Timolol effects on erythrocyte deformability and nitric oxide metabolism

Abstract

Timolol maleate is a compound used in treatment for reducing increased intra-ocular pressure by limiting aqueous humor production. Decreased erythrocyte deformability (ED), increased activity of erythrocyte acetylcholinesterase (AChE), increased values of nitrosoglutathione (GSNO) and nitic oxide (NO) and decreased plasma levels of NO metabolites, were described in primary open angle glaucoma patients. In healthy human red blood cells (RBCs), timolol is an inhibitor of AChE and induces NO efflux and GSNO efflux from that blood component in lower concentration than those obtained in presence of the natural AChE substrate, acetylcholine (ACh). The signal transduction pathway in RBCs described for NO in dependence of AChE-ACh active complex involves Gi protein, protein tyrosine kinase (PTK like Syk and p53/56Lyn), protein tyrosine phosphatase (PTP) and adenylyl cyclase (AC).

The aim of this in vitro study was to verify the effect of timolol maleate in ED, NO efflux and NO derivatives molecules (NOx) like nitrite (NO₂^–), nitrate (NO₃^–, peroxynitrite (^–ONOO) and GSNO under the presence of PTK, PTP, AC and guanylyl cyclase (GC) enzyme proteins inhibitors.

Blood samples from healthy donors were each one divided and were performed aliquots in absence (control aliquots) and presence of timolol or timolol plus each inhibitor and G_i protein uncoupling. No significant differences in erythrocyte NO efflux, GSNO, peroxynitrite, nitrite and nitrate concentrations in response to timolol when compared with the untreated blood samples aliquots were obtained.

It was observed an increase in erythrocyte deformability at high shear stresses induced by the simultaneous presence of timolol and band 3 protein dephosphorylation by PTK syk inhibitor. No significant differences where verified in peroxynitrite levels in the blood aliquots in presence of timolol plus each enzyme inhibitor and G_i protein uncoupling in relation to the control aliquots. No variation of GSNO concentration occurs under the presence of timolol and AMGT (PTK lyn inhibitor) besides the significant higher values observed with each one of the other inhibitors. Nitrate concentration increases significantly in all aliquots with timolol plus each one of the inhibitors. The same was observe with nitrite levels with exception of the aliquots with timolol plus AMGT or timolol plus G_i protein uncoupling showing no significant values in relation to the control aliquots.

Besides the changes in NO derivative molecules and NO efflux from RBCs obtained in this study with blood samples of healthy donors under the effect of timolol plus each inhibitor of the proteins participants in NO signal transduction mechanism, further analogue studies must be promoted with blood samples of patients with glaucoma or any other inflammatory vascular disease.

Keywords

Erythrocyte deformability nitric oxide acetylcholinesterase timolol

1 Introduction

Erythrocytes are blood components that scavenge the oxygen and nitric oxide (NO) when the perfused tissue partial oxygen pressure (PaO₂) is high and liberate both at lower PaO₂ values [1]. The capture and donation of both gases is associated respectively, with relax and tense states of haemoglobin [2, 3].

As we evidenced erythrocyte membrane acetylcholinesterase (AChE) is a partner of the NO signal transduction mechanism through its active and inactive complex forms of the enzyme, in presence of the natural substrate acetylcholine (ACh) and the strong inhibitor velnacrine maleate (VM) respectively [4, 5].

Red blood cells (RBCs) AChE, known as a marker of membrane integrity, showed increased enzyme activity in patients with glaucoma under timolol topically therapy [6, 7]. Timolol maleate is used for topical application in eyes of patients with glaucoma to reduce intra-ocular pressure [8].

Decreased values of AChE enzyme activity were verified in blood samples of healthy donors and patients with ocular diseases (without glaucoma and normal values of RBCs AChE enzyme activity), when incubated in presence of timolol [9]. So, the higher AChE erythrocyte membrane enzyme activity founded in primary open glaucoma (POAG) patients is independent of the inhibitory action of timolol [7, 9].

