RESEARCH ARTICLE


Involvement of Signaling Molecules on Na+/H+ Exchanger-1 Activity in Human Monocytes



Maria Sarigianni 1, 2, Apostolos Tsapas 1, 3, Dimitri P Mikhailidis 2, Martha Kaloyianni 4, George Koliakos 5, Konstantinos Paletas *, 1
1 Metabolic Diseases Unit, Second Department of Internal Medicine, Medical School, Aristotle University of Thessaloniki, Greece
2 Department of Clinical Biochemistry (Vascular Disease Prevention Clinics), Royal Free Hospital campus, University College London Medical School, University College London (UCL), London NW3 2QG, UK
3 University of Oxford, The Tseu Medical Institute, Harris Manchester College, Oxford, United Kingdom
4 Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, Greece
5 Department of Biological Chemistry, Medical School, Aristotle University of Thessaloniki, Greece


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© Sarigianni et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to this author at the 15 Ag. Sofias str, 54623, Thessaloniki, Greece; Tel: 00302310892751 Fax: 00302310992937 E-mail: paletas@med.auth.gr
# The paper has not been presented orally in any professional meeting.

This paper is part of the 03ED29 research project, implemented within the framework of the “Reinforcement Programme of Human Research Manpower” (PENED) and cofinanced by National and European Community Funds (25% from the Greek Ministry of Development - General Secretariat of Research and Technology and 75% from the EU - European Social Fund).



Abstract

Background:

Sodium/hydrogen exchanger-1 (NHE-1) contributes to maintaining intracellular pH (pHi). We assessed the effect of glucose, insulin, leptin and adrenaline on NHE-1 activity in human monocytes in vitro. These cells play a role in atherogenesis and disturbances in the hormones evaluated are associated with obesity and diabetes.

Methods and Results:

Monocytes were isolated from 16 healthy obese and 10 lean healthy subjects. NHE-1 activity was estimated by measuring pHi with a fluorescent dye. pHi was assessed pre- and post-incubation with glucose, insulin, leptin and adrenaline. Experiments were repeated after adding a NHE-1 inhibitor (cariporide) or an inhibitor of protein kinase C (PKC), nitric oxide synthase (NOS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, phosphoinositide 3-kinases (PI3K) or actin polymerization. Within the whole study population, glucose enhanced NHE-1 activity by a processes involving PKC, NOS, PI3K and actin polymerization (p = 0.0006 to 0.01). Insulin-mediated activation of NHE-1 (p = <0.0001 to 0.02) required the classical isoforms of PKC, NOS, NADPH oxidase and PI3K. Leptin increased NHE-1 activity (p = 0.0004 to 0.04) through the involvement of PKC and actin polymerization. Adrenaline activated NHE-1 (p = <0.0001 to 0.01) by a process involving the classical isoforms of PKC, NOS and actin polymerization. There were also some differences in responses when lean and obese subjects were compared. Incubation with cariporide attenuated the observed increase in NHE-1 activity.

Conclusions:

Selective inhibition of NHE-1 in monocytes could become a target for drug action in atherosclerotic vascular disease.

Keywords: Na+/H+ exchanger-1, obesity, monocytes, intracellular pH..



INTRODUCTION

The sodium/hydrogen exchanger-1 (NHE-1) is a ubiquitous integral membrane protein expressed in mammalian cells [1]. Its main role is intracellular pH (pHi) maintenance, achieved by exchanging 1 intracellular H+ for 1 extracellular Na+ [1]. It is also important in cell volume maintenance and cytoskeletal reorganization. Furthermore, it takes part in cell proliferation, apoptosis and migration [1]. NHE-1 is stimulated by intracellular acidosis, hormones and growth factors and inhibited by amiloride derivatives [2].

The role of NHE-1 inhibitors in cardiovascular disease has been investigated during the past decade. In animal models, cariporide in combination with metoprolol decreased myocardial infarction size [3]. However, human clinical trials did not show clinical benefit of NHE-1 inhibition [4].

NHE-1 overactivity is documented in overweight and obese subjects and correlated well with body mass index (BMI) but not with plasma insulin levels [5]. In obese animal models inhibition of NHE-1 improved insulin sensitivity and endothelial function [6]. Furthermore, human erythrocytes obtained from obese subjects NHE-1 were activated by insulin [7], leptin [8] and adrenaline [9] and by glucose [10] in healthy individuals. In monocytes obtained from healthy subjects, NHE-1 was activated by glucose [11] and leptin [12]. However, the influence of obesity on NHE-1 activity and its signaling pathway in human monocytes remains poorly documented. NHE-1 is involved in monocyte adhesion, migration and oxidized low density lipoprotein (oxLDL) phagocytosis under the influence of mediators such as glucose, insulin, leptin and adrenaline [13-17]. In addition to obesity, NHE-1 activation could contribute to the increased cardiovascular risk associated with insulin resistance and type 2 diabetes mellitus [18, 19].

We investigated the effect of high concentrations of glucose, insulin, leptin and adrenaline on the activity of NHE-1 by measuring the pHi in monocytes obtained from lean and obese healthy subjects. Furthermore, we investigated the role of protein kinase C (PKC), nitric oxide (NO) synthase (NOS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, phosphoinositide 3-kinases (PI3K) and actin polymerization on NHE-1 activity.

