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Effects of AlCl3 on toad skin, human erythrocytes, and model cell membranes
Brain Research Bulletin, Vol. 55, No. 2, pp. 205–210, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/01/$–see front matter
PII S0361-9230(01)00505-6
Effects of AlCl3 on toad skin, human erythrocytes, and model cell membranes M. Suwalsky,1* B. Ungerer,1 F. Villena,2 B. Norris,2 H. Ca´rdenas2 and P. Zatta3 1
Faculty of Chemical Sciences, 2Faculty of Biological Sciences, University of Concepcio´n, Concepcio´n, Chile; and 3CNR Center on Metalloproteins, University of Padoa, Padoa, Italy
ABSTRACT: Aluminum, a very abundant metal, could play a toxic role in several pathological processes, including neurodegeneration. Although the effects of Al(III) on biological membranes have been extensively described, direct information concerning the molecular basis of its biological activity is rather scanty. To examine aluminum challenges on cell membranes, various concentrations of AlCl3 in aqueous solutions were incubated with human erythrocytes, isolated toad skin, and molecular models of biomembranes. The latter consisted of multilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representing phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane. These specimens were studied by scanning electron microscopy, electrophysiological measurements, and x-ray diffraction. The results indicate that Al(III) in the concentration range of 10 –100 M induced the following structural and functional effects: (i) change in the normal discoid shape of human eryhrocytes to echinocytes due to the accumulation of Al(III) ions in the outer moiety of the red cell membrane; (ii) perturbation of dimyristoylphosphatidylcholine, and to a lesser extent of dimyristoylphosphatidylethanolamine bilayers, and (iii) decrease in the short-circuit current and in the potential difference of the isolated toad skin, effects that are in accordance with a time-dependent modulation of ion transport in response to changes in the molecular structure of the lipid bilayer. © 2001 Elsevier Science Inc.
incubated with metal ions they can become more or less rigid, depending on the type of membrane, the nature and concentration of the metal, and the pH. Corain et al. [5] found that 0.34 – 8 mM Al(III) caused an increase in the microviscosity of rabbit erythrocytes; Van Rensburg et al. [34] reported that 0.01–10 M Al(III) at pH 5.5 increased the microviscosity of erythrocyte membranes, but decreased that of platelet membranes. On the other hand, Al(III) salts injected in vivo into the brains of rabbits resulted in fragmentation of the endoplasmic reticulum, and in cats in the formation of monofilamentous neurofibrillary tangles [34]. In renal dialysis patients suffering from aluminum encephalopathy, postmortem examination showed immature plaques [34]. While none of these plaques conformed with the paired helical filamentous neurofibrillary tangle or classical senile plaque found in Alzheimer’s disease patients, aluminum was found to be responsible for these neuropathological aberrations [34]. High Al and manganese (Mn) concentrations in the drinking water in the Guam islands in the Western Pacific, together with low dietary calcium (Ca) and magnesium (Mg) have been implicated in the etiology of the so-called Parkinson-dementia complex of Guam [18]. Additional effects of Al(III) on membrane structure and function have been summarized by Banks and Kastin [2]. Thus, whereas the effects of Al(III) on biological membranes have been extensively described, direct information concerning the molecular basis of its biological activity is rather scanty. We recently reported the effect of aluminum acetylacetonate (Al(acac)3), a neutral, chemically well defined, hydrolytically stable and lipophilic compound on cell and model membranes [22]. The results showed that Al(acac)3 interacted with the human erythrocyte membrane modifying its normal discoid morphology to both echinocytic and stomatocytic shapes [22]. This finding indicated that the Al complex was inserted in both the outer and inner monolayers of the red cell membrane, a conclusion supported by the results of x-ray diffraction on membrane molecular models [22]. On the other hand, electrophysiological measurements performed on toad skin revealed a significant decrease in the potential difference (PD) and short-circuit current (Isc), effects interpreted to reflect inhibition of the active transport of ions [22]. As either Al(III) or its hydrated form [Al(H2O)6]3⫹ represent the principal reactive form in biology [8,12,13], we thought that it was of interest to carry out, by means of aqueous solutions of AlCl3, studies similar to those performed with Al(acac)3 in order to detect the structural and functional effects induced by the metal ion in cell membranes. In particular, we took into account that Al(III) salts are commonly used in water
KEY WORDS: Aluminum, Phospholipid bilayer, Alzheimer’s disease.
