Effects of phenylpropanolamine (PPA) on in vitro human erythrocyte membranes and molecular models

Norephedrine, also called phenylpropanolamine (PPA), is a synthetic form of the ephedrine alkaloid. After reports of the occurrence of intracranial hemorrhage and other adverse effects, including several deaths, PPA is no longer sold in USA and Canada. Despite the extensive information about PPA toxicity, reports on its effects on cell membranes are scarce. With the aim to better understand the molecular mechanisms of the interaction of PPA with cell membranes, ranges of concentrations were incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of cell membranes. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, respectively. The capacity of PPA to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). This study presents evidence that PPA affects human red cell membranes as follows: (a) in SEM studies on human erythrocytes it was observed that 0.5 mM PPA induced shape changes; (b) in IUM PPA induced a sharp decrease in the fluorescence anisotropy in the lipid bilayer acyl chains in a concentration range lower than 100 μM; (c) X-ray diffraction studies showed that PPA in the 0.1–0.5 mM range induced increasing structural perturbation to DMPC, but no effects on DMPE multibilayers were detected.     

Transmembrane migration (''flip-flop'') of cholesterol in erythrocyte membranes.

After exchange with [14C]cholesterol-labelled plasma lipoproteins for 0.5-4h, erythrocytes were extracted with bile-salt solutions. The extracted cholesterol (mainly from the outside of the erythrocyte membrane) had the same specific radioactivity as the residual sterol. Thus cholesterol equilibrates rapidly (half-time less than 1 h) between the two sides of the membrane.     

Mechanical stresses in fast locomotion of buffalo (Syncews coffer) and elephant (Loxodonta africana)

Films of buffalo and elephant running, and detailed measurements on dissected legs, have been used to estimate the maximum stresses which occur in locomotion, in certain muscles, tendons and bones. These stresses are similar to stresses previously determined for some other, smaller mammals.     

Decrease in 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) EPR signal in ozone-treated erythrocyte membranes

In ozone-treated erythrocyte membrane suspension a slow decrease occurs in the EPR signal of 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO). Because of the absence of such a phenomenon in control membranes and ozonized buffer, this effect must be caused by reaction of nitroxide radicals with products of ozone reactions with membrane components. To find out which components are responsible for the decrease in EPR signal we studied this effect in simple model systems. The same phenomenon was observed both in lipid and protein systems treated by ozone. For unsaturated fatty acids, the correlation between the rate of decrease in EPR signal and the number of double bonds in the lipid molecule was very strong. This suggests that the observed decrease in the nitroxide radical TEMPO EPR signal in ozone-treated erythrocyte membranes is a complex process, but probably the most important reaction is recombination of nitroxide radicals with organic free radicals produced both in the process of lipid peroxidation and ozonolysis of double bonds.     

Polyphosphoinositide synthesis in rabbit erythrocyte membranes

Incubation of rabbit erythrocyte ghosts at 25 °C with 1 mm [γ-³²P]ATP and MgCl₂ results in incorporation of ³²P into diphosphoinositide and triphosphoinositide with initial rates of 15.6 and 1.8 nmol ³²P/mg/h, respectively. Incorporation of ³²P into diphosphoinositide plateaus after 20 min whereas incorporation into triphosphoinositide did not plateau until after 80 min. Diphosphoinositide and triphosphoinositide, prelabeled with ³²P, did not undergo significant breakdown when incubated at 25 °C for 15 to 20 min. Turnover of ³²P-labeled diphosphoinositide and triphosphoinositide was insignificant in the presence of MgCl₂ and cold ATP. Diphosphoinositide is not phosphorylated to triphosphoinositide in the presence of Mg-ATP under conditions in which synthesis of these polyphosphoinositides can occur. In the presence of neomycin and Mg-ATP, labeled diphosphoinositide was rapidly phosphorylated to triphosphoinositide. Neomycin had no effect on labeled di- and triphosphoinositide content in the absence of ATP. Freeze-thawing the ghosts or the addition of Triton X-100 does not produce the same effect as neomycin. The results of this investigation suggest that diphosphoinositide and triphosphoinositide are normally synthesized from endogenous phosphatidylinositol in rabbit ghosts by separate enzymatic pathways. Neomycin an aminoglycoside which interacts with polyphosphoinositides may perturb the organization of substrates and kinase activities involved in polyphosphoinositide metabolism and alter these pathways.     

