Effect of glutamine supplementation on exercise-induced changes in lymphocyte function

The purpose of this study was toinvestigate the possible role of glutamine in exercise-inducedimpairment of lymphocyte function. Ten male athletesparticipated in a randomized, placebo-controlled, double-blindcrossover study. Each athlete performed bicycle exercise for 2 hat 75% of maximum O₂ consumption on 2 separate days.Glutamine or placebo supplements were given orally during and up to2 h postexercise. The trial induced postexercise neutrocytosisthat lasted at least 2 h. The total lymphocyte count increased bythe end of exercise due to increase of bothCD3⁺TCR⁺ andCD3⁺TCR⁺ T cells as well asCD3CD16⁺CD56⁺ naturalkiller (NK) cells. Concentrations of CD8⁺ andCD4⁺ T cells lacking CD28 and CD95 on their surfaceincreased more than those of cells expressing these receptors. Withinthe CD4⁺ cells, only CD45RA memory cells, butnot CD45RA⁺ naive cells, increased in response to exercise.Most lymphocyte subpopulations decreased 2 h after exercise.Glutamine supplementation abolished the postexercise decline in plasmaglutamine concentration but had no effect on lymphocyte trafficking, NKand lymphokine-activated killer cell activities, T cell proliferation,catecholamines, growth hormone, insulin, or glucose. Neutrocytosis wasless pronounced in the glutamine-supplemented group, but it is unlikelythat this finding is of any clinical significance. This study does notsupport the idea that glutamine plays a mechanistic role inexercise-induced immune changes.

     

Characterization of the CD14++CD16+ Monocyte Population in Human Bone Marrow

Numerous studies have divided blood monocytes according to their expression of the surface markers CD14 and CD16 into following subsets: classical CD14⁺⁺CD16⁻, intermediate CD14⁺⁺CD16⁺ and nonclassical CD14⁺CD16⁺⁺ monocytes. These subsets differ in phenotype and function and are further correlated to cardiovascular disease, inflammation and cancer. However, the CD14/CD16 nature of resident monocytes in human bone marrow remains largely unknown. In the present study, we identified a major population of CD14⁺⁺CD16⁺ monocytes by using cryopreserved bone marrow mononuclear cells from healthy donors. These cells express essential monocyte-related antigens and chemokine receptors such as CD11a, CD18, CD44, HLA-DR, Ccr2, Ccr5, Cx3cr1, Cxcr2 and Cxcr4. Notably, the expression of Ccr2 was inducible during culture. Furthermore, sorted CD14⁺⁺CD16⁺ bone marrow cells show typical macrophage morphology, phagocytic activity, angiogenic features and generation of intracellular oxygen species. Side-by-side comparison of the chemokine receptor profile with unpaired blood samples also demonstrated that these rather premature medullar monocytes mainly match the phenotype of intermediate and partially of (non)classical monocytes. Together, human monocytes obviously acquire their definitive CD14/CD16 signature in the bloodstream and the medullar monocytes probably transform into CD14⁺⁺CD16⁻ and CD14⁺CD16⁺⁺ subsets which appear enriched in the periphery.     