The less active complex AChE-timolol induces lower NO efflux from RBC and lower S-nitrosoglutathione (GSNO) levels inside erythrocyte when compared with the active complex AChE-ACh in blood samples obtained from healthy donors [9, 10]. However, RBCs in presence of the inactive complex AChE-VM showed higher GSNO concentration in relation to the active enzyme complex AChE-ACh [4]. Unequal erythrocytes ability to scavenge NO molecules content is evidenced to be dependent of the AChE enzyme complex status oscillating according the active, less active or inactive forms present in the RBCs membrane [4, 9].

The amount of GSNO efflux from erythrocyte was, for the first time described by us and, as far as we known, did not occurs in unstimulated blood samples [10]. GSNO efflux from erythrocyte of healthy donors is higher in presence of the AChE-ACh active complex form than under the influence of the AChE-timolol less active one [10].

The incubation of blood sample aliquots obtained from POAG patients with timolol does not affect NO efflux from erythrocyte neither its inside GSNO concentrations [11]. However, comparing with healthy donors, NO efflux is increased in absence and presence of timolol and GSNO levels increased in in POAG in relation to healthy donors only in presence of timolol [11].

Decreased levels of cyclic guanosine mono phosphate (cGMP) and of nitrite (NO₂^–) a NO derivative molecule, were obtained in plasma and in aqueous humor of patients with POAG [12].

High blood viscosity and high values of RBC filterability index (which indicate decreased of erythrocyte deformability) where observed in glaucoma patients [13].

It was verified that in erythrocytes from healthy donors in presence of AChE-ACh active complex ED is increased at high shear stresses [14]. Absence of changes was evidenced in presence of inhibitors of band3 protein phosphorylation (by protein tyrosine kinase, PTK like Syk and p53/56Lyn), dephosphorylation (by protein tyrosine phosphatase, PTP), adenylyl cyclase (AC) and guanylyl cyclase (GC) [14]. In addition to its participation in erythrocyte metabolism, band 3 protein plays a role in the maintenance of erythrocyte stability and shape by its interaction with cytoskeleton proteins [15 –19]. Cytoskeleton is responsible for the flexibility, erythrocyte deformability and cellular integrity [17 , 20–23].

The aim of this study was to verify the effect of timolol maleate in ED, NO efflux and NO derivatives molecules (NOx) like NO₂^–, nitrate (NO₃^–, peroxynitrite (^–ONOO) and GSNO under the presence of PTK, PTP, AC and GC enzyme proteins inhibitors.

2 Material and methods

2.1 Chemicals

Acetycholine chloride, p72syk inhibitor, aminogenistein (AMGT, p53/56lyn inhibitor), adenylyl cyclase inhibitor (MDL hydrochloride) guanylyl cyclase inhibitor (ly) and Pertussis toxin (PTX) caused uncoupling of adenylyl cyclase inhibition from receptor stimulation by Giα, were purchase from Sigma (St Louis, MO, USA). For protein tyrosine kinase (PTK) inhibition Syk, a PTK p72syk inhibitor and calpeptin (PTP inhibitor) was purchased from Calbiochem (Darmstadt, Germany). Nitrate reductase from Aspergillus Niger, NADPH (tetra sodium salt), sodium nitrate, sodium nitrite and atropine were all from Sigma Chemical Co., St Louis, MO, USA. The Griess Reagent kit was purchased from Molecular Probes, Eugene, USA. Sodium chloride was purchased from AnalaR (UK) and chloroform and ethanol 95% from MERCK, Darmstadt, Germany. Blood samples were collected into tubes BD Vacutainer TM with Lithium heparin (17UI/mL) as an anticoagulant. This — in vitro study was performed under the protocol established with the Portuguese Institute of Blood in Lisbon. All males donors (N = 10; aged between 30 and 40 years old) were duly informed and signed their agreement.

