Red blood cell microrheological changes and drug transport efficiency

2.1Preparation of blood samples

Venous blood samples (15 ml) were drawn via sterile venipuncture using heparin (5 IU/ml) as anticoagulant. The study was approved by the local ethic committee at the Yaroslavl State Pedagogical University, and an informed consent of all the subjects were obtained according to the recommendations of the Declaration of Helsinki (The International Response to Helsinki VI, The WMA’s Declaration of Helsinki on Ethical Principles for Medical Research Involving Human Subjects, as adopted by the 52nd WMA General Assembly, Edinburgh, October, 2000).

Red blood cells (RBCs) were separated by centrifugation at 1,400 g for 20 min and washed 3 times with 10 mM phosphate buffered saline (PBS) (pH = 7.4). The washed RBCs were then resuspended in PBS at a hematocrit (Hct) of approximately 40% for incubation with drugs or chemicals (experimental panel) and without any drugs (control panel). After an incubation period the supernatant was removed by centrifugation and cells were resuspended in autologous plasma at Hct = 40% for measurement of RBC aggregation and deformability.

2.2Protocols for in vitro aggregation studies

It was organized fourth research sessions. In the first one RBC suspension was divided into four aliquots and exposed to: 1) Isoproterenol (1.0 μM); 2) Insulin 0.1 μM); 3) Prostaglandins (PGE₁, 0.01 μM); 4) Prostacyclin (PGI₂, 0.01 μM).

In the second research session RBC suspension aliquots exposed to: 1) 5-fluorouracil (5-FU, 5 μM); 2) Inhibitor of the protein kinase C (PKC) activity, phorbol 12-myristate 13-acetate (PMA, 3 μM; n = 24); 3) PDE₄ inhibitor rolipram (10 μM; n = 24).

In the third research session RBC suspension aliquots exposed to: 1) prostaglandin F_2a (PGF_2a, 0.01 μM); 2) phenylephrine – alpha-1-adrenoceptor agonist (1.0 μM); 3) clonidine alpha-2-adrenoceptor agonist (1.0 μM).

In the fourth research session RBC suspension aliquots exposed to: 1) calcium ionophore A23187 (3 μM); 2) verapamil – Ca²⁺ channel blocker (10 μM). In this research session the Ringer’s solution was used as suspension medium, containing Ca²⁺.

To study the role of phosphodiesterases (PDEs) in red cell microrheology alterations the cells were incubated with: PDE₁ inhibitor vinpocetine (10 μM; PDE₃ inhibitor cilostazol (10 μM) and PDE4 inhibitor rolipram (10 μM). The cell shape was controlled with the light microscopy. The cell incubation was performed at 37°C for 15 min. The red blood cells, incubated in the phosphate buffered saline (or in Ringer’s solution) without any drugs, were used as a control. Stock solutions of drugs were prepared in DMSO or water. All analyses were completed within 4 h after the blood collection. Drugs and chemical compounds were mainly purchased from Sigma.

2.3Red blood cell aggregation measurement

Red blood cell aggregation (RBCA) in native plasma was assessed by the Myrenne Aggregometer which provides an index of RBC aggregation facilitated by low shear. In brief, the suspension was subjected to a short period of high shear to disrupt pre-existing aggregates, following which the shear was abruptly reduced to 3 s ⁻¹ and light transmission through the suspension was integrated for 5 seconds; the resulting index, termed “M₅” by the manufacturer and “RBCA” herein, increased with enhanced RBC aggregation.

To estimate the deformability of RBCs the latter were placed into a flow microchamber. The cells were attached to the bottom part of the chamber with “one point” and then were deformed by shear flow under constant shear stress (τ). At a given volume flow rate Q (determined by weighing the amount of saline which flows through the flow channel in a given time period) the shear stress at the surfaces τ, is given by [2]:

where η is the viscosity of the perfusate, w is the width and h is the height of the flow passageway. In our experiments, η= 1.07 mPa·s, w = 0.90 cm, h = 0.01 cm.

2.4Miscellaneous techniques

The whole blood and red cell suspension hematocrite was determined via the microhematocrit method (i.e., 12,000×g for 7 minutes).

2.5Statistics and data presentation

The results are presented as mean SEM. The differences between the mean values were evaluated using an ANOVA test. The values of p <  0.05 indicate statistical significance.

3.1The relatively positive microrheological effect of the drugs

Catecholamines as stress hormones ensure an effective adaptation to various factors of the environmental. It is important to note that human erythrocyte membranes contain both α₁ - and β₂ - adrenergic receptors. In the presence of 1.0 μM Isoproterenol the red blood cell aggregation was decreased to some extent versus control but not statistically (Table 1), while RBCD was significantly increased by 26% (p <  0.05) under these conditions. It is well known that insulin is a functional antagonist of the catecholamines, and erythrocytes possess insulin receptors. Therefore it may be assume that the red cell microrheology will be altered after the cell incubation with insulin.