Voltage-induced conductance in human erythrocyte membranes

Isotonic suspensions of erythrocytes were exposed to intense electric fields for a duration in microseconds. Time-dependent increase in the conductivity of the suspension was observed under fields greater than a threshold of about 1.5 kV/cm. The threshold was independent of the ionic strength of the medium, and changed little with temperature or with the rise time of the applied field. Under fields greater than 3 kV/cm, the time course of the conductivity increase consisted of a rapid (approx. 1 μs) and a slow (approx. 100 μs) phases. The increase is attributed primarily to large membrane conductance induced by the applied field. The membrane conductance is in the order of $\text{10 Ω}{}^{\mathrm{?1}}\text{/}\text{cm}{}^2$ in the rapid phase and $\text{10}{}^2\text{Ω}{}^{\mathrm{?1}}\text{/}\text{cm}{}^2$ in the slow phase. Comparison with previous results indicates that this induced membrane conductance corresponds to the formation of aqueous pores in the cell membrane. After the applied field was removed, the conductivity of the suspension returned nearly to its initial value, indicating that the induced membrane conductance is strongly dependent on the membrane potential. The conductivity then increased again in the time range of 10 s. This is attributed to the diffusional efflux of intracellular ions through the voltage-induced pores. From the rate of the efflux, number of the pores/cell is estimated to be in the order of 10². Final stage of the conductivity change was a slow decrease, corresponding to the colloid osmotic swelling of the perforated cells.     

Decrease in 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) EPR signal in peroxynitrite-treated erythrocyte membranes.

The treatment of erythrocyte membranes with peroxynitrite (ONOO-), a cytotoxic species formed in vivo by the almost completely diffusion controlled reaction of nitric oxide (NO*) and the superoxide anion (O2*-), led to the loss of the EPR signal of the nitroxide radical 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO). The decrease in the TEMPO EPR signal was peroxynitrite concentration dependent in the studied peroxynitrite concentration range (100-1000 microM). The absence of such a phenomenon in the control membranes (not treated with peroxynitrite) and in a buffer treated with peroxynitrite indicates that the effect must be caused by nitroxide radicals reacting with the products of peroxynitrite reactions with membrane components. To find out which membrane components are responsible for the decrease in EPR signal, this effect was studied in simple model systems (protein and lipid suspensions). The same phenomenon was observed in both lipid and protein systems treated with peroxynitrite, but in protein solutions the effect was greater and occurred for lower peroxynitrite concentrations. A clear effect of the loss of the EPR signal was observed for both erythrocyte membranes and bovine serum albumin (BSA) solution for a peroxynitrite concentration of 100 microM, while in the case of linolenic acid suspension, a significant difference between control and peroxynitrite-treated samples was achieved for a peroxynitrite concentration of 1000 microM. A comparison of the results obtained for the lipid and protein systems suggests that the reaction of nitroxide radicals with protein derived species plays the main role in the observed decrease in the TEMPO EPR signal in peroxynitrite treated erythrocyte membranes.     

Proteins of marsupial erythrocyte membranes

The proteins of erythrocyte membranes from the red kangaroo, western grey kangaroo, eastern grey wallaroo (euro), red-necked wallaby, Tammar wallaby, and brush-tail possum have been fractionated on acrylamide gels in the presence of sodium dodecyl sulfate. The pattern of proteins was remarkably similar between the different marsupial species. The pattern of Coomassie blue-staining proteins in the membranes of these species was also very similar to that of the human erythrocyte membrane. However, the glycoproteins in the marsupial erythrocyte membranes were markedly less conspicuous than those of the human erythrocyte membrane. Furthermore, the mobilities of the glycoproteins from the marsupials were different from those of the human erythrocyte membrane. The erythrocytes of the western grey kangaroo, the eastern wallaroo and the red-necked wallaby showed pronounced resistance to hypotonic lysis compared with those of the Tammar wallaby and the human. This effect seems to be related to the size of the erythrocytes rather than to differences in their protein composition.     