INTRODUCTION Aluminum is the most abundant metal and the third most common element. However, despite its abundance, no useful biological function for it has been discovered. On the contrary, it is recognized as a toxic metal. In fact, compelling evidence has shown that abnormally high Al(III) levels are linked to pathologies such as dialysis dementia, iron-adequate microcytic anemia, osteomalacia, and possibly Alzheimer’s disease [4]. The goal of understanding the cellular and eventually the molecular basis of aluminum toxicity has stimulated enormous efforts to develop animal, cellular, and molecular models. The cell membrane is a diffusion barrier which protects the cell interior. Therefore, its structure and functions are susceptible to alterations as a consequence of interactions with metal ions. In general, metal ions can influence the viscosity of cell membranes both in vitro and in vivo. When membrane suspensions in vitro are
* Address for correspondence: Dr. Mario Suwalsky, Faculty of Chemical Sciences, University of Concepcio´n, Casilla 160-C, Concepcio´n, Chile. Fax: ⫹56-41-245-974; E-mail: [email protected]
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treatment plants [1,8,20]. With this aim, various concentrations of AlCl3 in aqueous solutions were incubated with human erythrocytes, isolated toad skin and molecular models of biomembranes, i.e., systems that have been already used to determine the interaction and membrane perturbing effects of drugs [23,24], pesticides [25,26], and metal ions [27,28]. The molecular models utilized in our experiments consisted of multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers, respectively, of the human erythrocyte membrane [7]. The capacity of AlCl3 to perturb the bilayer structures of DMPC and DMPE was determined by x-ray diffraction. In epithelia such as toad skin, the two membrane hypothesis [19,33] has served as an excellent model in the study of Na⫹ transport. According to this hypothesis, Na⫹ diffuses into cells at the outer (mucosal or apical) membrane and is actively extruded across the inner (serosal or basolateral) membrane by Na⫹/K⫹-ATPase in exchange for K⫹. The Isc is the amount of current necessary to bring the PD across the skin down to zero and measures active Na⫹ transport. It has been found that Al(III) reversibly affects the Na⫹ current [11]. This observation prompted us to test the effect of AlCl3 on Na⫹ transport in toad skin by measuring the Isc and PD parameters. MATERIALS AND METHODS Scanning Electron Microscope Studies of Human Erythrocytes Blood was obtained from a healthy male by aspiration into a syringe containing 1 ml of saline solution (0.9% NaCl) from which the following samples were prepared: (a) control, by mixing 0.1 ml of blood stock with 0.9 ml of saline, and (b) 0.1 and 1 mM AlCl3 by mixing the blood with saline containing AlCl3 (Titrisol; Merck, Darmstadt, Germany) in adequate concentrations. Samples were incubated at 37°C for 1 h. They were then fixed overnight at 5°C by adding one drop of each sample to plastic tubes containing 1 ml of 2.5% glutaraldehyde in saline, reaching a final fixation concentration of about 2.4%. The fixed samples were directly placed on Al stubs, air dried at 37°C for 30 min to 1 h and gold-coated for 3 min at 10⫺1 Torr in a sputter device (Edwards S150, Sussex, England). Resulting specimens were examined in an Etec Autoscan SEM (Etec Corp., Hayword, CA, USA). Electrophysiological Measurements of Isolated Toad Skin Samples of abdominal skins were dissected from pithed Pleurodema thaul toads (8 –15 g) kept in tap water 24 h prior to sacrifice. Skin samples were carefully rinsed in Ringer’s solution (pH 7.4) and mounted between two halves of an Ussing chamber. A circular area of 1.0 cm2 was exposed to 3.0 ml Ringer’s solution on each side; the ion composition of the solution was (mM): Na⫹ 114, K⫹ 2.5, Cl⫺ 117.5, Ca2⫹ 2.0, HCO3⫺ 2.3 and glucose 11, and was oxygenated with a Model Elite Hagen aerator. The Isc was monitored with non-polarizable Ag/AgCl electrodes placed at a distance of 15 mm from the epithelium and connected to a voltageclamp circuit (G. Me´traux Electronique, Crissier, Switzerland) set to maintain the PD across the skin at zero mV. The PD was measured with calomel-agar electrodes for 4 s at intervals of 2 min. Both parameters were monitored on a two-channel Cole-Parmer recorder. Experiments were started 60 min after the bioelectric parameters of the skin had reached a steady level. AlCl3 was applied to the outer and inner surface of the skin in the final concentrations specified below. The experiments were carried out at room temperature (18 –22°C). Statistical treatment was performed by means of Student’s t-test for paired data.