MATERIALS AND METHODS

Materials

Source of reagents were described elsewhere [13, 14] apart from the following: 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluoresceinacetoxymethyl ester (BCECF/AM) was purchased from AppliChem (Darmstadt, Hesse, Germany). 4,4΄-di-isothiocyanato stilbene-2,2΄-disulfonic acid (DIDS), nigericin, methazolamide, iodoacetic acid, DPI (diphenyleneiodonium chloride), L-NAME (Nω-Nitro-L_arginine methyl ester hydrochloride) were obtained from Sigma (St. Louis, MO, USA). GF109203X and Gö6976 were purchased by Alexis (Lausen, Switzerland). Cytochalasin-D was obtained by Fluka (Seelze, Germany). All other reagents were of analytical grade and were obtained from commercial sources.

Subjects

Healthy obese [n = 16; BMI ≥30 Kg/m2] subjects aged between 18-35 years (13 female) attending the obesity outpatient clinic and 10 healthy lean (BMI <25 Kg/m2) age-matched subjects from the hospital staff (8 female), were enrolled in the study. None of the participants were taking any medication. All participants gave their written informed consent in accordance with the Declaration of Helsinki. This patient population was used in a previous study to assess the effect of leptin, adrenaline, insulin and glucose on monocyte function (adhesion, migration, CD36 expression, oxidized low density lipoproteins phagocytosis) [13, 14, 17].

Anthropometric measurements (body weight, height and waist circumference) and blood pressure were recorded by the same examiner and were reported elsewhere [13, 14, 17].

Study Protocol

Blood was collected after an overnight fast and distributed as previously described [13, 14, 17]. Measurement of pHi (as an estimate of NHE-1 activity) was assayed in isolated monocytes pre- and post- incubation with glucose, insulin, leptin or adrenaline. The glucose (20 mmol/l), insulin (50 μU/ml), leptin (160 ng/ml) and adrenaline (520 pmol/l) concentrations used were similar to those in previous studies [11, 13, 14, 16, 17]. All experiments were repeated after adding cariporide, a NHE-1 inhibitor. In order to investigate the signaling molecules involved the experiments were repeated after adding inhibitors of classical isoforms of PKC (Gö6976), all isoforms of PKC (GF10923X), NOS (L-NAME), NADPH oxidase (DPI), PI3K (wortmannin) and actin polymerization (cytochalasin-D).

Each experiment included 10 lean subjects and 12 obese subjects. The 12 obese subjects were randomly selected from the 16 available for each experiment. In the obese group, 8 subjects were insulin sensitive and 8 were insulin resistant as defined by euglycemic hyperinsulinemic clamp [14]. In order to investigate the influence of obesity, we performed a subgroup analysis of the lean (n = 10) and obese (n = 12) subjects.

Monocyte Isolation

Monocytes were isolated as previously described [13, 14] (Ficoll-Paque Plus followed by 46% iso-osmotic Percoll in CM). Monocyte purity measured using a Beckman Coulter (Brea, California, USA) EPICS XL-MCL flow cytometer and CD14 antibody was > 85%.

Measurement of pHi

pHi was measured using BCECF-AM [10, 11, 20]. Monocytes were washed 3 times with PBS (phosphate buffer solution) and then suspended in HCO3-free buffer NaCl (135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 20 mM HEPES; pH 7.3) to deplete them of HCO3-. This buffer was used for all determinations of pH unless otherwise stated. The monocytes were then loaded with BCECF-AM (1 mg/ml per 106 cells) and incubated for 30 min at 37oC in the dark. After incubation with the fluorescent dye monocytes were washed 5 times at room temperature at 1500 rpm with NaCl medium in order to remove the unbound fluorescent dye and suspended to the desired concentration (106 cells/ml). DIDS (0.125 mM) and methazolamide (0.4 mM) were added in order to avoid HCO3-/Cl- anion exchanger interference. Iodoxic Na (1 mM) was added to suppress glycolysis. The 10 μl of the inhibitor [Gö6976 (500 nM) inhibits α, β and γ isoforms of PKC, GF109203X (10 μM) inhibits all isoforms of the PKC, L-NAME (100 μM) inhibits NOS, DPI (10 μM) inhibits NADPH oxidase, wortmannin (50 nM) inhibits PI3K, cytochalasin D (2 μM) inhibits actin polymerization, cariporide (20 nM) inhibits the action of NHE-1] were added, when appropriate. An incubation for 30 min at 37oC in the dark followed. Then 10 μl of the mediactors [glucose (20 mmol/l), insulin (50 μU/ml), leptin (160 ng/ml) and adrenaline (520 pmol/l)] were added and incubated for 15 min at 37oC. Fluorescence was measured in a FL WINLab luminescence spectrometer (PerkinElmer, Waltham, MA, USA) in a 96-well black polystyrene plate with excitation and emission wavelengths set at 495 and 530 nm respectively, using 2.5 nm slit. Routinely, fluorescence was also measured with excitation wavelength set at 440 nm. At this wavelength fluorescence is proportional to intracellular dye concentration and is relatively pH-insensitive. Data were obtained as the ratio of the fluorescence at pH-sensitive excitation wavelength 495 nm and the fluorescence at the pH-insensitive excitation wavelength 440 nm.