Mechanism of the exercise hyperkalemia: an alternate hypothesis

Wasserman, Karlman, William W. Stringer, Richard Casaburi,and Yong-Yu Zhang. Mechanism of the exercisehyperkalemia: an alternate hypothesis. J. Appl.Physiol. 83(2): 631-643, 1997.A progressivehyperkalemia is observed as exercise intensity increases. The currentmost popular hypothesis for the hyperkalemia is that theNa⁺-K⁺pump cannot keep pace with the K⁺efflux from muscle during the depolarization-repolarization process ofthe sarcolemmal membrane during muscle contraction. In this report, wepresent data that suggest an alternate hypothesis to those previouslydescribed. Because phosphocreatine (PCr) is a highly dissociated acidand creatine is neutral at cell pH, the concentration of nondiffusibleanions decreases, and an alkaline reaction takes place when PCrhydrolyzes. This creates a state of cation(K⁺) excess andH⁺ depletion in the cell. Toexamine the balance of K⁺ andH⁺ for exercising muscle duringthe early period of exercise when PCr changes most rapidly, catheterswere inserted into the brachial artery and femoral vein (FV) in fivehealthy subjects who performed two 6-min cycle ergometer exercise testsat 40 and 85% of peak oxygen uptake. FV blood was sampled every 5 sduring the first 2 min, then every 30 s for the remaining 4 min ofexercise and the first 3 min of recovery, and then less frequently forthe next 12 min. Arterial sampling was every 30 s during exercise andsimultaneous with FV sampling during recovery. ArterialK⁺ concentration([K⁺]) increase laggedFV [K⁺]increase. The hyperkalemia observed during early exerciseresults from K⁺ release fromskeletal muscle. FV[K⁺] increased by 5 sof the start of exercise and followed the rate ofH⁺ loss from the FV blood for thefirst 30 s of exercise. FV lactate andNa⁺ kinetics differed fromK⁺ kinetics during exercise andrecovery. As predicted from the PCr hydrolysis reaction, the exercisinglimb took up H⁺ and releasedK⁺ at the start of exercise (first30 s) at both exercise intensities, resulting in a FV metabolicalkalosis. K⁺ release wasessentially complete by 3 min, the time at which oxygen uptake (and,presumably, PCr) reached its asymptote. These findings lead us tohypothesize that the early K⁺release by the cell takes place withH⁺ exchange and that the majormechanism for the exercise hyperkalemia is the reduction innondiffusible intracellular anions in the myocyte as PCr hydrolyzes.

     

Mesenchymal stromal cells impair the differentiation of CD14(++) CD16(-) CD64(+) classical monocytes into CD14(++) CD16(+) CD64(++) activate monocytes

Background aimsMesenchymal stromal cells (MSC) possess immunomodulatory activity both in vitro and in vivo. However, little information is available regarding their function during the initiation of immunologic responses through their interactions with monocytes. While many studies have shown that MSC impair the differentiation of monocytes into dendritic cells and macrophages, there are few articles showing the interaction between MSC and monocytes and none of them has addressed the question of monocyte subset modulationMethodsTo understand better the mechanism behind the benefit of MSC infusion for graft-versus-host treatment through monocyte involvement, we performed mixed leucocyte reactions (MLR) in the presence and absence of MSC. After 3 and 7 days, cultures were analyzed by flow cytometry using different approachesResultsMSC induced changes in monocyte phenotype in an MLR. This alteration was accompanied by an increase in monocyte counting and CD14 expression. MSC induced monocyte alterations even without contact, although the parameters above were more pronounced with cell–cell contact. Moreover, the presence of MSC impaired major histocompatibility complex (MHC) I and II, CD11c and CCR5 expression and induced CD14 and CD64 expression on monocytes. These alterations were accompanied by a decrease in interleukin (IL)-1β and IL-6 production by these monocytes, but no change was observed taking into account the phagocytosis capacity of these monocytesConclusionsOur results suggest that MSC impair the differentiation of CD14⁺⁺ CD16^? CD64⁺ classical monocytes into CD14⁺⁺ CD16⁺ CD64⁺⁺ activated monocytes, having an even earlier role than the differentiation of monocytes into dendritic cells and macrophages.     

The CD14+CD16+ Inflammatory Monocyte Subset Displays Increased Mitochondrial Activity and Effector Function During Acute Plasmodium vivax Malaria

Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax–infected patients display significant increase in circulating monocytes, which were defined as CD14⁺CD16⁻ (classical), CD14⁺CD16⁺ (inflammatory), and CD14^loCD16⁺ (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16⁺ cells, in particular the CD14⁺CD16⁺ monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14⁺ were distinguished from CD14^lo monocytes by displaying larger and more active mitochondria. CD14⁺CD16⁺ monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-α and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14⁺CD16⁺ cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection.     

Dopamine Increases CD14+CD16+ Monocyte Migration and Adhesion in the Context of Substance Abuse and HIV Neuropathogenesis

Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14⁺CD16⁺ monocytes, a mature subpopulation of peripheral blood monocytes, are key mediators of HIV neuropathogenesis. Infected CD14⁺CD16⁺ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy, continued influx of CD14⁺CD16⁺ monocytes, both infected and uninfected, contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain, CD14⁺CD16⁺ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14⁺CD16⁺ monocytes and their contribution to HIV neuropathogenesis are not known. In this study, we showed that CD14⁺CD16⁺ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R, D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist, {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393, increased CD14⁺CD16⁺ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion, live cell imaging was used to monitor the accumulation of CD14⁺CD16⁺ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14⁺CD16⁺ monocytes in suspension settled to the dish surface. In a spreading assay, dopamine increased the area of CD14⁺CD16⁺ monocytes during the early stages of cell adhesion. In addition, adhesion assays showed that the overall total number of adherent CD14⁺CD16⁺ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14⁺CD16⁺ monocytes in dopamine rich brain regions.     

