS Rb⁺ uptake was largely due to an augmented K⁺-Cl^? cotransport which exhibits a low affinity for Rb⁺_o and is blocked by 1 mM furosemide but not by 10 μM bumetanide. Red cells of PHR and PNR strains did not differ in either Na⁺ or Rb⁺ leaks. A slight increase of red cell Na⁺ content in PHR was evaluated in terms of the pump-leak concept. The present study did not reveal any obvious kinetic abnormalities of red cell cation transport the presence of which in tissues involved in blood pressure regulation would favor the development or the maintenance of genetic hypertension in PHR.     

Origin of first cells at terrestrial, anoxic geothermal fields

All cells contain much more potassium, phosphate, and transition metals than modern (or reconstructed primeval) oceans, lakes, or rivers. Cells maintain ion gradients by using sophisticated, energy-dependent membrane enzymes (membrane pumps) that are embedded in elaborate ion-tight membranes. The first cells could possess neither ion-tight membranes nor membrane pumps, so the concentrations of small inorganic molecules and ions within protocells and in their environment would equilibrate. Hence, the ion composition of modern cells might reflect the inorganic ion composition of the habitats of protocells. We attempted to reconstruct the "hatcheries" of the first cells by combining geochemical analysis with phylogenomic scrutiny of the inorganic ion requirements of universal components of modern cells. These ubiquitous, and by inference primordial, proteins and functional systems show affinity to and functional requirement for K(+), Zn(2+), Mn(2+), and phosphate. Thus, protocells must have evolved in habitats with a high K(+)/Na(+) ratio and relatively high concentrations of Zn, Mn, and phosphorous compounds. Geochemical reconstruction shows that the ionic composition conducive to the origin of cells could not have existed in marine settings but is compatible with emissions of vapor-dominated zones of inland geothermal systems. Under the anoxic, CO(2)-dominated primordial atmosphere, the chemistry of basins at geothermal fields would resemble the internal milieu of modern cells. The precellular stages of evolution might have transpired in shallow ponds of condensed and cooled geothermal vapor that were lined with porous silicate minerals mixed with metal sulfides and enriched in K(+), Zn(2+), and phosphorous compounds.     

Effect of Lipopolysaccharide on Small Intestinal l-Leucine Transport in Rabbit

In the present study, we have investigated whether the lipopolysaccharide (LPS) endotoxin from Escherichia coli is able to alter the jejunal transport of l-leucine when the tissue is exposed to endotoxin. The results have shown that the LPS at 3 × 10^-5 g/ml decreases the uptake of l-leucine into the enterocyte, as well as the mucosal to serosal flux of l-leucine. The secretagogue effect of LPS on the gut did not affect the inhibitory effect of LPS on the intestinal absorption of the amino acid. The endotoxin did not modify amino acid diffusion across the intestinal epithelium. However, from the mediated transport, only the Na⁺-dependent transport system was affected by LPS with a diminution of the transporter affinity (the apparent K_m was increased). In addition, we found a reduction of the Na⁺, K⁺-ATPase activity, which could explain the l-leucine Na⁺-dependent transport inhibition.     

Age-Dependent Alterations in Na+,K+-ATPase Activity in the Central Nervous System of Spontaneously Hypertensive Rats: Relationship to the Development of High Blood Pressure