2.2 Blood sampling and experimental model

Blood was supplied, according protocol, by the Portuguese Institute of Blood, Lisbon. Blood samples were collected into tubes with lithium heparin (17 IU/ml) as anticoagulant. Total blood was divided into nine aliquots of 1 ml each and centrifuged at 11 000 rcf (Biofuge 15 centrifuge, Heraeus) during 1minute at room temperature. Then, 10 μL of plasma were replaced by the same volume of either physiological serum (control aliquot) or timolol 10^–5 M or timolol plus each inhibitor, so that the final concentration of the inhibitor in the whole blood aliquots was 10^–5 M. Besides other concentrations (5×10^–5, 10^–6 and 5×10^–6 M) were tested, no significant alterations were observed with these concentrations in relation to 10^–5 M. Then the blood sample was homogenized by gently inversion incubated for 15 minutes at room temperature. Then deformability in all aliquots were assessed and after the NO efflux from RBC and the other NOx were determined following the methods described next.

2.3 Measurement of erythrocyte NO efflux, nitrite, nitrate, GSNO and peroxynitrite

Following incubation, blood samples were centrifuged and sodium chloride 0.9% at pH 7.0 was added on to compose a hematocrit of 0.05%. The suspension was mixed by gently inversion of tubes.

For amperometric NO quantification we used the amino-IV sensor (Innovative Instruments Inc. FL, USA), according to the method described previously [24]. NO diffuses through the gas-permeable membrane tripleCOAT of the sensor probe and it is then oxidized at the working platinum electrode, resulting on an electric current. The redox current is proportional to the NO concentration outside the membrane and it was continuously monitored with a computerized inNOTM system, (with a software version 1.9, Innovative Instruments Inc., Tampa, FL, USA) connected to a computer. Calibration of the NO sensor was performed daily. For each experiment, the NO sensor was immersed vertically in the erythrocyte suspension vials, allowed to stabilize for 30 min to achieve NO basal levels. 30 μl of acetylcholine (ACh) was added to erythrocyte suspension samples in order to achieve the final concentrations of 10 μM of ACh and NO. Data were recorded from constantly stirred suspensions at room temperature.

The measurement of nitrite/nitrate concentration was performed using the spectrophotometric Griess method as described in [25], after submitting the pellet of each centrifuged blood sample to haemolysis and haemoglobin precipitation. Haemolysis was induced with distilled water and hemoglobin precipitation with a mixture of ethanol and chloroform (5v/3v). The nitrite concentrations were measured with the spectrophotometric Griess reaction, at 548 nm. For nitrate measurement, this compound was first reduced to nitrites in presence of nitrate reductase [26].

For measurement of S-nitrosoglutathione (GSNO) colorimetric solutions containing a mixture of sulfanilic acid (B component of Griess reagent) and NEDD (A component of Griess reagent), consisting of 57.7 mM of sulfanilic acid and 1 mg/mL of NEDD, were dissolved in phosphate-buffered solution (PBS; pH 7.4). To constitute the 10 mM HgCl2 (Aldrich) mercury ion stock solutions were prepared in 0.136 g/50mL of dimethyl sulfoxide (DMSO) (Aldrich). GSNO was diluted to the following desired concentrations: 7,5 μM; 15 μM; 30 μM; 45 μM; 60 μM; 120 μM; 240 μM; 300 μM in the colorimetric analysis solutions. Various concentrations of mercury were then added to a final concentration of 100 μM. Following gentile shaking the solution was let to stand for twenty minutes. A control spectrum was measured by spectrophotometry at 496 nm against a solution without mercury ion. 300 μL of erythrocyte suspensions were added to the reaction mixture and GSNO concentrations were obtained as described [27].

For determinations of peroxynitrite levels the erythrocyte suspensions (1 mL) were incubated with 2, 7-dichlorofluorescein diacetate (DCFC-DA) 15 μM, in 3 mL buffer (Pi 155 mM, pH 7.4) during 30 min, at room temperature. Suspensions were rinsed several times and diluted in the working solution with 1.8 mL of the same buffer. The pellets were rinsed in the same buffer and used for fluorescence measurement with a Microplate Reader TECAN Infinite F500 (TECAN Trading AG, Switzerland) with excitation and emission wavelengths at 485 and 535 nm, respectively. The concentration of peroxynitrite was finally calculated through a calibration curve previously performed [28].