X-ray Diffraction Analysis of Phospholipid Multilayers Synthetic DMPC (lot 80H-8371 A grade MW 677.9) and DMPE (lot 68F-8350 A grade MW 635.9) from Sigma (St. Louis, MO, USA) were used without further purification. About 1 mg of each phospholipid was introduced into special glass capillaries (1 mm in diameter; Glas Technik & Konstruktion, Berlin, Germany), which were then filled with 200 l of distilled water or aqueous solutions of AlCl3 (10⫺2 mM to 400 mM). The specimens were x-ray diffracted 2 days after preparation in flat plate cameras provided with rotating devices. Specimen-to-film distances were 8 and 14 cm, standardized by sprinkling calcite powder on the capillary surface. Ni-filtered CuK␣ radiation from a Philips PW 1140 x-ray generator (Eindhoven, The Netherlands) was used. The relative reflection intensities on films were measured by peakintegration using a Bio-Rad GS-700 (Hercules, CA, USA) microdensitometer and Bio-Rad Molecular Analyst/PC image software; no correction factors were applied. The experiments were performed at 17 ⫾ 2°C, which is below the main phase transition temperature of both DMPC and DMPE. Each experiment was repeated three times and in case of doubts additional experiments were carried out. RESULTS Study of Human Erythrocytes Phase contrast and scanning electron microscopy observations revealed that human red blood cells incubated with 0.1 mM AlCl3 lost their normal discoid shapes. Figure 1 shows that AlCl3 induced echinocytosis, an alteration characterized by the development of blebs and/or protuberances on the red cell surfaces. Virtually all the cells underwent this shape change but with different intensities, i.e., some of them showed marked shape alterations, while such changes were minimal in other cells. Electrophysiological Measurements of Isolated Toad Skin The electrical response of the toad skin to AlCl3 (outer bathing solution) was represented by a decrease in the electrical parameters in respect to control values (Fig. 2A). For the maximal concentration used (0.1 mM) such decrease was 32 ⫾ 3% and 16 ⫾ 4% for Isc and PD, respectively, reaching a trough in 50 ⫾ 5 min (n ⫽ 7). The decrease was only partially reversible after washout of the compound. Furthermore, skin resistance increased by 20 ⫾ 5% (p ⬍ 0.01). The addition of 0.1 mM AlCl3 to the inner bathing solution decreased slightly, although significantly, the electrical parameters (Fig. 2B). In five out of seven experiments, after incubation with 0.1 mM AlCl3 applied to both surfaces of the skin, it was found that the decrease in the electrical parameters was preceded by a transient (22 ⫾ 3 min) and significant (p ⬍ 0.05) increase in Isc and PD (15 ⫾ 2%) (Fig. 2C). X-ray Diffraction Analysis of Phospholipid Multilayers The molecular interactions of AlCl3 with multilayers of the phospholipids DMPC and DMPE were investigated in an aqueous media. Figure 3A shows a comparison of the diffraction pattern of DMPC alone and that of DMPC incubated with AlCl3 in a 10⫺2 mM to 400 mM concentration range. As expected, pure water altered the structure of DMPC: its bilayer width increased from about 55 Å in its dry crystalline form [29] to 63 Å when immersed in water, and its reflections were reduced to only the first three orders of the bilayer width. On the other hand, a new and strong reflection of 4.2 Å appeared indicative of the fluid state reached by DMPC bilayers, and corresponded to the average distance between the fully extended acyl chains organized with rotational disorder in
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FIG. 1. Effect of AlCl3 on morphology of human erythrocytes. Images (obtained by scanning electron microscopy) of untreated erythrocytes (A), and erythrocytes incubated with 0.1 mM AlCl3 (B). Scale bar in (A): 3.8 m, in (B): 3.5 m.