We have previously demonstrated that cerebroventricular administrations (i.c.v) of potassium chloride solutions (KCl; 0.375–1.25 μmoles/5 μl) elicit ouabain-sensitive, concentration-dependent decreases in the blood pressure and heart rates of anesthetized, normotensive Sprague-Dawley (SD) rats. These studies have suggested an inverse relationship between Na⁺-pump activity in the central nervous system (CNS) and central sympathetic outflow. Such a view is further supported by the present studies showing that i.c.v. injections of KCl failed to produce any alterations in the blood pressures of rats pretreated with an autonomic ganglionic blocker, chlorisondamine. In the present studies, depressor responses to i.c.v. potassium chloride were considered as functional indices for evaluation of neuronal Na⁺-pump activity in 8 and 12 week old (8 wk and 12 wk) SHR, WKY and Sprague-Dawley (SD) rats. Basal arterial blood pressures of 8 wk-old SD and SHR, and the responsiveness of these two groups to i.c.v. potassium chloride solutions are similar and they both are significantly greater than that of age matched WKY However, in the 12 wk-old groups, arterial pressure of SHR was significantly greater than that of WKY as well as SD, whereas the depressor responses to KCl in SHR were significantly greater than that of only WKY. Pretreatment of the rats with i.c.v. ouabain abolished the differences in the hypotensive responses to i.c.v. potassium chloride that existed between various groups but not the differences in the basal blood pressures. Evaluation of these data suggest that a) the centrally mediated hypotensive responses to K⁺ in various groups could depend upon Na⁺,K⁺-pump activity in C.N.S. and/or on basal central sympathetic discharge; b) central sympathetic activity is greater in SHR only when compared to WKY but not to SD; c) since the central Na⁺-pump activity and sympathetic tone appears to be similar in SHR and SD, mecahnisms other than the increases in sympathetic activity must play a prominent role in the development of spontaneous hypertension; d) attenuation of neuronal Na⁺-pump activity cannot account for greater sympathetic tone in SHR and SD-rats when compared to WKY.     

Pharmacological induction of delayed and prolonged cardiac protection: The role of prostanoids

In 1983, a delayed and prolonged cardioprotection induced by drugs was described. This pharmacologically induced adaptation to stress represents a new trend in cardioprotection as opposed to the classical drug treatment that was based on the presence of drug-receptor binding. Such a long-lasting, delayed adaptation can be induced by non-injurious pharmacological stimuli (eg, prostacyclin and its stable analogues, catecholamines and other substances) and manifests as a marked protection against the severe consequences of ischemia; attenuation of early morphological changes (limitation in infarct size) and reduction in ventricular arrhythmias as results of coronary artery occlusion and reperfusion or ouabain toxication. The protection is time- and dose-dependent; the maximum effects occur 24 h and 48 h after drug treatment. These effects can be prolonged for a longer period by the periodic administration of maintenance doses. Concerning the mechanism of this marked delayed protection, the findings show that these adaptive stresses stimulate the adenylate cyclase/cyclic AMP (cAMP) system and result in elevation in cardiac cAMP level. This triggers the induction of Na⁺/K⁺-ATPase and activates phosphodiesterase (PDE) isoforms, most likely PDE1 and PDE4. The increased amount of PDE isoforms and activated Na⁺/K⁺-ATPase moderates ischemic myocardial potassium loss, and reduces sodium and calcium accumulation during myocardial ischemia. This also attenuates ouabain toxicity. Induction of PDE isoforms may lead to a reduction in the accumulation of excess cAMP and contribute to a lessened response to beta-adrenergic stimuli. The antiarrhythmic effects can be explained by electrophysiological changes, such as prolongations of the effective refractory period and the action potential duration during ischemia and reperfusion. The advantages of pharmacologically induced adaptation to stress in preventive therapy are that an exact dosage can be applied, the risk of the harmful effects is minimal, the protection can be prolonged, and it can be induced under pathological conditions (eg, atherosclerosis, hypercholesterolemia). Pharmacologically induced long-term protection may represent a new approach in the therapy of cardiovascular diseases.     

Cell shrinkage activates Na+/H+ exchange in dog red cells by relieving inhibition of exchange by Na+ in isotonic medium

Na⁺/H⁺ exchangers (NHE) are widely distributed transporters responsible for regulation of cell volume and pH. In isotonic medium, NHE is often low or negligible, and is strongly activated by osmotic cell shrinkage. It is reported here that extracellular Na⁺ inhibits NHE in isotonic medium, and cell shrinkage stimulates NHE by relieving this inhibition. There is more than one inhibitory Na⁺ site on each transporter. Shrinkage activates NHE by decreasing the apparent affinity for Na⁺ at the inhibitory sites. Shrinkage has no effect on the apparent affinity for Na⁺ at the substrate sites for activation of NHE.     