2.4 Erythrocyte deformability

The erythrocyte deformability (ED) for different shear stress (0.30, 0.60, 1.20, 3.00, 12.00, 30.00 and 60.00 Pa) was determined by using the Rheodyn SSD shear stress diffractometer from Myrenne GMBH (Roentgen, Germany), and erythrocyte deformability is expressed as the elongation index (EI) in percentage [29]. The Rheodyn SSD diffractometer determines RBC deformability by simulating the shear forces exerted by the blood flow and vascular walls on the erythrocytes. Erythrocytes are suspended in a viscous medium and placed between a rotating optical disk and a stationary disk. A well-defined shear force is exerted upon the suspension which forces the erythrocytes to deform to ellipsoids and align with the fluid shear stresses. If a laser beam is allowed to pass through the erythrocyte suspension a diffraction pattern appears on the opposite end. That diffraction pattern will be circular with resting erythrocytes, but becomes elliptical when the erythrocytes are deformed by shear. The light intensity of the diffraction pattern are measured at two different points (A and B), equidistant from the center of the image. The erythrocyte elongation index (EEI), in percentage, is obtained according the following formula: EEI(%) = [(A – B)/ (A + B)] × 100.

2.5 Statistical analysis

Data are expressed as mean values±SD. Student’s paired t-tests were used to compare values between different samples of erythrocyte suspensions. Statistical analysis was conducted using the Statistical Package from the Social Sciences (SPSS; version 16.0). One-way analysis of variance and paired t-tests were applied to assess statistical significance between samples. Bonferroni post-hoc tests were conducted when appropriate. Statistical significance was set at a p < 0.05 level.

3 Results

3.1 In vitro effect of Timolol on erythrocyte deformability under the presence of PTX, AMGT, MDL, Ly and Syk inhibitor

Table 1 shows that the values of erythrocyte elongation indexes measured at different shear stresses did not change with the presence of PTX, AMGT, MDL or Ly in the blood aliquots when comparing with the blood control aliquot. When the timolol and inhibitor of PTK Syk are present and at an higher shear stress of 60 Pa a significant increase of ED in relation to their absence 48.4±3.90 versus 46.848.4±4.7 (p < 0.05) is observed.

Table 1
Values (Mean±SD) of erythrocyte deformability index (%) obtained in absence and presence of timolol under PTX (G_i protein uncoupling), PTK (Syk inib, AMGT is Lyn inhibitor, adenylyl cyclase (MDL), guanylyl cyclase (Ly) inhibitors and (PTX) Gi Protein uncoupling

Samples

Shear stress (Pa) Control Timolol+PTX Timolol+AMGT Timolol+MDL Timolol+Ly Timolol+Syk inb

0.3 2.45±1.13 2.52±0.88 2.14±2.04 2.22±1.43 2.35±1.24 2.20±1.23

0.6 5.01±1.42 5.65±1.13 4.87±2.29 5.21±1.20 5.26±1.05 5.18±1.59

1.2 13.5±2.10 15.0±2.14 13.7±2.37 13.9±1.71 14.1±1.65 13.9±1.74

3 28.2±3.49 29.5±1.80 28.0±2.18 28.7±1.63 28.6±2.03 28.3±1.82

6 37.8±2.91 39.0±2.36 37.6±1.74 37.9±1.78 38.1±1.9 37.9±1.81

12 44.2±4.02 45.0±3.72 44.5±2.57 44.4±3.01 44.8±2.93 44.6±3.05

30 47.4±4.07 47.5±4.11 47.9±3.58 47.5±3.45 48.2±3.36 48.4±3.71

60 46.8±4.26 46.9±3.86 48.1±3.8 47.4±3.61 48.1±3.50 48.4±3.90^*

	Samples
0.3	2.45±1.13	2.52±0.88	2.14±2.04	2.22±1.43	2.35±1.24	2.20±1.23
0.6	5.01±1.42	5.65±1.13	4.87±2.29	5.21±1.20	5.26±1.05	5.18±1.59
1.2	13.5±2.10	15.0±2.14	13.7±2.37	13.9±1.71	14.1±1.65	13.9±1.74
3	28.2±3.49	29.5±1.80	28.0±2.18	28.7±1.63	28.6±2.03	28.3±1.82
6	37.8±2.91	39.0±2.36	37.6±1.74	37.9±1.78	38.1±1.9	37.9±1.81
12	44.2±4.02	45.0±3.72	44.5±2.57	44.4±3.01	44.8±2.93	44.6±3.05
30	47.4±4.07	47.5±4.11	47.9±3.58	47.5±3.45	48.2±3.36	48.4±3.71
60	46.8±4.26	46.9±3.86	48.1±3.8	47.4±3.61	48.1±3.50	48.4±3.90^*

^*p < 0.05 in relation to the Control.

