Ana Santos Silva-Herdade

Modulation of erythrocyte deformability by PKC activity

The interactions between membrane, peripheral and cytoskeleton proteins are responsible for the maintenance of erythrocyte deformability (EEI) and some of these interactions are modulated by PKC activity. Protein band 3 of the erythrocyte membrane is phosphorylated by phosphotyrosine kinases (PTK) and dephosphorylated by phosphotyrosine phosphatase (PTP). It was previously described by us a signal transduction mechanism that describes a possible pathway connecting an erythrocyte external membrane protein, acetylcholinesterase (AChE), with protein band 3. So how does PKC activity modulate EEI when protein band 3 is phosphorylated or dephosphorylated in absence or presence of AChE effectors?To answer this we used phorbol 12-myristate 13-acetate (PMA) as an activator and chelerythrine chloride as inhibitor of PKC and also band 3 modulators of band 3 phosphorylation degree, in presence and absence of AChE effectors in order to measure in whole blood samples EEI. Our results showed that erythrocyte deformability was significantly (i) decreased by inhibition of PKC, in absence and presence of AChE inhibitor velnacrine (ii) increased with PMA in absence and presence of ACh and (iii) decreased in presence of calpeptin in absence and presence of either chelerythrine or PMA. These results establish dependence between cytoskeleton proteins, PKC activity, band 3 phosphorylation degrees and EEI. Better understanding of those proteins interactions on transduction mechanisms might trigger possible targets for drug action that would modulate EEI.

Erythrocyte deformability - A partner of the inflammatory response.

We aim to establish an in vivo animal model of acute inflammation using PAF (platelet activating factor) as inflammatory agent and to study the erythrocyte deformability changes induced by the inflammatory response. Counting the number of rolling and adherent neutrophils to endothelium after 2, 4 and 6h of intrascrotal injection of PAF we showed the induction of an inflammatory state. Blood samples are collected in order to measure the erythrocyte deformability and to quantify NO efflux from the red blood cells (RBCs). The results show an increased number of rolling and adherent neutrophils after 2h and 4h of inflammation as well as decreased values of erythrocyte deformability in the same time-points. This result is in line with the need of a low blood viscosity to the recruitment process that will improve leukocyte migration towards the endothelial wall. NO efflux from RBCs is also affected by the inflammatory response at the first hours of inflammation. This animal model demonstrates in vivo the association between an acute inflammatory response and the rheological properties of the blood, namely the RBCs deformability. For those reasons we consider this as an adequate model to study acute inflammatory responses as well as hemorheological parameters.

Redox thiol status plays a central role in the mobilization and metabolism of nitric oxide in human red blood cells

We assessed the redox thiol status influence on nitric oxide (NO) metabolism and efflux in erythrocytes stimulated with acetylcholinesterase substrate (acetylcholine, ACh) and inhibitor (velnacrine maleate, VM). Erythrocyte suspensions from healthy donors were incubated with increasing concentrations of dithiothreitol (1–50 μM), in the presence and absence of acetylcholine/velnacrine (10 μM). Levels of NO, nitrite/nitrate, S‐nitrosohemoglobin, peroxynitrite and S‐nitrosoglutathione were determined by spectrofluorimetric and spectrophotometric methods.

Application of a Nitric Oxide Sensor in Biomedicine

In the present study, we describe the biochemical properties and effects of nitric oxide (NO) in intact and dysfunctional arterial and venous endothelium. Application of the NO electrochemical sensor in vivo and in vitro in erythrocytes of healthy subjects and patients with vascular disease are reviewed. The electrochemical NO sensor device applied to human umbilical venous endothelial cells (HUVECs) and the description of others NO types of sensors are also mentioned.

Leukocytes dynamics in microcirculation under shear-thinning blood flow

We present detailed simulation results of localised hemodynamics for a cluster of rolling leukocytes under shear-thinning blood flow using a lattice Boltzmann model. Leukocytes were modelled as hard spheres moving through a venule of rigid walls. The used hemorheological parameters were obtained from in vivo measurements in blood samples of Wistar rats. Velocities, shear stresses and torques were computed and visualised for each individual cell, for the cluster and for the fluid. We have found that the flow is mainly three-dimensional due to the swirling and the asymmetry of the formed vortices during the recruitment process. The shear stress is maximum on a cap covering the cell and a cone with its base on the endothelial wall at the contact region. The leukocyte is recruited to the wall with the aid of trapping vortices and four stagnant regions surrounding the cell in addition to lateral motion towards the wall. We suggest that these phenomena are highly dependent on the angular velocity of the leukocyte and on the attractive force between the leukocyte and the endothelial wall. For a moving cluster of recruited leukocytes, velocities and shear stresses as well as torques are computed. It was found that the shear stress at the endothelium gets higher as the cluster moves in the main stream enabling early initialisation of the rolling process.

Erythrocyte deformability and nitric oxide mobilization under pannexin-1 and PKC dependence.

The erythrocyte adenosine triphosphate (ATP) is utilised for protein phosphorylation and exported through the pannexin 1 hemichannel (Px1) in the microcirculation. The physiological stimuli for ATP release are dependent of blood shear rate level and of the tissue oxygen content. The deoxygenated and oxygenated states of haemoglobin are respectively bound and unbound to N terminal domain of the protein band 3 of the erythrocyte membrane in dependence of its degree of phosphorylation. The protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) contribute to the phosphorylation degree of band 3 and are modulated by protein kinase C (PKC). Chelerythrine (Che) is a competitive inhibitor of ATP for PKC and a negative modulator of erythrocyte deformability. The aim of this study was to assess the mobilization of nitric oxide (NO) in erythrocyte in absence and presence of Che and Px1 inhibitor (carbenoxolone). Erythrocyte deformability was evaluated in presence of carbenoxolone (Carb). Regarding the effects observed in the erythrocyte by presence of Che or Carb, the values of efflux of NO and the concentration of nitrosogluthatione are similar and with no changes in relation to their absence. Px1inhibition by Carb 10 μM ameliorates the erythrocyte deformability at a shear force of 0.6 and 1.2 Pa. The PKC inhibitor shows similar effects to the Carb on the mobilization of nitric oxide in erythrocyte. The blockage of ATP release by Carb from erythrocytes suggests a possible benefit to develop in ischemia reperfusion or in inflammatory response where will be needed to rescue the excess of NO present and ameliorate the red blood cell deformability at low shear rates.