Routinely calibration of fluorescence to pH was carried out by suspending the cells in K+ solutions (KCl 130 nM, MgCl2 1 mM, HEPES 30 mM) at 3 different pH values (6.7, 7 and 7.3) [21].The concentration of cells was 106 cells/ml. The cells were then washed with the same buffer and nigericin (13 μM) was added to each well. Nigericin promotes ion-channel opening equalizing pHi to the extracellular pH. Fluorescence was measured 5 min after nigericin addition. The curve obtained between the ratio of fluorescence and pH was linear. The pH of each solution was measured by a pH meter (Accumet, Fischer Scientific, Hampton, New Hampshire, USA).

Statistical Analysis

Statistical analysis was performed using SPSS v 15.0 for Windows (SPSS Inc., Chicago, Illinois). All values are expressed as mean ± standard deviation (SD), unless otherwise noted. Normality was tested with the Shapiro-Wilk test. Differences in continuous variables were assessed by means of 1-way analysis of variance, Student’s t-test or paired t-test where appropriate. When necessary, non-parametric tests were used. A 2-tailed p < 0.05 was considered significant.

RESULTS

Anthropometric Characteristics

As previously described [13, 14], the mean age of the subjects was 29 ± 3 years; there was no difference between the lean (n = 10) and the obese group (n = 16) (p > 0.1). The BMI was 23.8 ± 0.9 kg/m2 for the lean and 37.9 ± 7.1 kg/m2 for the obese subjects. The mean waist circumference was 86 ± 2 cm for the lean and 112 ± 13 cm for the obese subjects.

Measurement of pHi

Incubation with Glucose in the Overall Population

Glucose significantly increased pHi (p <0.0001). Cariporide inhibited glucose-induced increase in pHi (p = 0.003). Monocyte incubation with Gö6976, GF109203X, L-NAME, wortmannin or cytochalasin-D inhibited the glucose-induced increase in pHi (p = 0.008, p = 0.002, p = 0.0006, p= 0.01 and p = 0.001, respectively). The addition of DPI inhibited the glucose-induced increase in pHi but this was not significant (p = 0.056).

Incubation with Glucose: Subgroup Analysis, Lean and Obese

Glucose significantly increased pHi in both groups (p = 0.003 in lean and p = 0.006 in obese subjects). Cariporide inhibited glucose-induced increase in pHi in both groups (p = 0.005 in lean and p = 0.03 in obese subjects). Monocyte incubation with GF109203X, L-NAME or cytochalasin-D inhibited the glucose-induced increase in pHi in both groups (p <0.04). However, after adding Gö6976 or wortmannin the glucose-induced increase in pHi was significantly attenuated only in lean subjects (p = 0.008 and p = 0.034, respectively). The addition of DPI inhibited the glucose-induced increase in pHi only in obese subjects but this was not significant (p = 0.061).

Incubation with Insulin in the Overall Population

Insulin significantly increased pHi (p <0.0001). Cariporide inhibited insulin-induced increase in pHi (p = 0.002). Monocyte incubation with Gö6976, L-NAME, DPI or wortmannin inhibited insulin-induced increase in pHi (p = 0.002, p = 0.008, p = 0.02 and p = 0.005, respectively). Cytochalasin-D inhibited insulin-induced increase in pHi but this was not significant (p = 0.07). The addition of GF109203X had no effect on pHi.

Incubation with Insulin: Subgroup Analysis, Lean and Obese

Insulin significantly increased pHi in both groups (p = 0.02 in lean and p = 0.003 in obese subjects). Cariporide inhibited insulin-induced increase in pHi in both groups (p = 0.03 in lean and p = 0.039 in obese subjects). Monocyte incubation with Gö6976 or L-NAME inhibited the insulin-induced increase in pHi in both groups (p <0.05). After adding wortmannin insulin-induced increase in pHi was significantly attenuated only in lean subjects (p = 0.04). Incubation with DPI inhibited insulin-induced increase in pHi only in obese subjects (p = 0.037). The addition of GF109203X or cytochalasin-D had no effect on pHi.

Incubation with Leptin in the Overall Population

Leptin significantly increased pHi (p = 0.0004). Cariporide inhibited leptin-induced increase in pHi (p = 0.001). Monocyte incubation with Gö6976 or cytochalasin-D inhibited leptin-induced increase in pHi (p = 0.003 and p = 0.04, respectively). The addition of GF109203X, L-NAME, DPI or wortmannin had no effect on pHi.

Incubation with Leptin: Subgroup Analysis, Lean and Obese

Leptin significantly increased pHi in both groups (p = 0.016 in lean and p = 0.04 in obese subjects). Cariporide inhibited leptin-induced increase in pHi in both groups (p = 0.03 in lean and p = 0.02 in obese subjects). Monocyte incubation with Gö6976 inhibited leptin-induced increase in pHi in both groups (p = 0.001 in lean and p = 0.033 in obese subjects). However, after adding cytochalasin-D leptin-induced increase in pHi was attenuated only in obese subjects (p = 0.032). The addition of GF109203X or L-NAME did not significantly inhibit leptin-induced increase in pHi only in obese subjects (p = 0.052 and p = 0.058, respectively). The addition of DPI or wortmannin had no effect on pHi.