hexagonal packing. The response to 10⫺2 mM AlCl3 consisted of a marked decrease in the phospholipid reflection intensities, all of which almost disappeared after exposure to 1 mM and 10 mM AlCl3. These results imply that the salt induced a strong molecular disorder of the DMPC bilayer in a concentration-dependent manner. However, this effect was reversed at 100 mM AlCl3 as the intensities increased to values somewhat similar to those shown by DMPC in pure water. The finding suggests that this concentration of the salt induced a molecular reordering of DMPC. The intensity of the 4.2 Å reflection was unchanged with higher AlCl3 concentration; however, a marked decrease of the remaining intensities was observed. This result implies that only the polar head region was structurally affected. The results of an x-ray diffraction analysis of DMPE incubated with water and AlCl3 is shown in Fig. 3B. As reported elsewhere [30], water did not significantly affect the bilayer structure of DMPE. However, 10⫺2 mM AlCl3 produced a considerable decrease of the reflection intensities. They remained almost unchanged with higher concentrations of the salt, although an increase of the low angle reflection intensity was observed when it reached a 400-mM concentration. DISCUSSION The present results indicate that Al(III) ions interact with the human erythrocyte membrane changing its normal discoid shape to an echinocytic form, characterized by the formation of blebs and/or protuberances over the cell surface. According to the bilayer couple hypothesis [21], shape changes induced in erythrocytes by foreign molecules are due to a differential expansion of the two monolayers of the membrane. Thus, the spiculated shape (echinocyte) arises when the added compound locates in the outer monolayer, whereas a cup shape (stomatocyte) is induced when the compound is inserted in the inner monolayer. The fact that 0.1 mM AlCl3 induced the formation of echinocytes is an indication that Al(III) ions accumulated in the outer moiety of the red cell membrane. This conclusion is supported by x-ray diffraction of bilayers
composed of DMPC and DMPE. They represent phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively [7]. These phospholipids differ only in their terminal amino groups, these being ⫹N(CH3)3 in DMPC and ⫹NH3 in DMPE. Moreover, both molecular conformations are very similar in their dry crystalline phases [29,30] with the hydrocarbon chains mostly parallel and extended, and the polar groups lying perpendicularly to them. However, the gradual hydration of DMPC results in water filling the highly polar interbilayer spaces. Thus, its bilayer width increases from 54.5 Å (when dry) up to about 63 Å (when fully hydrated). This phenomenon allows the incorporation of Al(III) ions into DMPC bilayers producing its structural perturbation at a 10⫺2 mM concentration and complete destruction when it is 1 mM. On the other hand, DMPE molecules pack tighter than those of DMPC due to their smaller polar group and higher effective charge, resulting in a very stable bilayer system that is not significantly affected by water [29,30] or by a number of compounds [23–26]. This organization did not prevent a low concentration of Al(III) (10⫺2 mM) from interacting with and perturbing its structure. However, these effects were much milder than those observed in DMPC bilayers. At relatively high Al(III) concentrations (100 mM in DMPC and 400 mM in DMPE), a reordering effect takes place in both phospholipid bilayers. Although these ordering-disordering effects might seem surprising, we have reported similar effects with Al(acac)3 [22] and other cations such as Zn(II) [27], Cu(II) [28], and Hg(II) [31]. The structural fluctuations observed with Al(III) ions can be explained as follows: as described previously, the lipid polar head groups lie perpendicularly to the extended acyl chains; contacts between monolayers to form bilayers occur via the acyl chain terminal amino groups. This arrangement is stabilized through hydrophobic interactions among the acyl chains and electrostatic interactions between the negatively charged phosphates and positively charged amino groups of neighboring polar heads [29,30]. At low AlCl3 concentrations Al(III) ions bind electrostatically to a