Alanine Prevents the Decrease of Na+,K+-ATPase Activity in Experimental Phenylketonuria

Our objective was to investigate the effect of alanine administration on Na⁺,K⁺-ATPase activity in cerebral cortex of rats subjected to chemically-induced phenylketonuria. Wistar rats were treated from the 6th to the 28th day of life with subcutaneous injections of either 2.6 mol alanine or 5.2 mol phenylalanine plus 2.6 mol -methylphenylalanine per g body weight or phenylalanine plus -methylphenylalanine plus alanine in the same doses or equivalent volumes of 0.15 M saline. The animals were killed on the 29th or 60th day of life. Synaptic plasma membrane from cerebral cortex was prepared for Na⁺,K⁺-ATPase activity determination. The results showed that alanine injection prevents the decrease of Na⁺,K⁺-ATPase activity in animals subjected to experimental phenylketonuria. Therefore, in case the same effects are achieved with ingested alanine, it is possible that alanine supplementation may be an important dietary adjuvant for phenylketonuric patients.     

In silico identified CCR4 antagonists target regulatory T cells and exert adjuvant activity in vaccination

Adjuvants are substances that enhance immune responses and thus improve the efficacy of vaccination. Few adjuvants are available for use in humans, and the one that is most commonly used (alum) often induces suboptimal immunity for protection against many pathogens. There is thus an obvious need to develop new and improved adjuvants. We have therefore taken an approach to adjuvant discovery that uses in silico modeling and structure-based drug-design. As proof-of-principle we chose to target the interaction of the chemokines CCL22 and CCL17 with their receptor CCR4. CCR4 was posited as an adjuvant target based on its expression on CD4(+)CD25(+) regulatory T cells (Tregs), which negatively regulate immune responses induced by dendritic cells (DC), whereas CCL17 and CCL22 are chemotactic agents produced by DC, which are crucial in promoting contact between DC and CCR4(+) T cells. Molecules identified by virtual screening and molecular docking as CCR4 antagonists were able to block CCL22- and CCL17-mediated recruitment of human Tregs and Th2 cells. Furthermore, CCR4 antagonists enhanced DC-mediated human CD4(+) T cell proliferation in an in vitro immune response model and amplified cellular and humoral immune responses in vivo in experimental models when injected in combination with either Modified Vaccinia Ankara expressing Ag85A from Mycobacterium tuberculosis (MVA85A) or recombinant hepatitis B virus surface antigen (rHBsAg) vaccines. The significant adjuvant activity observed provides good evidence supporting our hypothesis that CCR4 is a viable target for rational adjuvant design.     

Microfluidic glucose stimulation reveals limited coordination of intracellular Ca2+ activity oscillations in pancreatic islets

The pancreatic islet is a functional microorgan involved in maintaining normoglycemia through regulated secretion of insulin and other hormones. Extracellular glucose stimulates insulin secretion from islet beta cells through an increase in redox state, which can be measured by NAD(P)H autofluorescence. Glucose concentrations over approximately 7 mM generate synchronous oscillations in beta cell intracellular Ca2+ concentration ([Ca2+]i), which lead to pulsatile insulin secretion. Prevailing models assume that the pancreatic islet acts as a functional syncytium, and the whole islet [Ca2+]i response has been modeled in terms of islet bursting and pacemaker models. To test these models, we developed a microfluidic device capable of partially stimulating an islet, while allowing observation of the NAD(P)H and [Ca2+]i responses. We show that beta cell [Ca2+]i oscillations occur only within regions stimulated with more than approximately 6.6 mM glucose. Furthermore, we show that tolbutamide, an antagonist of the ATP-sensitive K+ channel, allows these oscillations to travel farther into the nonstimulated regions of the islet. Our approach shows that the extent of Ca2+ propagation across the islet depends on a delicate interaction between the degree of coupling and the extent of ATP-sensitive K+-channel activation and illustrates an experimental paradigm that will have utility for many other biological systems.