Incubation with Adrenaline in the Overall Population

Adrenaline significantly increased pHi (p <0.0001). Cariporide inhibited adrenaline-induced increase in pHi (p = 0.0004). Monocyte incubation with Gö6976, L-NAME or cytochalasin-D inhibited adrenaline-induced increase in (p = 0.001, p = 0.01 and p = 0.01, respectively). The addition of GF109203X, DPI or wortmannin had no effect on pHi.

Incubation with Adrenaline: Subgroup Analysis, Lean and Obese

Adrenaline significantly increased pHi in both groups (p = 0.006). Cariporide inhibited adrenaline-induced increase in pHi in both groups (p = 0.01 in lean and p = 0.047 in obese subjects). Monocyte incubation with Gö6976 inhibited adrenaline-induced increase in pHi in both groups (p = 0.024 in lean and p = 0.017 in obese subjects). However, adding L-NAME, wortmannin or cytochalasin-D significantly attenuated adrenaline-induced increase in pHi only in lean subjects (p = 0.004, p = 0.003 and p = 0.031, respectively). The addition of GF109203X or DPI had no effect on pHi.

DISCUSSION

Similar to our findings, high concentrations of glucose increased NHE-1 activity in human umbilical vein endothelial cells [22] and in human monocytes [11]. There is evidence that a higher level of glucose (5 vs 20 mM) results in significantly (p < 0.001) greater increase in pHi in monocytes obtained from healthy subjects [11]. This effect was assessed by inhibition of ethylisopropyl amiloride (EIPA) [11]. Furthermore, glucose induced hypertrophy of cardiomyocytes through the involvement of NHE-1 [23].

Inhibition of NHE-1 prevented insulin-induced glucose uptake by rat ventricular cardiomyocytes [24]. In contrast, in insulin resistant whole animal models cariporide improved insulin sensitivity [6]. Furthermore, insulin phosphorylated NHE-1 in 3T3-L1 adipocytes [25] and increased its activity in cardiomyocytes [24] and in human erythrocytes [7]. A controversial finding was reported in vascular endothelial and smooth muscle cells, where insulin inhibited NHE (mainly NHE-1) activity [26]. In our study, insulin activated NHE-1 and was inhibited by cariporide.

Leptin increased NHE-1 activity, a finding supported by previous studies in human erythrocytes [8]. In human monocytes obtained from healthy donors, leptin-induced increase in adhesion, migration, CD36 expression and oxLDL phagocytosis was mediated by NHE-1 (i.e. inhibited by cariporide) [16]. In the latter study, the role of several signaling molecules was also assessed. However, pHi was not measured. In another study, leptin-induced increase in pHi showed a dose-response pattern [12]. However, the role of signaling molecules was not assessed [12].

Another hormone that activated NHE-1 is adrenaline, which was also reported to increase NHE-1 activity in erythrocytes [9] and in cardiomyocytes [27].

We have previously shown that cariporide (a NHE-1 inhibitor) eliminated leptin-, adrenaline- glucose- and insulin-induced increase in adhesion, migration of monocytes, CD36 expression and monocyte phagocytosis of oxidized-LDL in some patient groups [11, 13-17]. Furthermore, cariporide may exert anti-atherogenic effects [15, 28] by inhibiting monocyte adhesion and expression of intercellular adhesion molecule (ICAM) [29]. The potential effect of cariporide on monocytes could be mediated through NHE-1 inhibition [13, 14, 17] or another action. However, these promising experimental results were not supported by the findings of a clinical trial. In the EXPEDITION (for Na+/H+ Exchange inhibition to Prevent coronary Events in acute cardiac condition) trial cariporide administration to coronary artery bypass graft patients (n = 5761) resulted in significantly less death + myocardial infarction compared with the placebo group (20.3% vs 16.6%; p = 0.0002) [4]. However, there was a significant increase in mortality mainly due to cerebrovascular events [1.5% in the placebo group vs 2.2% with cariporide (p = 0.02)] [4]. Therefore, it is unlikely that cariporide will be further investigated it is unlikely that cariporide will be further investigated [4].

We found that PKC and NOS were involved in the NHE-1 signaling pathway in all subjects. Similarly, PKC inhibition decreased NHE-1activity in bovine neutrophils [30]. Furthermore, in animal cardiomyocytes, PKC was involved in NHE-1 inhibition [31]. Ex vivo studies in human monocytes and erythrocytes indicate an involvement of PKC in NHE-1 activation by glucose [10] and insulin [7]. An interaction between NOS and NHE-1 is supported by a previous animal study where inhibition of NHE-1 resulted in decreased activity of neuronal NOS [32]. In contrast, inhibition of NOS was associated with increased NHE-3 mRNA and protein in other cells [33]. However, monocytes do not express NHE-3 [34]. Therefore, different isoforms of NHE may be inhibited/stimulated by the same mediator. The response may also depend on the location of the NHE.

PI3K was involved in NHE-1 activation by glucose, insulin and adrenaline only in lean subjects which could reflect a dysfunction of this kinase in the obese subjects. PI3K was involved in NHE-1 activation pathway in monocytes [16] and its activity was decreased in human skeletal muscle cells from insulin resistant obese subjects [35]. Furthermore, there is a regulatory disruption between the subunits of PI3K in obesity resulting in alteration of PI3K function [36]. The disrupted function of PI3K was also found in animal models of diet-induced obesity [37]. Alternatively, our findings may reflect the low numbers of subjects in the subgroup analysis.