Effects of hepatic portal infusion of deionized water on metabolic and hormonal responses to exercise in rats

The present study was conducted to investigatethe in vivo effects of an intrahepatic infusion of deionized waterduring exercise in rats. Adrenodemedullated male Sprague-Dawley ratswere continuously infused for 30 min either at rest or during treadmillexercise (26 m/min, 0% grade). Rats were randomly assigned to one ofthree infusion conditions (52 µl/min) with either deionized water(PW) or saline (PS; NaCl; 0.9%) via the hepatic portal vein ordeionized water through the jugular vein (JW). The exercise periodcaused a significant (P < 0.05)decrease in liver glycogen and relative liver water content andperipheral and portal blood glucose and insulin while increasingperipheral and portal glucagon andK⁺ plasma concentrations. Theseresponses, with the exception of K⁺, were not influenced by thedifferent types of infusions. The increase inK⁺ during exercise wassignificantly (P < 0.05) higher inJW rats than in the PW and PS groups. Both the infusion and exerciseprotocols did not significantly alter the liver weight-to-body weightratio, plasma osmolality, free fatty acids, -hydroxybutyrate,Na⁺,Cl, vasopressin, andcatecholamine concentrations. It is concluded that an hepatic portalinfusion of deionized water does not specifically alter the metabolicand hormonal responses to exercise in rats.

     

The significance of CD14+ monocytes in peripheral blood stem cells for the treatment of rat liver cirrhosis

Background aimsCirculating monocytes have been exploited as an important progenitor cell resource for hepatocytes in vitro and are instrumental in the removal of fibrosis. We investigated the significance of monocytes in peripheral blood stem cells (PBSC) for the treatment of liver cirrhosis.MethodsRat CD14⁺ monocytes in PBSC were mobilized with granulocyte-colony-stimulating factor (G-CSF) and harvested by magnetic cell sorting (MACS). Female rats with carbon tetrachloride (CCl₄)-induced liver cirrhosis were injected CM-DiI-labeled monocytes, CD14^? cells (1 × 10⁷ cells/rat) or saline via the portal vein.ResultsRat CD14⁺ and CD11b⁺ monocytes in PBSC were partly positive for CD34, CD45, CD44, Oct3/4 and Sox2, suggesting monocytes with progenitor capacity. Compared with CD14^? cell-infused and saline-injected rats, rats undergoing monocyte transplantation showed a gradually increased serum albumin level and decreased portal vein pressure, resulting in a significantly improved survival rate. Meanwhile, monocyte transplantation apparently attenuated liver fibrosis by analysis for fibronectin, α2-(1)-procollagen, α-smooth muscle aorta (SMA) and transforming growth factor (TGF)-β. Transplanted monocytes mainly clustered in periportal areas of liver, in which 1.8% cells expressed hepatocyte marker albumin and CK18. The expression level of hepatocyte growth factor (HGF), TGF-α, extracellular matrix (EGF) and vascular endothelial growth factor (VEGF) increased, while monocyte transplantation enhanced hepatocyte proliferation. On the other hand, the activities and expression of matrix metalloproteinases (MMP) increased while tissue inhibitor of metalloproteinase (TIMP)-1 expression significantly reduced in monocyte-transplanted livers. Some transplanted monocytes expressed MMP-9 and -13.ConclusionsThe data suggest that CD14⁺ monocytes in PBSC contribute to hepatocyte regeneration and extracellular matrix (ECM) remodeling in rat liver cirrhosis much more than CD14^? cells, and might offer a therapeutic alternative for patients with liver cirrhosis.     

