Cell membrane fluctuations (CMF) of human erythrocytes, measured by point dark field microscopy, were shown to depend, to a large extent, on intracellular MgATP (Levin, S.V., and R. Korenstein. 1991. Biophys. J. 60:733–737). The present study extends that investigation and associates CMF with F-actin's ATPase activity. MgATP was found to reconstitute CMF in red blood cell (RBC) ghosts and RBC skeletons to their levels in intact RBCs, with an apparent K d of 0.29 mM. However, neither non-hydrolyzable ATP analogues (AMP-PNP, ATPγS) nor hydrolyzable ones (ITP, GTP), were able to elevate CMF levels. The inhibition of ATPase activity associated with the RBC's skeleton, carried out either by the omission of the MgATP substrate or by the use of several inhibitors (vanadate, phalloidin, and DNase I), resulted in a strong decrease of CMF. We suggest that the actin's ATPase, located at the pointed end of the short actin filament, is responsible for the MgATP stimulation of CMF in RBCs.

Extracellular f luid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body f luids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical f luctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase f luid viscosity. The parameters of cell membrane f luctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane f luctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATPdepleted red blood cells elevation of EMF did not affect cell membrane f luctuations. This study demonstrates that (i) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (ii) cell membrane f luctuations are driven by a metabolic driving force in addition to the thermal one.The viscosity of body fluids is determined by the level of macromolecules consisting of proteins, lipoproteins, and polysacharides (1). Accordingly, elevated plasma viscosity has been observed in various diseases associated with increased levels of proteins and lipoproteins, such as diabetes, hyperlipidemia, macroglobulinemia, multiple myeloma, nephrosis, and others (1-5). Various studies have shown that solvent viscosity affects protein dynamics and reactions (6-10). However, in these studies the solvent viscosity was modified by the addition of high concentrations of small cosolvents such as glycerol and sucrose, producing relatively high viscosity levels. This is incompatible with physiological and pathological states, where fluid viscosity is altered by small concentrations of large macromolecules (1). Other studies, in which the viscosity was elevated by macromolecular cosolvents, have shown that extracellular fluid macroviscosity (EFM) is a regulator of cellular processes, such as secretion of renin (11) and lipoproteins (12), phospholipase A 2 activity at the cell membrane (13, 14), and ganglioside metabolism (15). In search of the mechanism of this phenomenon, the effect of macroviscosity, as modified by macromolecules, on isolated proteins in aqueous solutions was examined (16,17). It was found that the effect of solvent viscosity decreases with increasing molecular weight of the cosolvent and is practically diminished when the cosolvent molecular weight exceeds that of the protein. Because the activity of cell membrane enzymes is known to be sensitive to the physical properties of the membrane (18), we considered the possibility that the...

The observation of low-frequency fluctuations of the cell membrane in erythrocytes and in several nucleated cells suggests that this phenomenon may be a general property of the living cell. A study of these fluctuations in human erythrocytes and its ghosts has now been carried out using a novel optical method based on point dark field microscopy. We have demonstrated that the reestablishment of membrane fluctuations in erythrocyte ghosts is dependent on MgATP but does not necessarily require the restoration of the biconcave shape. The results imply that the dominant component of membrane fluctuations are metabolically dependent and suggest the existence of a dynamic mechano-chemical coupling within the membrane skeleton network induced by MgATP.

This paper describes transverse oscillations, within the range 0.2-30 Hz, of the surface of different animal cells: human and frog erythrocytes, human lymphocytes and monocytes, cultured 3T6 fibroblasts, and rat cardiomyocytes. The minimal area of the cell surface which undergoes unidirectional transverse movement is equal to or less than 0.5 x 0.5 microns. The amplitude of the oscillations recorded on larger surface areas is lower than on the smaller ones because of the averaging of solitary oscillations. The oscillation amplitude is different in different cells. The highest amplitude is recorded in human erythrocytes (350-400 nm), the lowest one, in fibroblasts, lymphocytes and monocytes (20-30 nm). The oscillations of the human erythrocyte are suppressed on hypotonic swelling, after hardening of the cell membrane owing to adsorption at the surface of the impermeable dye Heliogen Blue, by treatment of the cell with 0.01% glutaraldehyde, by treatment with 0.5 mM 4-hydroxy-mercurybenzoate, and after crenation caused by 1-2 mM 2,4-dinitrophenol. The amplitude of the surface oscillations is decreased in spectrin deficient erythrocytes obtained from patients with hereditary spherocytosis, which indicates an essential role for spectrin in the rapid oscillations of the erythrocyte surface.

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 (≈0.25 μm2) of the cell surface by a method based on point dark field microscopy. 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 β‐adrenergic agonists yielded an EC50 of 5 × 10−9 and 1 × 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. 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 β‐agonist effect on CMFs is transduced via a cAMP‐dependent pathway. Deoxygenation decreased CMFs and filterability of erythrocytes by ≈30 %. The stimulatory effect of isoproterenol on CMFs was 2.2‐fold higher in deoxygenated RBCs than in oxygenated cells. 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 β‐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.