Hemorheological Effects of Valsartan in L-NAME Induced Hypertension in Rats

Animal models are a useful tool, for example, in the study of hypertension, but its use requires robust knowledge and well characterized models in order to extrapolate the obtained results. Nitric oxide is an important regulator of vascular function and blood pressure. The chronic administration of nitric oxide synthase (NOS) inhibitors provides an animal experimental model of hypertension. Our study aims to investigate the hemorheological effects of (i) N  -nitro-L-arginine methyl ester (L-NAME) added daily to Sprague-Dawley rats in drinking water and (ii) valsartan, an angiotensin II AT1-receptor antagonist, administration when the Sprague-Dawley rats acquired the hypertensive state. In three experimental groups (Control, Hypertensive and Valsartan) systolic blood pressure was measured and blood samples were collected for determination of erythrocyte membrane deformability and fluidity as well as blood and plasma viscosities. L-NAME intake induces an increase in the systolic blood pressure indicating the development of systemic hypertension. Concerning the hemorheological parameters, the erythrocyte deformability is decreased in the hypertensive animal group and on contrary the erythrocyte membrane fluidity increased being both parameters values reverted after valsartan administration. Valsartan also decreases the plasma and blood viscosities values obtained in the animal group which have acquired systemic hypertension after L-NAME intake. In conclusion, the angiotensin II AT1-receptor antagonist, valsartan, restores in hypertensive Sprague-Dawley rats L- NAME- dependent their systolic blood pressure to the physiological values, as well as, normalized their hemorheological parameters values; due to the similarity effects to human essential hypertension we conclude that this can be a suitable animal model for hemorheological studies in the field of hypertension.

Fibrinogen modulates leukocyte recruitment in vivo during the acute inflammatory response

Besides playing an important role in blood hemostases, fibrinogen also regulates leukocyte function in inflammation. Our previous in vitro studies showed that the adhesive behaviour of the neutrophil is modulated by soluble fibrinogen when present at a physiological concentration. This led us to propose that this plasma glycoprotein might further influence leukocyte recruitment in vivo and thus contribute to the inflammatory response. To address this in vivo, leukocyte recruitment was here investigated under acute inflammatory conditions in the absence of soluble fibrinogen in the blood circulation. For such, intravital microscopy on mesentery post-capillary venules was performed on homozygous fibrinogen α chain-deficient mice ((α-/-) mice). Acute inflammatory states were induced by perfusing platelet activating factor (PAF) over the exposed tissue. As control animals, two groups of mice expressing soluble fibrinogen in circulation were used, namely, C57BL/6 wild type animals and heterozygous fibrinogen α chain-deficient mice ((α+/-) mice). Under acute inflammatory conditions, an abnormal pattern of recruitment was observed for leukocytes in homozygous (α-/-) mice in comparison to both control groups. In fact, the former exhibited a significantly decreased number of rolling leukocytes that nevertheless, migrated with increased rolling velocities when compared to leukocytes from control animals. Consistently, homozygous mice further displayed a diminished number of adherent leukocytes than the other groups. Altogether our observations led us to conclude that leukocyte recruitment in homozygous (α-/-) mice is compromised what strongly suggests a role for soluble fibrinogen in leukocyte recruitment in inflammation.

Signal transduction pathways in erythrocyte nitric oxide metabolism under high fibrinogen levels

Previous studies show that the fibrinogen molecule modulates the metabolism of nitric oxide (NO) in erythrocyte. The in vitro induced hiperfibrinogenemia interferes in the metabolism of the NO in the erythrocyte in dependence of the phosphorylation degree of the band 3. The soluble form of fibrinogen binds into CD47 protein present in the erythrocyte membrane. The soluble thrombomodulin is an inflammatory marker that binds to the erythrocyte CD47 in a site with a sequence peptide known as 4N1K. A study done in vitro shows that when hiperfibrinogenemia was induced in the presence of the peptide 4N1K agonist of CD47 it were observed variations in the efflux of NO from erythrocyte and an increase in the concentrations of GSNO, peroxinitrite, nitrite and nitrate of the erythrocytes. The aim of this work was to study the influence of the peptide 4N1K, on the metabolism of NO in the erythrocyte under high fibrinogen concentration and in the presence of inhibitors of the status of phosphorylation of protein band 3. In this in vitro study, whole blood samples were harvested from healthy subjects and NO, peroxynitrite, nitrite, nitrate and S-nitro-glutathione (GSNO) were determined in presence of 4N1K, calpeptine, Syk inhibitor and under high fibrinogen concentrations. The results obtained in erythrocytes under high fibrinogen levels when 4N1K is present with the Syk inhibitor or with calpeptine, showed in relation to the control samples increased significant concentrations of efflux of NO and of peroxynitrite, nitrite, nitrate and GSNO. In conclusion it was verified that in the in vitro model of hiperfibrinogenemia the peptide 4N1K, agonist of CD47, induces mobilization of NO in the erythrocyte in dependence of the status of phosphorylation of protein band 3.

The Ubiquity Nature of Acetylcholine

© 2014 Saldanha C, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.