Proposed Dynamics of CDB3 Activation in Human Erythrocytes by Nifedipine Studied with Scanning Flow Cytometry

Abstract

Nifedipine is calcium channels and pumps blocker widely used in medicine. However, mechanisms of nifedipine action in blood are not clear. In particular, the influence of nifedipine on erythrocytes is far from completely understood. In this work, applying scanning flow cytometry, we observed experimentally for the first time the dynamics behind a significant increase of HCO 3 − /Cl − transmembrane exchange rate of CDB3 (main anion exchanger, AE1, Band 3, SLC4A1) of human erythrocytes in the presence of nifedipine in blood. It was found that the rate of CDB3 activation is not limited by the rate of nifedipine binding and/or Ca 2+ transport. In order to explain the experimental data, we suggested a kinetic model assuming that the rate of CDB3 activation is limited by the dynamics of the balance between two intracellular processes (1) the activation of CDB3 limited by its interaction with intracellular Ca 2+ , and (2) the spontaneous deactivation of CDB3. Thus the use of scanning flow cytometry allowed to clarify quantitatively the molecular kinetic mechanism of nifedipine action on human erythrocytes. In particular, the efficiency (~30) and rates of activation (~0.3 min −1 ) and deactivation (~10 −3 min −1 ) of CDB3 in human erythrocytes was evaluated for two donors.Nifedipine is widely used in medicine under the name of Adalat and other drugs. One common usage is the treatment of cardiovascular pathology such as atherosclerosis, coronary heart disease, hypertension, cerebral ischemia, and others (1). Other uses include painful spasms of the esophagus (2,3), Raynaud's phenomenon (4), tocolytic therapy (5). However, the effect of the treatment by nifedipine is not always positive (6). In particular, it is not clear (7) the influence of nifedipine on the rate of HCO 3− /Cl − transmembrane CDB3 protein (AE1, Band 3), which plays an important role in bicarbonate metabolism (8). Overall, a more complete understanding of nifedipine action in blood is needed.Nifedipine belongs to the subclass of dihydropyridines (DHPs), which are known as inhibitors of Ca 2+ transport across the erythrocyte membrane in both pathways: (1) inward leak through Ca 2+ entry channels (9-11), and (2) extrusion by plasma membrane Ca 2+ pumps (PMCA) (12,13). It was reported (13) that about 50% inhibition of PMCA was obtained at extracellular nifedipine concentration of 300 μM. Interestingly, the same extracellular concentration of 300 μM nifedipine was reported (14) to cause less (about 30%) inhibition of Ca 2+ entry channels of erythrocyte membrane. Stronger inhibition of PMCA than entry channels leads to the accumulation of intracellular Ca 2+ in erythrocytes in the presence of extracellular

Erythrocyte lysis and angle‐resolved light scattering measured by scanning flow cytometry result to 48 indices quantifying a gas exchange function of the human organism

“…Concerning to the indices, which relate to an entire cell population, the reliability in this case is expressed by errors of indices retrieved from the nonlinear fitting of the experimentally measured kinetics of hemolysis by the molecular‐kinetic model of lysis developed early [3,30,31]. There is the example that demonstrates the typical kinetic curves measured with SFC and calculated from the model in Figure 3.…”

“…The high meanings of this index correspond to more tensile membrane and increased ability to osmotic lysis resistance as you can see in Equation (5). Here K S is the membrane dilation modulus, S and S 0 are final and start erythrocyte surface area, correspondingly, during hemolysis. Taking into account our experimental results with the enhancement of an anion permeability of erythrocyte membrane by magnesium sulfate [30] and Nifedipine [31] we defined the ultimate effective number of Band 3 proteins per a cell as the saturation level of anion permeability under Nifedipine activation. The effective numbers were used in the calculation of total concentration of Band 3 proteins in the serum and Band 3 protein density on a surface.…”

“…Taking into account our experimental results with the enhancement of an anion permeability of erythrocyte membrane by magnesium sulfate [30] and Nifedipine [31] we defined the ultimate effective number of Band 3 proteins per a cell as the saturation level of anion permeability under Nifedipine activation. The effective numbers were used in the calculation of total concentration of Band 3 proteins in the serum and Band 3 protein density on a surface.…”

“…In order to retrieve these indices we have measured the kinetics of the erythrocyte lysis initiated by ammonium chloride according to the method developed a few years ago [3]. Recently we have extended this approach with measurement of the ultimate membrane permeability using magnesium sulfate [30] or Nifedipine [31] activation. The indices of the aging category came from the well-documented observation that erythrocytes lose a part of their volume and hemoglobin during their 115-day lifespan in the bloodstream [32,33].…”