Acid-base regulation after maximal exercise testing in late gestation

Kemp, Justin G., Felicia A. Greer, and Larry A. Wolfe.Acid-base regulation after maximal exercise testing in late gestation. J. Appl. Physiol. 83(2):644-651, 1997.This study employed Stewart's physicochemicalapproach to quantify the effects of pregnancy and strenuous exercise onthe independent determinants of plasmaH⁺ concentration([H⁺]). Subjects werenine physically active pregnant women [mean gestational age = 33 ± 1 (SE) wk] and 14 age-matched nonpregnant controls. Venousblood samples and respiratory data were obtained at rest and during 15 min of recovery from a maximal cycle ergometer test that involved 20 W/min increases in work rate to exhaustion. Mean values for[H⁺],PCO₂, and total protein increased,whereas those for bicarbonate concentration([HCO₃]) and the strong ion difference ([SID]) decreased in the transition fromrest to maximal exercise within both groups. At rest and throughoutpostexercise recovery, the pregnant group exhibited significantly lowermean values for PCO₂,[HCO₃], and total protein,whereas [SID] was significantly lower at rest and early recovery from exercise.[H⁺] was also lower atall sampling times in the pregnant group, but this effect wassignificant only at rest. Our results support the hypothesis thatreduced PCO₂ and weak acidconcentration are important mechanisms to regulate plasma[H⁺] and to maintain aless acidic plasma environment at rest and after exercise in lategestation compared with the nonpregnant state. These effects areestablished in the resting state and appear to be maintained aftermaximal exertion.

     

Mechanisms of HIV Entry into the CNS: Increased Sensitivity of HIV Infected CD14+CD16+ Monocytes to CCL2 and Key Roles of CCR2, JAM-A,and ALCAM in Diapedesis

As HIV infected individuals live longer, the prevalence of HIV associated neurocognitive disorders is increasing, despite successful antiretroviral therapy. CD14⁺CD16⁺ monocytes are critical to the neuropathogenesis of HIV as they promote viral seeding of the brain and establish neuroinflammation. The mechanisms by which HIV infected and uninfected monocytes cross the blood brain barrier and enter the central nervous system are not fully understood. We determined that HIV infection of CD14⁺CD16⁺ monocytes resulted in their highly increased transmigration across the blood brain barrier in response to CCL2 as compared to uninfected cells, which did not occur in the absence of the chemokine. This exuberant transmigration of HIV infected monocytes was due, at least in part, to increased CCR2 and significantly heightened sensitivity to CCL2. The entry of HIV infected and uninfected CD14⁺CD16⁺ monocytes into the brain was facilitated by significantly increased surface JAM-A, ALCAM, CD99, and PECAM-1, as compared to CD14⁺ cells that are CD16 negative. Upon HIV infection, there was an additional increase in surface JAM-A and ALCAM on CD14⁺CD16⁺ monocytes isolated from some individuals. Antibodies to ALCAM and JAM-A inhibited the transmigration of both HIV infected and uninfected CD14⁺CD16⁺ monocytes across the BBB, demonstrating their importance in facilitating monocyte transmigration and entry into the brain parenchyma. Targeting CCR2, JAM-A, and ALCAM present on CD14⁺CD16⁺ monocytes that preferentially infiltrate the CNS represents a therapeutic strategy to reduce viral seeding of the brain as well as the ongoing neuroinflammation that occurs during HIV pathogenesis.     

Soluble form of the endothelial adhesion molecule CD146 binds preferentially CD16+ monocytes

The cell adhesion molecule CD146 is normally located at the endothelial cell-to-cell junction and colocalizes with actin cytoskeleton. The soluble form of CD146 (sCD146) has been identified in the endothelial cell supernatant and in normal human plasma, and is increased in pathologic conditions with altered endothelial function. Soluble CD146 binding to monocytes promotes their transendothelial migration, which represents a central step in the development of atherosclerotic plaque. Since peripheral blood monocytes are characterized by a phenotypic and functional heterogeneity, with different transendothelial migration capacity, we hypothesized that monocyte subsets differently bind sCD146. Based on surface CD14 and CD16 expression monocytes were distinguished by flow cytometry (FACS) into three subsets: CD14++/CD16−, CD14++/CD16+ and CD14+/CD16+. CD16+ monocytes have been found to possess higher transendothelial migration ability. FACS analysis on blood monocytes from 30 healthy subjects revealed that higher percentages of CD14++/CD16+ (median, first and third quartile: 2.26, 1.62–3.87) and of CD14+/CD16+ (2.59, 1.28–4.80) were positive for CD146 (both p < 0.01), in comparison to CD14++/CD16− (0.66, 0.47–1.01). Moreover, in vitro treatment of ficoll separated monocytes with recombinant CD146 showed that both CD16+ subsets increased their percentage of CD146-positive events compared to CD16− monocytes (p < 0.01). Soluble CD146 levels were evaluated by ELISA in plasma samples of subjects from our study group and showed a correlation with percentage of CD146-positive CD14+/CD16+ monocyte subset. In this work we have demonstrated that monocyte subsets behave differently with regard to their sCD146 binding activity; because binding of CD146 influences transendothelial migration of monocytes, modulation of monocyte-CD146 interaction may represent a potential target to limit atherosclerotic plaque development.     