Actin polymerization is involved in NHE-1 activation by leptin. The interaction of NHE-1 with cytoskeleton is well established [38] and seems to be bidirectional. NHE-1 is important for de novo assembly of actin filaments [39, 40] and its activation may contribute to actin-filaments organization [41, 42]. On the other hand, NHE-1 localization in the plasma membrane requires an intact actin cytoskeleton [43]. The cytoskeleton may differ in obese subjects since in animal models a high fat meal decreased the mRNA of cytoskeleton [44]. Furthermore, obesity commonly coexists with insulin resistance and hyperinsulinemia. Altered structure of F-actin was found in insulin resistance [45] and exposure to insulin led to actin filament rearrangement in skeletal muscle cells [46] and 3T3-L1 adipocytes [47].

Leptin is another hormone that is increased in obesity and it influences actin cytoskeletal dynamics in hippocampal neurons [48] and in vascular tissue [49]. It is therefore of interest that in our subgroup analysis, the leptin-induced increase in NHE-1 activity was reduced by cytochalasin-D only in the obese patients.

NADPH oxidase is involved in NHE-1 activation by insulin. It is known that NHE-1 and NADPH oxidase interact in a reciprocal way. NADPH oxidase stimulation produces acid thus activating NHE-1 which alkalinizes the pH [50]. Furthermore, in animal models pH neutralization is essential for NADPH oxidase action [51]. DPI inhibited NADPH oxidase and the activity of NHE-1 [52]. NADPH oxidase activity could be different in obese subjects since obese animal models exhibit increased NADPH oxidase-production of superoxide [53]. Furthermore, in monocytes obtained from obese subjects a prolonged production of reactive oxygen species was reported [54].

The differences we observed between lean and obese subjects suggest that obese subjects could have signaling defects in NHE-1 activation pathways. Alternatively, these differences could reflect the small sample size and the fact that among the obese group some individuals were insulin sensitive while others were insulin resistant. This is a limitation of our study but our findings allow power calculations for future studies.

In previous studies we found that rosiglitazone inhibited some NHE-1-mediated actions in monocytes. It was suggested that rosiglitazone could have an ion-transport action [55] and act on NHE-1 [13]. Furthermore, there is a peroxisome proliferator-activated receptor γ (PPARγ) element in the promoter region of NHE-1 [56] indicating a possible interaction between them. It would also be of interest in future studies to investigate whether rosiglitazone can directly influence NHE-1 activity.

It would be of interest to assess the effect of other mediators that are abnormal in obesity (e.g. adiponectin, resistin, ghrelin and visfatin) [57, 58] on NHE-1 activity.

Glucose, insulin, leptin and adrenaline may be increased in obese subjects [59, 60] and monocytes are involved in atherogenesis [61]. Inhibition of the action of these mediators on NHE-1 in lean and obese subjects may be beneficial in the prevention and treatment of atherogenesis. NHE-1 and the signaling molecules involved in its activation are potential therapeutic targets in obesity.

Fig. (1).

Intracellular pH (pHi) in human monocytes. Glucose was added and pHi was estimated. Monocytes were pre-incubated with cariporide or one of the inhibitors (Gö6976 inhibits α, β and γ isoforms of PKC, GF109203X inhibits all isoforms of the PKC, L-NAME inhibits NOS, DPI inhibits NADPH oxidase, wortmannin inhibits PI3K, cytochalasin D inhibits actin polymerization) and then glucose was added. Error bars indicate standard deviation (SD).

* p < 0.05 vs the respective baseline sample (control sample)

# p < 0.05 vs the respective glucose sample


Fig. (2).

Intracellular pH (pHi) in human monocytes. Insulin was added and pHi was estimated. Monocytes were pre-incubated with cariporide or one of the inhibitors (Gö6976 inhibits α, β and γ isoforms of PKC, GF109203X inhibits all isoforms of the PKC, L-NAME inhibits NOS, DPI inhibits NADPH oxidase, wortmannin inhibits PI3K, cytochalasin D inhibits actin polymerization) and then insulin was added. Error bars indicate standard deviation (SD).

* p < 0.05 vs the respective baseline sample (control sample)

# p < 0.05 vs the respective insulin sample


Fig. (3).

Intracellular pH (pHi) in human monocytes. Leptin was added and pHi was estimated. Monocytes were pre-incubated with cariporide or one of the inhibitors (Gö6976 inhibits α, β and γ isoforms of PKC, GF109203X inhibits all isoforms of the PKC, L-NAME inhibits NOS, DPI inhibits NADPH oxidase, wortmannin inhibits PI3K, cytochalasin D inhibits actin polymerization) and then leptin was added. Error bars indicate standard deviation (SD).

* p < 0.05 vs the respective baseline sample (control sample)

# p < 0.05 vs the respective leptin sample


Fig. (4).