3.2 “In vitro” effect of Timolol on erythrocyte, NO, GSNO, peroxynitrite, nitrite and nitrate under the presence of Syk inhibitor, calpetin, PTX, AMGT, Ly and MDL

Table 2 shows that the values of NO efflux from erythrocytes, GSNO, peroxynitrite, nitrites and nitrates concentrations obtained in presence of timolol do not show significant changes in relation to the control blood aliquots. The exception is when timolol is in presence of syk inhibitor or calpeptine, or MDL the values of NO efflux from erythrocytes in those blood aliquots increased significantly.

Table 2
Values (Mean±SD) of erythrocyte nitric oxide efflux (nM) GSNO (μM), peroxynitrite (μM), nitrite (μM) and nitrate(μM) obtained in absence of timolol [control aliquot) and in presence of timolol without or under the addition of PTP (Calpeptin), PTK (Syk inib; AMGT is Lyn inhibitor), adenylyl cyclase (MDL), guanylyl cyclase (Ly) inhibitors and (PTX) Gi protein uncoupling

Blood samples Nitric oxide GSNO Peroxynitrite Nitrate Nitrite

Control 1.38±0.03 8.27±0.56 218.4±35.42 8.40±089 8.55±0.80

Timolol 1.37±0.14 8.88±0.54 228.6±51.47 9.55±0.93 9.00±1.05

Timolol+Syk inib 1,60±0.27^* 11.51±0.95^** 179.3±47.20 11.35±1.14^* 11.35±1.14^*

Calpeptine 1.17±0.20 9.47±0.89^* 192.5±71.37 10.25±0.41^** 9.65±0.76^*

Timolol+Calpeptine 1.68±0.19^* 10.44±1.00^*** 221.7±77.50 11.30±0.75^* 10.75±0.97^*

Timolol+PTX 1.36±0.17 9.77±0.76^ 193.8±45.20 9.40±0.089^ 8.95±0.91

Timolol+AMGT 1.41±0.25 9.34±1.39 186.7±52.94 10.45±0.85^ 9.80±0.02

Timolol+Ly 1.37±0.23 11.51±0.95^* 178.6±29.86 12.00±0.46^* 11.45±1.14^***

Timolol+MDL 1.60±0.23^* 10.42±0.93^*** 180.2±30.00 10.90±1.14^* 10.20±1.17^**

Blood samples	Nitric oxide	GSNO	Peroxynitrite	Nitrate	Nitrite
Control	1.38±0.03	8.27±0.56	218.4±35.42	8.40±089	8.55±0.80
Timolol	1.37±0.14	8.88±0.54	228.6±51.47	9.55±0.93	9.00±1.05
Timolol+Syk inib	1,60±0.27^*	11.51±0.95^**	179.3±47.20	11.35±1.14^*	11.35±1.14^*
Calpeptine	1.17±0.20	9.47±0.89^*	192.5±71.37	10.25±0.41^**	9.65±0.76^*
Timolol+Calpeptine	1.68±0.19^*	10.44±1.00^***	221.7±77.50	11.30±0.75^*	10.75±0.97^***
Timolol+PTX	1.36±0.17	9.77±0.76^**	193.8±45.20	9.40±0.089^**	8.95±0.91
Timolol+AMGT	1.41±0.25	9.34±1.39	186.7±52.94	10.45±0.85^**	9.80±0.02
Timolol+Ly	1.37±0.23	11.51±0.95^***	178.6±29.86	12.00±0.46^***	11.45±1.14^***
Timolol+MDL	1.60±0.23^*	10.42±0.93^***	180.2±30.00	10.90±1.14^*	10.20±1.17^**

In relation to control sample: ^*p < 0.05 ^*p < 0.001; ^**p < 0.005; ^***p < 0.0001.