Contribution of Na(+)-K(+)-Cl(-) cotransporter to high-[K(+)](o)- induced swelling and EAA release in astrocytes

We hypothesized that highextracellular K⁺ concentration([K⁺]_o)-mediated stimulation ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) may result in a net gain of K⁺ and Cland thus lead to high-[K⁺]_o-induced swellingand glutamate release. In the current study, relative cell volumechanges were determined in astrocytes. Under 75 mM[K⁺]_o, astrocytes swelled by 20.2 ± 4.9%. This high-[K⁺]_o-mediated swelling wasabolished by the NKCC1 inhibitor bumetanide (10 µM, 1.0 ± 3.1%; P < 0.05). Intracellular³⁶Cl accumulation was increased from acontrol value of 0.39 ± 0.06 to 0.68 ± 0.05 µmol/mgprotein in response to 75 mM [K⁺]_o. Thisincrease was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na⁺ concentration([Na⁺]_i) was reduced from 19.1 ± 0.8 to16.8 ± 1.9 mM by bumetanide (P < 0.05).[Na⁺]_i decreased to 8.4 ± 1.0 mM under75 mM [K⁺]_o and was further reduced to5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of[Na⁺]_i on return to 5.8 mM[K⁺]_o was decreased by 40% in the presenceof bumetanide (P < 0.05). Bumetanide inhibitedhigh-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release by ~50% (P < 0.05).These results suggest that NKCC1 contributes tohigh-[K⁺]_o-induced astrocyte swelling andglutamate release.

     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

Effect of adrenergic blockade on lymphocyte cytokine production at rest and during exercise

To examine the effect of exercise andadrenergic blockade on lymphocyte cytokine production, six men ingestedeither a placebo (control) or an - (prazosin hydrochloride) and-adrenoceptor antagonist (timolol malate) capsule (blockade, or BLK)2 h before performing 19 ± 1 min of supine bicycle exerciseat 78 ± 3% peak pulmonary uptake. Blood was collected before andafter exercise, stimulated with phorbol 12-myristate 13-acetate andionomycin, and surface stained for T (CD3⁺) and naturalkiller [NK (CD3CD56⁺)] lymphocyte surfaceantigens. Cells were permeabilized, stained for the intracellularcytokines interleukin (IL)-2 and interferon (IFN)-, and analyzedusing flow cytometry. BLK had no effect on the resting concentration ofstimulated cytokine-positive T and NK lymphocytes or the amount ofcytokine they were producing. Exercise resulted in an increase (P< 0.05) in the concentration of stimulated T and NK lymphocytesproducing cytokines in the circulation, but these cells produced less(P < 0.05) cytokine post- compared with preexercise.BLK attenuated (P < 0.05) the elevation in theconcentration of lymphocytes producing cytokines during exercise;however, BLK did not affect the amount of IL-2 and IFN- produced.These results suggest that adrenergic stimulation contributes to theexercise-induced increase in the concentration of lymphocytes in thecirculation; however, it does not appear to be responsible for theexercise-induced suppression in cytokine production.

     

CD14<Superscript>++</Superscript>CD16<Superscript>−</Superscript> and CD14<Superscript>+</Superscript>CD16<Superscript>+</Superscript> human monocyte adhesion to endothelial cells

Based on the difference in the CD14 and CD16 expression, two subsets of monocytes were identified in human and other mammalian blood. These subsets have different patterns of adhesion molecules and chemokine receptors that suggests the different mode of their interaction with endothelium and tissue traffic. Here, we investigated the ability of CD14⁺CD16⁺ and CD14⁺⁺CD16⁻ monocytes to adhere to endothelial cell monolayer in presence or absence of pro- and anti-inflammatory cytokines. We demonstrated that CD14⁺CD16⁺ monocytes had a higher level of adhesion to intact monolayer of endothelial cells than CD14⁺⁺CD16⁻ monocytes. Adhesion of CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes significantly increased in the presence of TNFα or its combination with other cytokines. IFNγ and IL-4 alone did not affect the adhesion of monocytes. These results show that CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes can be recruited to the inflamed endothelium, but CD14⁺CD16⁺ monocytes adhere to endothelial cells without inflammations twice as strongly as CD14⁺⁺CD16⁻ monocytes.     

