Beta-adrenergic agonists regulate cell membrane fluctuations of human erythrocytes

Abstract

1. Mechanical fluctuations of the cell membrane (CMFs) in human erythrocytes reflect the bending deformability of the membrane-skeleton complex. These fluctuations were monitored by time-dependent light scattering from a small area ( approximately 0. 25 microm2) of the cell surface by a method based on point dark field microscopy. 2. Exposure of red blood cells (RBCs) to adrenaline (epinephrine) and isoproterenol (isoprenaline) resulted in up to a 45 % increase in the maximal fluctuation amplitude and up to a 35 % increase in the half-width of the amplitude distribution. The power spectra of membrane fluctuations of control and treated cells revealed that adrenaline stimulated only the low frequency component (0.3-3 Hz). Analysis of the dose-response curves of beta-adrenergic agonists yielded an EC50 of 5 x 10-9 and 1 x 10-11 M for adrenaline and isoproterenol, respectively. Propranolol had an inhibitory effect on the stimulatory effect of isoproterenol. These findings show a potency order of propranolol > isoproterenol > adrenaline. 3. The stimulatory effect of adrenaline was a temporal one, reaching its maximal level after 20-30 min but being abolished after 60 min. However, in the presence of 3-isobutyl-1-methylxanthine, a partial stimulatory effect was maintained even after 60 min. Pentoxifylline and 8-bromo-cAMP elevated CMFs. However, exposure of ATP-depleted erythrocytes to adrenaline or 8-bromo-cAMP did not yield any elevation in CMFs. These findings suggest that the beta-agonist effect on CMFs is transduced via a cAMP-dependent pathway. 4. Deoxygenation decreased CMFs and filterability of erythrocytes by approximately 30 %. The stimulatory effect of isoproterenol on CMFs was 2.2-fold higher in deoxygenated RBCs than in oxygenated cells. 5. Exposure of RBCs to adrenaline resulted in a concentration-dependent increase in RBC filterability, demonstrating a linear relationship between CMFs and filterability, under the same exposure conditions to adrenaline. These findings suggest that beta-adrenergic agonists may improve passage of erythrocytes through microvasculature, enhancing oxygen delivery to tissues, especially under situations of reduced oxygen tension for periods longer than 20 min.

Figure 2. Effect of adrenaline on the amplitude distribution of CMFs

RBCs were incubated in the presence of 10⁻⁷ M adrenaline (○) and in its absence (Control; •) for 20 min, at 37 °C. The amplitude distributions of control and exposed RBCs are an average of 16 and 21 time series of CMFs in different RBCs, respectively.

Figure 1. Dependence of CMF amplitude on adrenaline concentration at 24 and 37 °C

RBCs were incubated with adrenaline for 30 min at 24 °C (○) and 37 °C (•). Data are given in terms of the time-averaged maximal peak-to-peak amplitude of CMFs in the frequency range 0.3-35 Hz. The amplitudes are given by δI_max/I (%; means ±s.e.m.); see Methods. Each independent experiment involved measurements of 15-25 RBCs.

Figure 3. Effect of adrenaline on the power spectrum of CMFs

•, control RBCs, mean of 16 spectra; ○, RBCs exposed to 10⁻⁷ M adrenaline, mean of 21 spectra. The frequency resolution of the power spectra is 0.3 Hz. Inset: power spectra in the frequency range 0.3-4.0 Hz. Error bars represent the s.d. of each data point. All incubations were carried out for 20 min, at 37 °C. a.u., arbitrary units.

Figure 4. Dependence of CMF amplitude on isoproterenol concentration in the absence and presence of propranolol

RBCs were incubated with isoproterenol in the absence (•) and presence (○) of 10⁻⁹ M propranolol for 20 min, at 37 °C. Data are given in terms of the maximal CMF in the frequency range 0.3-35 Hz. The amplitudes are given by δI_max/I (%; means ±s.e.m.) Each independent experiment involved measurements of 15-25 RBCs.

Figure 5. Effect of isoproterenol on CMF amplitudes in oxygenated and deoxygenated RBCs

RBCs were incubated in the absence (Control; □) and presence () of 10⁻⁹ M isoproterenol for 20 min, at 37 °C. The amplitudes are given by δI_max/I (%; means ±s.e.m.), where n (given in parentheses) is the number of RBCs tested.

Figure 6. Effect of 8-bromo-cAMP and adrenaline on CMF amplitudes in normal and ATP-depleted RBCs

Normal (□) and ATP-depleted () RBCs were incubated for 20 min with 10⁻⁴ M 8-bromo-cAMP and 10⁻⁷ M adrenaline, at 37 °C. The amplitudes are given by δI_max/I (%; means ±s.e.m.), where n (given in parentheses) is the number of RBCs tested.

Figure 7. Dependence of CMF amplitude on the concentration of pentoxifylline

RBCs were incubated with pentoxifylline for 15 min at 37 °C. Data are given in terms of the maximal CMF in the frequency range 0.3-35 Hz. The amplitudes are given by δI_max/I (%; means ±s.e.m.) Each independent experiment involved measurements of 15-25 RBCs.

Figure 8. Time dependence of CMF amplitude following exposure to adrenaline in the absence and presence of IBMX

RBCs were incubated with 10⁻⁷ M adrenaline in the absence (•) and presence (○) of 10⁻³ M IBMX (for different time periods) at 37 °C. The amplitudes are given by δI_max/I (%; means ±s.e.m.) Each independent experiment involved measurements of 15-25 RBCs.

Figure 9. Relationship between CMF amplitude and filterability in adrenaline-treated RBCs

Filterability is given in terms of t_s/t_c and expressed as means ±s.d.; n is the number of experiments: Control, n = 71; 10⁻⁹ M adrenaline, n = 19; 10⁻⁷ M adrenaline, n = 31; 10⁻⁵ M adrenaline, n = 13. The maximal amplitudes of CMF are given by δI_max/I (%; means ±s.d.), where n is the number of RBCs tested: control, n = 51; 10⁻⁹ M adrenaline, n = 23; 10⁻⁷ M adrenaline, n = 27; 10⁻⁵ M adrenaline, n = 24. All incubations were carried out for 20 min, at 37 °C.