The values of GSNO inside the erythrocytes increase significantly in all blood aliquots under the simultaneous presence of timolol and each one of all inhibitors and the G_αi uncoupling in relation to the control aliquots. The exception was no variation of GSNO concentration under the presence of timolol and AMGT.

Unchanged values of peroxynitrite concentration in relation to control aliquot were observed in all blood aliquots under the presence of timolol or timolol plus Syk inhibitor or calpeptine or PTX or AMGT or Ly and MDL (Table 2). The same absence of concentration changes in relation to control aliquot was observed in the presence of calpeptine (Table 2).

Regarding nitrate values in all blood aliquots under the presence of timolol plus each one of the used inhibitors or plus the G_αi uncoupling PTX in relation to the control aliquots they are all significantly increased (Table 2). The blood aliquots with timolol under the presence of the G_αi uncoupling PTX or AMGT do not induced variations in the values of nitrite concentrations in relation to the control aliquots (Table 2). The opposite was verified in the other blood aliquots with timolol plus Syk inhibitor or calpeptine or Ly or MDL where significant increased concentrations were obtained (Table 2).

4 Discussion

In this study we found that the presence of timolol 10^–5M in blood aliquots do not change erythrocyte deformability values at any shear stresses in relation to the control aliquot (Table 1). The same unchanged ED was verified when timolol is under the effect of band 3 phosphorylation (PTP inhibition by calpeptin) or low cAMP levels (by AC inhibition) or low cGMP concentration (by GC inhibition) or G_i protein uncoupling (Table 1). At opposite, timolol increases ED when band 3 is dephosphorylate by Syk inhibitor (Table 1). The increase of ED resulting from band 3 dephosphorylation present in the blood aliquot with the less active complex AChE-timolol plus Syk inhibitor is similar to the increase observed under AChE-VM plus PKC inhibitor (chelerythrine; Che) [29]. PKC phosphorylates PTK and PTP proteins promoting the activation and the inhibition of their enzymes activities respectively [30]. When PKC is inhibit by Che the PTP regains its active form and band 3 becomes dephosphorylated [30]. Membrane-bound band 3 protein is a multifunctional protein containing tyrosine residues able to become phosphorylated, through a highly regulated system of kinases [28]. Phosphorylation of band3 protein by PTK is a sequential process occurring in two points being PTK syk (p72^syk) the first to bind followed by Lyn (p53/56^lyn) [31]. This process explain the absence of effect on ED by timolol plus p53/56^lyn inhibitor, because the band 3 is partially phosphorylated by p72syk (Table 1).

Timolol do not modify the values of NO efflux from erythrocytes in absence and presence of GC inhibitor, or G_i protein uncoupling in relation to the control aliquots (Table 2). The increase of NO efflux from erythrocytes was obtained in blood aliquots in presence of timolol when band 3 protein is phosphorylated or dephosphorylated or when AC is inhibited (Table 2). The less active complex AChE– timolol influences NO efflux in similar way to the inactive complex AChE-VM when band 3 is phosphorylated but in opposite way when is dephosphorylated Table 2 [4]. The presence of MDL inhibiting AC increase the activity of PKC and PTP is phosphorylated becoming in inactive state [32]. Consequently, band 3 phosphorylated and the couple timolol plus MDL induced increase in NO efflux from erythrocytes (Table 2).

NO efflux from erythrocytes did not exclude others reactions inside where NO react with: (i) superoxide anion to form peroxynitrite (ONOO^–) which subsequently yields nitrite and nitrate, (ii) deoxyhemoglobin to form nitrosylhemoglobin, (iii) oxyhemoglobin to form methemoglobin and nitrate without methemoglobin concentration changes and (iv) thiol group of glutathione originating GSNO [31, 33].