Increased Protease-Activated Receptor-2 (PAR-2) Expression on CD14++CD16+ Peripheral Blood Monocytes of Patients with Severe Asthma

Background

Protease-Activated Receptor-2 (PAR-2), a G protein coupled receptor activated by serine proteases, is widely expressed in humans and is involved in inflammation. PAR-2 activation in the airways plays an important role in the development of allergic airway inflammation. PAR-2 expression is known to be upregulated in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells in patients with asthma has not been studied.

Methods

We recruited 12 severe and 24 mild/moderate asthmatics from the University of Alberta Hospital Asthma Clinics and collected baseline demographic information, medication use and parameters of asthma severity. PAR-2 expression on blood inflammatory cells was analyzed by flow cytometry.

Results

Subjects with severe asthma had higher PAR-2 expression on CD14⁺⁺CD16⁺ monocytes (intermediate monocytes) and also higher percentage of CD14⁺⁺CD16⁺PAR-2⁺ monocytes (intermediate monocytes expressing PAR-2) in blood compared to subjects with mild/moderate asthma. Receiver operating characteristics (ROC) curve analysis showed that the percent of CD14⁺⁺CD16⁺PAR-2⁺ in peripheral blood was able to discriminate between patients with severe and those with mild/moderate asthma with high sensitivity and specificity. In addition, among the whole populations, subjects with a history of asthma exacerbations over the last year had higher percent of CD14⁺⁺CD16⁺ PAR-2⁺ cells in peripheral blood compared to subjects without exacerbations.

Conclusions

PAR-2 expression is increased on CD14⁺⁺CD16⁺ monocytes in the peripheral blood of subjects with severe asthma and may be a biomarker of asthma severity. Our data suggest that PAR-2 -mediated activation of CD14⁺⁺CD16⁺ monocytes may play a role in the pathogenesis of severe asthma.     

Is urodilatin the missing link in exercise-dependent renal sodium retention?

Schmidt, W., A. Bub, M. Meyer, T. Weiss, D. Schneider, N. Maassen, and W. G. Forssmann. Is urodilatin the missing link inexercise-dependent renal sodium retention? J. Appl.Physiol. 84(1): 123-128, 1998.The purpose of thepresent study was to investigate the behavior of plasma atrialnatriuretic peptide [ANP-(99126)] concentration([ANP]) and renal urodilatin [Uro; ANP-(95126)] excretion during and after exercise and theirpossible effects on renal Na⁺retention. Ten male subjects performed a cycle ergometer test for 60 min at 60% of maximum workload. Blood and urine samples were collectedbefore, during, and up to 24 h after exercise. During exercise, plasma[ANP] and renal Uro excretion were oppositely affected:whereas [ANP] increased from 46.5 ± 5.1 to 124.1 ± 10.6 pg/ml, urinary Uro excretion decreased from 120.8 ± 16.0 to49.5 ± 9.8 fmol/min and remained at a lower level until 1 h afterexercise. Glomerular filtration rate showed lowest values duringexercise (from 164.9 ± 15.3 to 75.8 ± 10.1 ml/min), and urineflow and the fractional excretion rate ofNa⁺(FE_Na⁺) andCl()had their nadir during the first hour after exercise. Positiverelationships were observed between Uro excretion andFE_Na⁺(P < 0.05) and, whereas a tendency toward a negative correlation was obtained between[ANP] andFE_Na⁺. It seemspossible that Uro may be, among other factors, involved in theexercise-related regulation of renalNa⁺ retention. The specific rolesUro and ANP play during exercise, however, remain to be investigated.