Intracellular pH (pHi) in human monocytes. Adrenaline was added and pHi was estimated. Monocytes were pre-incubated with cariporide or one of the inhibitors (Gö6976 inhibits α, β and γ isoforms of PKC, GF109203X inhibits all isoforms of the PKC, L-NAME inhibits NOS, DPI inhibits NADPH oxidase, wortmannin inhibits PI3K, cytochalasin D inhibits actin polymerization) and then adrenaline was added. Error bars indicate standard deviation (SD).

* p < 0.05 vs the respective baseline sample (control sample)

# p < 0.05 vs the respective adrenaline sample


ACKNOWLEDGEMENTS

M.S. is supported by a grant awarded by the Hellenic Atherosclerosis Society. A.T. is a Senior Research Fellow of the Tseu Medical Institute within Harris Manchester College, Oxford, United Kingdom, and acknowledges funding support.

ABBREVIATIONS

BCECF-AM  = 2´,7´-Bis-(Carboxyethyl)-5(6)-Carboxy-fluoresceinacetoxymethyl Ester
BMI  = Body Mass Index
CM  = Culture Medium
CPDA  = Citrate Phosphate Dextrose Adenine
DIDS  = 4,4´-Di-Isothiocyanatostilbene-2,2´- Disulfonic Acid
DPI  = Diphenyleneiodonium Chloride
DTPA  = Diethylenetriamine-Pentaacetic Acids
FCS  = Fetal Calf Serum
HDL-C  = High Density Lipoprotein Cholesterol
HEPES  = N-2-Hydroxyethylpiperazine-N΄-2 Ethanesulfonic Acid
IMDM  = Iscove’s Modified Dulbecco’s Medium
L-NAME  = Nω-Nitro-L_Arginine Methyl Ester Hydrochloride
NADPH  = Nicotinamide Adenine Dinucleotide Phosphate
NHE-1  = Na+/H+ Exchanger-1
PBS  = Phosphate Buffered Saline
PI3K  = Phosphoinositide 3-Kinases
PKC  = Protein Kinase C