If auto-oxidation of hemoglobin occurs the peroxide anion will be produced, which generates peroxynitrite after reaction with NO [34]. In blood aliquots where timolol is present without or plus each of one of all inhibitors the peroxynitrite concentrations obtained are not significantly different from the control aliquots, Table 2. Timolol did not modify the oxidative status of erythrocyte but at variance the ability of scavenger NO by glutathione originating the GSNO molecules increased in all blood aliquots where timolol is present plus each one of all inhibitors (Table 2). However, GSNO levels do not show change in blood aliquots with timolol or timolol plus AMGT in relation to the control aliquots, Table 2, which is in opposition to the increased GSNO levels verified with the inactive AChE-VM complex [35]. The efflux o GSNO from RBCs suspensions induced by timolol as previous evidenced could seems not occur contributing for absence or increase of the GSNO concentration verified in the blood aliquots in the present study [11].

In blood aliquots where timolol is present without any inhibitors there are no variation in the levels of nitrate and nitrite in relation to the control aliquots (Table 2). Opposite behavior was showed by the inactive complex AChE-VM where nitrite levels increased and nitrate decreased [30]. In the blood aliquots where timolol is present plus G_i protein uncoupling or plus p53/56 lyn there are no changes in nitrite concentrations (Table 2). Significantly increased of nitrite levels were verified in blood aliquots with timolol plus band 3 phosphorylated or dephosphorylated and plus AC inhibited or plus CG inhibited. The levels of nitrate increased in all blood aliquots with timolol plus each one of inhibitors (Table 2).

The present study evidenced that timolol besides inhibits acetylcholinesterase enzyme activity do not perturb erythrocyte deformability and maintains the nitric oxide metabolism and the NO efflux from erythrocyte between the normal values obtained in healthy donors.

Besides the degree of phosphorylation of band 3 or inhibition of the enzymes responsible for the synthesis of cAMP or cGMP or compounds like PTX are biomolecules, which when in presence of timolol, modify ED, NO metabolism inside the erythrocytes and of the amount of NO efflux from RBCs of blood aliquots. So, those biomolecules are target points to further development of therapeutic compounds to be used in addition to timolol in glaucoma patients. The increase of erythrocyte deformability at high shear stress induced by the simultaneous presence of timolol and dephosphorylated band 3 protein (Table 1) may constitute a window of opportunity to normalize the lower ED founded in glaucoma patients [13]. Until now, we know that timolol did not change the higher ability to release NO from erythrocytes of POAG patients but increase GSNO concentration [11]. From the results evidenced, in Table 2, the increase of NO efflux from erythrocytes observed rise the hypothesis that erythrocyte from OAGP have higher amounts of band 3 protein phosphorylation which could explain the increase AChE enzyme activity evidenced [7].

So, more studies are needed with blood samples of POAG to know the effect of the same targets and its inhibitors herein studied in presence of timolol.

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Acknowledgments

This study was funded by Fundação para a Ciência e Tecnologia: LISBOA-01-0145-FEDER-007391, project cofunded by FEDER, through POR Lisboa 2020 - Programa Operacional Regional de Lisboa, PORTUGAL 2020. The author is also grateful to Emilia Alves for type-writing the references.

This study was performed in accordance with the ethical guidelines of Clinical Hemorheology and Microcirculation [].

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	Samples
Shear stress (Pa)	Control	Timolol+PTX	Timolol+AMGT	Timolol+MDL	Timolol+Ly	Timolol+Syk inb
0.3	2.45±1.13	2.52±0.88	2.14±2.04	2.22±1.43	2.35±1.24	2.20±1.23
0.6	5.01±1.42	5.65±1.13	4.87±2.29	5.21±1.20	5.26±1.05	5.18±1.59
1.2	13.5±2.10	15.0±2.14	13.7±2.37	13.9±1.71	14.1±1.65	13.9±1.74
3	28.2±3.49	29.5±1.80	28.0±2.18	28.7±1.63	28.6±2.03	28.3±1.82
6	37.8±2.91	39.0±2.36	37.6±1.74	37.9±1.78	38.1±1.9	37.9±1.81
12	44.2±4.02	45.0±3.72	44.5±2.57	44.4±3.01	44.8±2.93	44.6±3.05
30	47.4±4.07	47.5±4.11	47.9±3.58	47.5±3.45	48.2±3.36	48.4±3.71
60	46.8±4.26	46.9±3.86	48.1±3.8	47.4±3.61	48.1±3.50	48.4±3.90^*