Structural changes in erythrocyte membranes in nephropathy

Spin probes were used to study alteration of red cell membranes in nephropathy of varying degree of gravity. Iminoxyl radicals of the lipid nature were applied as spin probes, in particular 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyl (probe I). It was shown that in nephropathy, the orderliness parameter increases and the hydrophoby of probe I localized in a red cell suspension of nephropathy patients diminishes as compared with analogous parameters in healthy pregnant women. This attests to both immobilization of the fatty acid chains of phospholipids and to an increase in the polarity of the lipid bilayer in the area of probe localization. It was established that diminution of probe I hydrophoby is in a satisfactory agreement with the disease gravity and the degree of edema in patients. It was noted that alterations discovered in red cell membranes in nephropathy are similar to those seen during activation of lipid peroxidation in membranes. The possibility of lipid peroxidation involvement into the pathogenesis of nephropathy is discussed.     

Amiloride fluxes across erythrocyte membranes

Amiloride is known to inhibit both the influx of Na+ and the activation of mitogenesis in many cultured cell lines. This paper describes experiments in which the permeability coefficient of amiloride was determined from measurements of tracer fluxes across human erythrocytes and resealed ghosts. From an analysis of these fluxes, a permeability coefficient of 10(-7) cm/s for the uncharged form of amiloride was deduced. Based upon this measured permeability value, we present calculations of intracellular accumulation times of amiloride in cells of differing surface-to-volume ratio.     

Control of lecithin biosynthesis in erythrocyte membranes

The detailed relationship between the relative composition of the potential precursor acids, the esterification rates of their CoA thiol ester derivatives, and the relative composition of the fatty acids in the product, lecithin, which was isolated from normal erythrocytes, suggests that in humans the stromal acyltransferases could be the significant enzymatic factor controlling the fatty acid composition at the 2-position of lecithin in erythrocytes.     

A transmembranous NADH-dehydrogenase in human erythrocyte membranes

Evidence is presented for a transmembranous NADH-dehydrogenase in human erythrocyte plasma membrane. We suggest that this enzyme is responsible for the ferricyanide reduction by intact cells. This NADH-dehydrogenase is distinctly different from the NADH-cytochromeb₅ reductase on the cytoplasmic side of the membrane. Pretreatment of erythrocytes with the nonpenetrating inhibitor diazobenzene sulfonate (DABS) results in a 35% loss of NADH-ferricyanide reductase activity in the isolated plasma membrane. Since NADH and ferricyanide are both impermeable, the transmembrane enzyme can only be assayed in open membrane sheets with both surfaces exposed, and not in closed vesicles. The transmembrane dehydrogenase has affinity constants of 90 µM for NADH and 125 µM for ferricyanide. It is inhibited byp-chloromercuribenzoate, bathophenanthroline sulfonate, and chlorpromazine.     

Guanosine triphosphatase activity in human erythrocyte membranes

Human red cell membranes have the capacity to hydrolyze enzymatically GTD to GDP. The reaction requires magnesium, is not appreciably affected by sodium, potassium or calcium, and is not inhibited by ouabain. Kinetic analysis suggests that there are two separate enzymes in membranes which cleave GTP, a 'high Km' GTPase and a 'low Km' GTPase. Both enzymes are also ATPases, with an approximately equal affinity for GTP and ATP. GTPase activity did not extract from the membrane with spectrin and was not inactivated by antispectrin antibody. Activity was partially destroyed by 0.5% Triton X-100. It seems probable that the low Km GTPase is the sodium- and potassium-independent ATPase of red cell membranes. The identity of the high Km enzyme is not clear.     

Muscarinic cholinergic binding in human erythrocyte membranes

Human erythrocyte ghosts contain a small population of muscarinic cholinergic receptors, as evidenced by their high affinity binding of radiolabeled quinuclinidinyl benzilate ([³H]QNB). The apparent K_D is 1.3 × 10^?9 M and the receptor sites are saturated at a QNB concentration of 5 nM. The number of sites is 23 fmoles/mg membrane protein. The pharmacological profile of the specific binding is similar to that of neural membranes. The binding is not stereoselective for the d and 1 isomers of QNB, a situation which prevails in the muscarinic receptors of another peripheral cholinergic system, the rat iris, but not in the central nervous system.