REFERENCES

[1] Putney LK, Denker SP, Barber DL. The changing face of the Na+/H+ exchanger, NHE1: structure, regulation, and cellular actions Annu Rev Pharmacol Toxicol 2002; 42: 527-.
[2] Orlowski J, Grinstein S. Diversity of the mammalian sodium/proton exchanger SLC9 gene family Pflugers Arch 2004; 447: 549-65.
[3] Wang P, Zaragoza C, Holman W. Sodium-hydrogen exchange inhibition and beta-blockade additively decrease infarct size Ann Thorac Surg 2007; 83: 1121-7.
[4] Mentzer RM Jr, Bartels C, Bolli R, et al. Sodium-hydrogen exchange inhibition by cariporide to reduce the risk of ischemic cardiac events in patients undergoing coronary artery bypass grafting: results of the EXPEDITION study Ann Thorac Surg 2008; 85: 1261-70.
[5] Delva P, Pastori C, Provoli E, et al. Erythrocyte Na+-H+ exchange activity in essential hypertensive and obese patients: role of excess body weight J Hypertens 1993; 11: 823-30.
[6] Russell JC, Proctor SD, Kelly SE, et al. Insulin-sensitizing and cardiovascular effects of the sodium-hydrogen exchange inhibitor, cariporide, in the JCR: LA-cp rat and db/db mouse J Cardiovasc Pharmacol 2005; 46: 746-53.
[7] Kaloyianni M, Bourikas D, Koliakos G. The effect of insulin on Na+-H+ antiport activity of obese and normal subjects erythrocytes Cell Physiol Biochem 2001; 11: 253-8.
[8] Konstantinou-Tegou A, Kaloyianni M, Bourikas D, Koliakos G. The effect of leptin on Na(+)-H(+) antiport (NHE 1) activity of obese and normal subjects erythrocytes Mol Cell Endocrinol 2001; 183: 11-8.
[9] Bourikas D, Kaloyianni M, Bougoulia M, Zolota Z, Koliakos G. Modulation of the Na(+)-H(+) antiport activity by adrenaline on erythrocytes from normal and obese individuals Mol Cell Endocrinol 2003; 205: 141-50.
[10] Kaloyianni M, Tsagias N, Liakos P, et al. Stimulation of Na+/H+ antiport and pyruvate kinase activities by high glucose concentration in human erythrocytes Mol Cells 2004; 17: 415-21.
[11] Koliakos G, Zolota Z, Paletas K, Kaloyianni M. High glucose concentrations stimulate human monocyte sodium/hydrogen exchanger activity and modulate atherosclerosis-related functions Pflugers Arch 2004; 449: 298-306.
[12] Konstantinidis D, Paletas K, Koliakos G, Kaloyianni M. The ambiguous role of the Na+-H+ exchanger isoform 1 (NHE1) in leptin-induced oxidative stress in human monocytes Cell Stress Chaperones 2009; 14: 591-601.
[13] Sarigianni M, Bekiari E, Tsapas A, et al. Effect of epinephrine and insulin resistance on human monocytes obtained from lean and obese healthy subjects: a pilot study Angiology 2010. In press
[14] Sarigianni M, Bekiari E, Tsapas A, et al. Effect of leptin and insulin resistance on properties of human monocytes in lean and obese healthy subjects Angiology 2010. In press
[15] Kaloyianni M, Zolota Z, Paletas K, Tsapas A, Koliakos G. Cariporide counteracts atherosclerosis-related functions in monocytes from obese and normal individuals Obes Res 2005; 13: 1588-95.
[16] Konstantinidis D, Paletas K, Koliakos G, Kaloyianni M. Signaling components involved in leptin-induced amplification of the atherosclerosis-related properties of human monocytes J Vasc Res 2009; 46: 199-208.
[17] Sarigianni M, Bekiari E, Tsapas A, et al. Effect of glucose and insulin on oxidized low density lipoprotein phagocytosis by human monocytes: a pilot study Angiology 2010. in press
[18] Grundy SM. Obesity, metabolic syndrome, and cardiovascular disease J Clin Endocrinol Metab 2004; 89: 2595-600.
[19] Fox CS, Coady S, Sorlie PD, et al. Increasing cardiovascular disease burden due to diabetes mellitus: the Framingham Heart Study Circulation 2007; 115: 1544-50.
[20] Incerpi S, Spagnuolo S, Terenzi F, Leoni S. EGF modulation of Na+/H+ antiport in rat hepatocytes: different sensitivity in adult and fetal cells Am J Physiol 1996; 270: C841-7.
[21] Thomas JA, Buchsbaum RN, Zimniak A, Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ Biochemistry 1979; 18: 2210-8.
[22] Wang S, Peng Q, Zhang J, Liu L. Na+/H+ exchanger is required for hyperglycaemia-induced endothelial dysfunction via calcium-dependent calpain Cardiovasc Res 2008; 80: 255-62.
[23] Chen S, Khan ZA, Karmazyn M, Chakrabarti S. Role of endothelin-1, sodium hydrogen exchanger-1 and mitogen activated protein kinase (MAPK) activation in glucose-induced cardiomyocyte hypertrophy Diabetes Metab Res Rev 2007; 23: 356-67.
[24] Segalen C, Longnus SL, Baetz D, Counillon L, Van Obberghen E. 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside reduces glucose uptake via the inhibition of Na+/H+ exchanger 1 in isolated rat ventricular cardiomyocytes Endocrinology 2008; 149: 1490-8.
[25] Chen S, Mackintosh C. Differential regulation of NHE1 phosphorylation and glucose uptake by inhibitors of the ERK pathway and p90RSK in 3T3-L1 adipocytes Cell Signal 2009; 21: 1984-93.
[26] Boedtkjer E, Aalkjaer C. Insulin inhibits Na+/H+ exchange in vascular smooth muscle and endothelial cells in situ: involvement of H2O2 and tyrosine phosphatase SHP-2 Am J Physiol Heart Circ Physiol 2009; 296: H247-55.
[27] Leineweber K, Heusch G, Schulz R. Regulation and role of the presynaptic and myocardial Na+/H+ exchanger NHE1: effects on the sympathetic nervous system in heart failure Cardiovasc Drug Rev 2007; 25: 123-31.
[28] Li Z, Song T, Liu GZ, Liu LY. Inhibitory effects of cariporide on LPC-induced expression of ICAM-1 and adhesion of monocytes to smooth muscle cells in vitro Acta Pharmacol Sin 2006; 27: 1326-32.
[29] Wang SX, Sun XY, Zhang XH, et al. Cariporide inhibits high glucose-mediated adhesion of monocyte-endothelial cell and expression of intercellular adhesion molecule-1 Life Sci 2006; 79: 1399-404.
[30] Sandoval A, Trivinos F, Sanhueza A, et al. Propionate induces pH(i) changes through calcium flux, ERK1/2, p38, and PKC in bovine neutrophils Vet Immunol Immunopathol 2007; 115: 286-98.