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SUWALSKY ET AL. few lipid polar groups and as a consequence their stabilizing inter-head group interactions are disrupted and some head groups and acyl chains change their orientations. This type of structural perturbation would result in the fainting of the phospholipid reflection intensities. However, as the AlCl3 concentration increases, more Al(III) ions link to neighboring polar groups leading to a cooperative ordering of the lipid molecules. Experiments performed on isolated toad skin showed a moderate, although significant decrease in the Isc and in the PD after application of AlCl3, which could indicate inhibition of the active transport of ions. The effect, especially evident when the compound was applied to the outer surface, was far more pronounced than that of aluminum acetylcetonate, which at 0.15 mM concentration (outer surface) induced a 3% decrease in Isc [22], whereas 0.1 mM AlCl3 applied in the current study was followed by a 32% decline in Isc. The fact that AlCl3 was less active at the inner surface of the skin suggests that the main site of interaction is the outward facing epithelial membrane. It has been recently shown that one of the primary targets of aluminum is the plasma membrane [32], which is affected by the metal from the cell exterior. The initial transient but significant rise in the electric parameters found in several experiments on application of AlCl3 to both surfaces might be partly understood by the fact that 0.1 mM Al(III) is able to open a metal ion-activated channel [17] and that micromolar concentrations of AlCl3 were found to markedly enhance the voltage-activated sodium currents in the neurons of a pond snail [6]. Furthermore, Homma and Harris [10] found transient stimulation of Na⫹/K⫹/Cl⫺ co-transport in rat glomerular mesangial cells. On the other hand, these authors [10] also showed that the increase in co-transport was followed by a progressive decrease of its baseline activity, and Missiaen et al. [15] showed that AlF4⫺ inhibits the activity of cation-transport ATPases. Advances in the understanding of epithelial ion channels [9] argue for several putative mechanisms of action for metal toxicity, such as (a) disturbance of lipid bilayer integrity; (b) alterations in lipid structure which lead to membrane protein changes; (c) direct interaction with receptor proteins, enzymes or ion channels, which could involve specific receptor sites for metal ions. Mechanism (a) can be ruled out because it involves decreased resistance across the bilayer, whereas the maximal Al(III) concentration applied to the outer surface of the skin induced a significant increase in resistance. Evidence for mechanism (b) was found by x-ray diffraction which showed molecular disorder of DMPC after exposure to Al(III), thus altering protein activity, which could then affect apical Na⫹ channels. Micromolar concentrations of AlCl3 decreased the conductance of single outer membrane channels from rat brain mitochondria [14]. Taken together, these findings could link the biological activity of aluminum to changes in channel protein structure and function which lead to the impairment of ion fluxes; the metal ion is a potent specific blocker of calcium channel currents in mammalian neurons [3]. Such actions may be limited at higher AlCl3 concentrations due to a reordering effect of the phospholipid bilayers, which for the permeable toad skin may take place at far lower Al(III) concentrations than for a relatively rigid model membrane. Further evidence for mechanism (b) is provided by x-ray diffraction studies which revealed a structural perturba-
FIG. 2. (A) and (B) represent the effects of increasing concentration of AlCl3 on the electrical properties of the isolated toad skin. Results are expressed as percentage decrease of control values. Each point represents the mean ⫾ standard error of the mean; n ⫽ 7. Abbreviations: PD, potential difference; Isc, short-circuit current. Values for untreated skins were: PD,
35.8 ⫾ 3.4 mV, and Isc 45.5 ⫾ 4.9 A/cm2. (A) Outer surface, AlCl3 0.01, 0.03 and 0.1 mM. (B) Inner surface, AlCl3 0.03, 0.067 and 0.1 mM. In contrast with control values, *p ⬍ 0.05, **p ⬍ 0.01, ***p ⬍ 0.001; NS ⫽ not significant. (C) illustrates the tracing representative of five experimental runs showing the effect of 0.1 mM AlCl3 on the bioelectric parameters of the skin.
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FIG. 3. (A) Microdensitograms from x-ray diffraction patterns of dimyristoylphosphatidylcholine (DMPC) in water and aqueous solutions of AlCl3. (B) Microdensitograms from x-ray diffraction patterns of and dimyristoylphosphatidylethanolamine (DMPE) in water and aqueous solutions of AlCl3. In both (A) and (B), specimen-to-film distance: (a) 14 cm, (b) 8 cm.
tion of DMPE molecules after interaction with Al(III), a process which could alter membrane proteins or enzymes and affect ion transport at the inner facing membrane. The minimum Al(III) concentrations that in the present work induced structural and functional perturbations ranged from 10 M to 100 M. These concentrations are relevant since a level of 14 – 40 M aluminum was found in the plasma of uremic patients [16] and much higher levels in serum and brain of such patients [13]. ACKNOWLEDGEMENTS
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8.