We have tested this hypothesis and found the RBCA was decreased by 44% (p <  0.05) and RBCD was increased by 16% (p <  0.05) versus control. The similar positive effect was observed after RBC incubation with Prostaglandin E1 and Prostacyclin (Table 1).

3.2The relatively neutral microrheological effect of the drugs

5-fluorouracil is a drug for cancer chemotherapy and it employs for this purpose. It was found that this chemical has a neutral microrheological effect. Neither RBCA nor RBCD were changed significantly after the cell incubation with this drug (Table 2). PDE₄ inhibitor rolipram also possess the relatively neutral effect on RBC microrheological behavior. Changes of red cell microrheology (up to 6% ) were not significant (Table 2). PMA is an activator of cellular protein kinase, mainly PKC. It was shown minor alteration (by 2-3% ) of red cell microrheological properties, both RBCA and RBCD after PMA treatment (Table 2).

3.3The relatively negative microrheological effect of the drugs

The prostaglandin of F family – PGF_2α intensified the red cell aggregation. In presence of PGF_2α erythrocyte aggregation increased by 66% , p <  0.05 (Table 3). PGF_2α is known as a calcium cell entry stimulator [20]. Therefore the aggregation rise may be associated with an augmentation of Ca²⁺ influx.

In the presence of 1.0 μM α ₁- and α ₂-receptor agonists (phenylephrine and clonidine) the red blood cell aggregation was markedly increased – from 49 to 60% (vs. control; P <  0.05). The most significant aggregation effect had α ₂-agonist clonidine. After the cells incubation with this drug the RBCA was increased by 60% (Table 3). Therefore, it concerns mostly α-adrenergic receptor stimulation by epinephrine or specific α-agonists. On the contrary, these alpha-receptor agonists had the lowering RBCD effect (Table 3). On the whole, the obtained data make us believe that an activation of the red blood cell alpha-receptor is associated with a relative negative microrheological effect.

4.1Role of Ca²⁺ in red blood cell microrheology alteration

The measurement of the total red cell calcium concentration has yielded values between 5 and 50 nM [22], while only a few percent of total red cell Ca²⁺ is in an ionized form [27]. Any rise in intracellular Ca²⁺ in erythrocytes activates a specific K ⁺ channel (‘Gardos’ channel) which normally makes little contribution to K ⁺ fluxes [10]. RBC treatment with stimulators of Ca²⁺ influx as well as the inhibition of Ca²⁺ efflux resulted in a significant increase of their aggregation [17]. Contrasting with it, Ca²⁺ entry blocking into the red cells by verapamil led to a significant RBCA decrease and RBCD rise (Table 4). Our experimental data indicated that the stimulation of Ca²⁺ influx by A23187 caused a significant increase of red blood cell aggregation and some RBCD decrease (Table 4).

The changes in RBC Ca²⁺ and cAMP content are not simply two isolated linear signaling pathways, but they actually interact at multiple levels to form an effective signaling network.

4.2Effect of phosphodiesterase (PDE) activity inhibition on red blood cell microrheology

It is known that the intracellular cAMP level is regulated by phosphodiesterases (PDEs) [1, 5]. To study their role in red cell aggregation alterations the cells were incubated with: PDE₁ inhibitor vinpocetine; PDE₃ inhibitor cilostazol; PDE₄ inhibitor rolipram. The red cell aggregation was reduced after the cell incubation with the above-mentioned drugs, having the PDE inhibitory activity. The most significant effect was found under cell incubation with PDE₁inhibitor – vinpocetine. Taken as a whole RBCA reduction was on the averaged 25% under these conditions (Fig. 1). On the whole, the obtained data make us believe that PDEs are a part of the cell regulatory system “the cell membrane receptor – G-protein – AC-cAMP complex” and they involved in the RBC microrheological control mechanisms.

Although the concept that the cAMP and Ca²⁺ signaling pathways interact at multiple levels was appreciated for some time, an emerging body of data has begun to define specific molecular loci where the two pathways interface. Spotlighting these important molecular loci at which cAMP and Ca²⁺ signaling pathways converge in non-excitable cells can help us further understand and appreciate the complexity and specificity of stimulus–response coupling in these cells. In the capacity of the potential sites of the cross-talk between Ca²⁺ and cAMP signaling systems in red blood cells may be PDEs [14]. For example, the constitutive type 4 phosphodiesterase activity rapidly hydrolyzes cAMP so the Ca²⁺ inhibition of AC is difficult to resolve, indicating that high phosphodiesterase activity works coordinately with AC to regulate membrane-delimited cAMP concentrations, which is important for control of cell-cell apposition [5]. Moreover there are data that clearly indicate that the activation of AC and cAMP increase lead to the Ca²⁺ influx decrease and red cell aggregation and leukocyte adhesion lowering [17, 28]. The similar results were found under the blocking Ca²⁺ entry into cell or PDE activity inhibition [16].

Taken together the obtained data showed that the drugs infused in blood flow can interact with blood cells, including the most numerous cell population – red blood cells. This interaction can lead to an alteration of RBC microrheological properties and blood transport possibilities at least according to three abovementioned scenarios.