[31] Li L, Watanabe Y, Matsuoka I, Kimura J. Acidic preconditioning inhibits Na+/H+ and Na+/Ca2+ exchanger interaction via PKCepsilon in guinea-pig ventricular myocytes J Pharmacol Sci 2008; 107: 309-16.
[32] Kawada H, Yasuoka Y, Fukuda H, Kawahara K. Low [NaCl]-induced neuronal nitric oxide synthase (nNOS) expression and NO generation are regulated by intracellular pH in a mouse macula densa cell line (NE-MD) J Physiol Sci 2009; 59: 165-73.
[33] Coon S, Kekuda R, Saha P, Talukder JR, Sundaram U. Constitutive nitric oxide differentially regulates Na-H and Na-glucose cotransport in intestinal epithelial cells Am J Physiol Gastrointest Liver Physiol 2008; 294: G1369-75.
[34] Donowitz M, Li X. Regulatory binding partners and complexes of NHE3 Physiol Rev 2007; 87: 825-72.
[35] Bandyopadhyay GK, Yu JG, Ofrecio J, Olefsky JM. Increased p85/55/50 expression and decreased phosphotidylinositol 3-kinase activity in insulin-resistant human skeletal muscle Diabetes 2005; 54: 2351-9.
[36] Park SW, Zhou Y, Lee J, et al. The regulatory subunits of PI3K, p85alpha and p85beta, interact with XBP-1 and increase its nuclear translocation Nat Med 2010; 16: 429-37.
[37] Lee J, Xu Y, Lu L, et al. Multiple abnormalities of myocardial insulin signaling in a porcine model of diet-induced obesity Am J Physiol Heart Circ Physiol 2010; 298: H310-9.
[38] Fliegel L. The Na+/H+ exchanger isoform 1 Int J Biochem Cell Biol 2005; 37: 33-7.
[39] Patel H, Barber DL. A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis J Cell Biol 2005; 169: 321-9.
[40] Frantz C, Barreiro G, Dominguez L, et al. Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding J Cell Biol 2008; 183: 865-79.
[41] Rasmussen M, Alexander RT, Darborg BV, et al. Osmotic cell shrinkage activates ezrin/radixin/moesin (ERM) proteins: activation mechanisms and physiological implications Am J Physiol Cell Physiol 2008; 294: C197-212.
[42] Meima ME, Mackley JR, Barber DL. Beyond ion translocation: structural functions of the sodium-hydrogen exchanger isoform-1 Curr Opin Nephrol Hypertens 2007; 16: 365-72.
[43] Ilic D, Mao-Qiang M, Crumrine D, et al. Focal adhesion kinase controls pH-dependent epidermal barrier homeostasis by regulating actin-directed Na+/H+ exchanger 1 plasma membrane localization Am J Pathol 2007; 170: 2055-67.
[44] Bolduc C, Yoshioka M, St-Amand J. Acute molecular mechanisms responsive to feeding and meal constitution in mesenteric adipose tissue Obesity (Silver Spring) 2010; 18: 410-3.
[45] Bhonagiri P, Pattar GR, Horvath EM. Hexosamine biosynthesis pathway flux contributes to insulin resistance via altering membrane phosphatidylinositol 4,5-bisphosphate and cortical filamentous actin Endocrinology 2009; 150: 1636-45.
[46] McCarthy AM, Spisak KO, Brozinick JT, Elmendorf JS. Loss of cortical actin filaments in insulin-resistant skeletal muscle cells impairs GLUT4 vesicle trafficking and glucose transport Am J Physiol Cell Physiol 2006; 291: C860-8.
[47] Barres R, Gremeaux T, Gual P, et al. Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation Mol Endocrinol 2006; 20: 2864-75.
[48] O'Malley D, Irving AJ, Harvey J. Leptin-induced dynamic alterations in the actin cytoskeleton mediate the activation and synaptic clustering of BK channels FASEB J 2005; 19: 1917-9.
[49] Zeidan A, Paylor B, Steinhoff KJ, et al. Actin cytoskeleton dynamics promotes leptin-induced vascular smooth muscle hypertrophy via RhoA/ROCK- and phosphatidylinositol 3-kinase/protein kinase B-dependent pathways J Pharmacol Exp Ther 2007; 322: 1110-6.
[50] Hayashi H, Aharonovitz O, Alexander RT, et al. Na+/H+ exchange and pH regulation in the control of neutrophil chemokinesis and chemotaxis Am J Physiol Cell Physiol 2008; 294: C526-34.
[51] Morgan D, Capasso M, Musset B, et al. Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis Proc Natl Acad Sci U S A 2009; 106: 18022-7.
[52] Behe P, Segal AW. The function of the NADPH oxidase of phagocytes, and its relationship to other NOXs Biochem Soc Trans 2007; 35: 1100-3.
[53] Serpillon S, Floyd BC, Gupte RS, et al. Superoxide production by NAD(P)H oxidase and mitochondria is increased in genetically obese and hyperglycemic rat heart and aorta before the development of cardiac dysfunction. The role of glucose-6-phosphate dehydrogenase-derived NADPH Am J Physiol Heart Circ Physiol 2009; 297: H153-62.
[54] Patel C, Ghanim H, Ravishankar S, et al. Prolonged reactive oxygen species generation and nuclear factor-kappaB activation after a high-fat, high-carbohydrate meal in the obese J Clin Endocrinol Metab 2007; 92: 4476-9.
[55] Pavlov TS, Levchenko V, Karpushev AV, Vandewalle A, Staruschenko A. Peroxisome proliferator-activated receptor gamma antagonists decrease Na+ transport via the epithelial Na+ channel Mol Pharmacol 2009; 76: 1333-40.
[56] Kumar AP, Quake AL, Chang MK, et al. Repression of NHE1 expression by PPARgamma activation is a potential new approach for specific inhibition of the growth of tumor cells in vitro and in vivo Cancer Res 2009; 69: 8636-44.
[57] Athyros VG, Tziomalos K, Karagiannis A, Anagnostis P, Mikhailidis DP. Should adipokines be considered in the choice of the treatment of obesity-related health problems? Curr Drug Targets 2010; 11: 122-35.
[58] Filippatos TD, Randeva HS, Derdemezis CS, Elisaf MS, Mikhailidis DP. Visfatin/PBEF and atherosclerosis-related diseases Curr Vasc Pharmacol 2010; 8: 12-28.
[59] Paraskevas KI, Liapis CD, Mikhailidis DP. Leptin: a promising therapeutic target with pleiotropic action besides body weight regulation Curr Drug Targets 2006; 7: 761-1.
[60] Alvarez GE, Beske SD, Ballard TP, Davy KP. Sympathetic neural activation in visceral obesity Circulation 2002; 106: 2533-6.
[61] Lamon BD, Hajjar DP. Inflammation at the molecular interface of atherogenesis: an anthropological journey Am J Pathol 2008; 173: 1253-64.