The authors thank Sylvia Marchant, Fernando Neira, and Jose´ Morales for technical assistance. This work was supported by grants from FONDECYT (1990289), DIUC (95.24.09-1) and the CONICYT (Chile)-CNR (Italy) International Program of Scientific Cooperation (99091).
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PII S0361-9230(01)00505-6
Effects of AlCl3 on toad skin, human erythrocytes, and model cell membranes M. Suwalsky,1* B. Ungerer,1 F. Villena,2 B. Norris,2 H. Ca´rdenas2 and P. Zatta3 1
Faculty of Chemical Sciences, 2Faculty of Biological Sciences, University of Concepcio´n, Concepcio´n, Chile; and 3CNR Center on Metalloproteins, University of Padoa, Padoa, Italy
ABSTRACT: Aluminum, a very abundant metal, could play a toxic role in several pathological processes, including neurodegeneration. Although the effects of Al(III) on biological membranes have been extensively described, direct information concerning the molecular basis of its biological activity is rather scanty. To examine aluminum challenges on cell membranes, various concentrations of AlCl3 in aqueous solutions were incubated with human erythrocytes, isolated toad skin, and molecular models of biomembranes. The latter consisted of multilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representing phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane. These specimens were studied by scanning electron microscopy, electrophysiological measurements, and x-ray diffraction. The results indicate that Al(III) in the concentration range of 10 –100 M induced the following structural and functional effects: (i) change in the normal discoid shape of human eryhrocytes to echinocytes due to the accumulation of Al(III) ions in the outer moiety of the red cell membrane; (ii) perturbation of dimyristoylphosphatidylcholine, and to a lesser extent of dimyristoylphosphatidylethanolamine bilayers, and (iii) decrease in the short-circuit current and in the potential difference of the isolated toad skin, effects that are in accordance with a time-dependent modulation of ion transport in response to changes in the molecular structure of the lipid bilayer. © 2001 Elsevier Science Inc.
incubated with metal ions they can become more or less rigid, depending on the type of membrane, the nature and concentration of the metal, and the pH. Corain et al. [5] found that 0.34 – 8 mM Al(III) caused an increase in the microviscosity of rabbit erythrocytes; Van Rensburg et al. [34] reported that 0.01–10 M Al(III) at pH 5.5 increased the microviscosity of erythrocyte membranes, but decreased that of platelet membranes. On the other hand, Al(III) salts injected in vivo into the brains of rabbits resulted in fragmentation of the endoplasmic reticulum, and in cats in the formation of monofilamentous neurofibrillary tangles [34]. In renal dialysis patients suffering from aluminum encephalopathy, postmortem examination showed immature plaques [34]. While none of these plaques conformed with the paired helical filamentous neurofibrillary tangle or classical senile plaque found in Alzheimer’s disease patients, aluminum was found to be responsible for these neuropathological aberrations [34]. High Al and manganese (Mn) concentrations in the drinking water in the Guam islands in the Western Pacific, together with low dietary calcium (Ca) and magnesium (Mg) have been implicated in the etiology of the so-called Parkinson-dementia complex of Guam [18]. Additional effects of Al(III) on membrane structure and function have been summarized by Banks and Kastin [2]. Thus, whereas the effects of Al(III) on biological membranes have been extensively described, direct information concerning the molecular basis of its biological activity is rather scanty. We recently reported the effect of aluminum acetylacetonate (Al(acac)3), a neutral, chemically well defined, hydrolytically stable and lipophilic compound on cell and model membranes [22]. The results showed that Al(acac)3 interacted with the human erythrocyte membrane modifying its normal discoid morphology to both echinocytic and stomatocytic shapes [22]. This finding indicated that the Al complex was inserted in both the outer and inner monolayers of the red cell membrane, a conclusion supported by the results of x-ray diffraction on membrane molecular models [22]. On the other hand, electrophysiological measurements performed on toad skin revealed a significant decrease in the potential difference (PD) and short-circuit current (Isc), effects interpreted to reflect inhibition of the active transport of ions [22]. As either Al(III) or its hydrated form [Al(H2O)6]3⫹ represent the principal reactive form in biology [8,12,13], we thought that it was of interest to carry out, by means of aqueous solutions of AlCl3, studies similar to those performed with Al(acac)3 in order to detect the structural and functional effects induced by the metal ion in cell membranes. In particular, we took into account that Al(III) salts are commonly used in water
KEY WORDS: Aluminum, Phospholipid bilayer, Alzheimer’s disease.