     

Perturbation of the pump-leak balance for Na(+) and K(+) in malaria-infected erythrocytes

In humanerythrocytes infected with the mature form of the malaria parasitePlasmodium falciparum, the cytosolic concentration ofNa⁺ is increased and that of K⁺ is decreased.In this study, the membrane transport changes underlying thisperturbation were investigated using a combination of⁸⁶Rb⁺, ⁴³K⁺, and²²Na⁺ flux measurements and a semiquantitativehemolysis technique. From >15 h postinvasion, there appeared in theinfected erythrocyte membrane new permeation pathways (NPP) that causeda significant increase in the basal ion permeability of theerythrocyte membrane and that were inhibited by furosemide (0.1 mM). The NPP showed the selectivity sequenceCs⁺ > Rb⁺ > K⁺ > Na⁺, with the K⁺-to-Na⁺permeability ratio estimated as 2.3. From 18 to 36 h postinvasion, the activity of the erythrocyte Na⁺/K⁺ pumpincreased in response to increased cytosolic Na⁺ (aconsequence of the increased leakage of Na⁺ via the NPP)but underwent a progressive decrease in the latter 12 h of theparasite's occupancy of the erythrocyte (36-48 h postinvasion). Incorporation of the measured ion transport rates into a mathematical model of the human erythrocyte indicates that the induction of the NPP,together with the impairment of the Na⁺/K⁺pump, accounts for the altered Na⁺ and K⁺levels in the host cell cytosol, as well as predicting an initial decrease, followed by a lytic increase in the volume of the host erythrocyte.

     

Receptor-mediated inhibition of renal Na+-K+-ATPase is associated with endocytosis of its alpha - and beta -subunits

The mechanisms involved in receptor-mediated inhibition ofNa⁺-K⁺-ATPaseremain poorly understood. In this study, we evaluate whether inhibitionof proximal tubuleNa⁺-K⁺-ATPaseactivity by dopamine is linked to its removal from the plasma membraneand internalization into defined intracellular compartments.Clathrin-coated vesicles were isolated by sucrose gradientcentrifugation and negative lectin selection, and early and lateendosomes were separated on a flotation gradient. Inhibition ofNa⁺-K⁺-ATPaseactivity by dopamine, in contrast to its inhibition by ouabain, wasaccompanied by a sequential increase in the abundance of the-subunit in clathrin-coated vesicles (1 min), early endosomes (2.5 min), and late endosomes (5 min), suggesting its stepwise translocationbetween these organelles. A similar pattern was found for the-subunit. The increased incorporation of both subunits in allcompartments was blocked by calphostin C. The results demonstrate thatthe dopamine-induced decrease inNa⁺-K⁺-ATPaseactivity in proximal tubules is associated with internalization of its- and -subunits into early and late endosomes via aclathrin-dependent pathway and that this process is protein kinase Cdependent. The presence ofNa⁺-K⁺-ATPasesubunits in endosomes suggests that these compartments may constitutenormal traffic reservoirs during pump degradation and/orsynthesis.

     

Astrocytes from Na(+)-K(+)-Cl(-) cotransporter-null mice exhibit absence of swelling and decrease in EAA release

We reported previously that inhibition ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K⁺concentration ([K⁺]_o)-induced swelling andintracellular Cl accumulation in rat cortical astrocytes.In this report, we extended our study by using cortical astrocytes fromNKCC1-deficient (NKCC1^/) mice. NKCC1 protein andactivity were absent in NKCC1^/ astrocytes.[K⁺]_o of 75 mM increased NKCC1 activityapproximately fourfold in NKCC1^+/+ cells (P < 0.05) but had no effect in NKCC1^/ astrocytes.Intracellular Cl was increased by 70% inNKCC1^+/+ astrocytes under 75 mM[K⁺]_o (P < 0.05) butremained unchanged in NKCC1^/ astrocytes. Baselineintracellular Na⁺ concentration([Na⁺]_i) in NKCC1^+/+ astrocyteswas 19.0 ± 0.5 mM, compared with 16.9 ± 0.3 mM[Na⁺]_i in NKCC1^/ astrocytes(P < 0.05). Relative cell volume ofNKCC1^+/+ astrocytes increased by 13 ± 2% in 75 mM[K⁺]_o, compared with a value of 1.0 ± 0.5% in NKCC1^/ astrocytes (P < 0.05).Regulatory volume increase after hypertonic shrinkage was completelyimpaired in NKCC1^/ astrocytes.High-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release was reduced by ~30% inNKCC1^/ astrocytes. Our study suggests that stimulationof NKCC1 is required for high-[K⁺]_o-inducedswelling, which contributes to glutamate release from astrocytes underhigh [K⁺]_o.