INTRODUCTION Aluminum is the most abundant metal and the third most common element. However, despite its abundance, no useful biological function for it has been discovered. On the contrary, it is recognized as a toxic metal. In fact, compelling evidence has shown that abnormally high Al(III) levels are linked to pathologies such as dialysis dementia, iron-adequate microcytic anemia, osteomalacia, and possibly Alzheimer’s disease [4]. The goal of understanding the cellular and eventually the molecular basis of aluminum toxicity has stimulated enormous efforts to develop animal, cellular, and molecular models. The cell membrane is a diffusion barrier which protects the cell interior. Therefore, its structure and functions are susceptible to alterations as a consequence of interactions with metal ions. In general, metal ions can influence the viscosity of cell membranes both in vitro and in vivo. When membrane suspensions in vitro are
* Address for correspondence: Dr. Mario Suwalsky, Faculty of Chemical Sciences, University of Concepcio´n, Casilla 160-C, Concepcio´n, Chile. Fax: ⫹56-41-245-974; E-mail: [email protected]
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treatment plants [1,8,20]. With this aim, various concentrations of AlCl3 in aqueous solutions were incubated with human erythrocytes, isolated toad skin and molecular models of biomembranes, i.e., systems that have been already used to determine the interaction and membrane perturbing effects of drugs [23,24], pesticides [25,26], and metal ions [27,28]. The molecular models utilized in our experiments consisted of multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers, respectively, of the human erythrocyte membrane [7]. The capacity of AlCl3 to perturb the bilayer structures of DMPC and DMPE was determined by x-ray diffraction. In epithelia such as toad skin, the two membrane hypothesis [19,33] has served as an excellent model in the study of Na⫹ transport. According to this hypothesis, Na⫹ diffuses into cells at the outer (mucosal or apical) membrane and is actively extruded across the inner (serosal or basolateral) membrane by Na⫹/K⫹-ATPase in exchange for K⫹. The Isc is the amount of current necessary to bring the PD across the skin down to zero and measures active Na⫹ transport. It has been found that Al(III) reversibly affects the Na⫹ current [11]. This observation prompted us to test the effect of AlCl3 on Na⫹ transport in toad skin by measuring the Isc and PD parameters. MATERIALS AND METHODS Scanning Electron Microscope Studies of Human Erythrocytes Blood was obtained from a healthy male by aspiration into a syringe containing 1 ml of saline solution (0.9% NaCl) from which the following samples were prepared: (a) control, by mixing 0.1 ml of blood stock with 0.9 ml of saline, and (b) 0.1 and 1 mM AlCl3 by mixing the blood with saline containing AlCl3 (Titrisol; Merck, Darmstadt, Germany) in adequate concentrations. Samples were incubated at 37°C for 1 h. They were then fixed overnight at 5°C by adding one drop of each sample to plastic tubes containing 1 ml of 2.5% glutaraldehyde in saline, reaching a final fixation concentration of about 2.4%. The fixed samples were directly placed on Al stubs, air dried at 37°C for 30 min to 1 h and gold-coated for 3 min at 10⫺1 Torr in a sputter device (Edwards S150, Sussex, England). Resulting specimens were examined in an Etec Autoscan SEM (Etec Corp., Hayword, CA, USA). Electrophysiological Measurements of Isolated Toad Skin Samples of abdominal skins were dissected from pithed Pleurodema thaul toads (8 –15 g) kept in tap water 24 h prior to sacrifice. Skin samples were carefully rinsed in Ringer’s solution (pH 7.4) and mounted between two halves of an Ussing chamber. A circular area of 1.0 cm2 was exposed to 3.0 ml Ringer’s solution on each side; the ion composition of the solution was (mM): Na⫹ 114, K⫹ 2.5, Cl⫺ 117.5, Ca2⫹ 2.0, HCO3⫺ 2.3 and glucose 11, and was oxygenated with a Model Elite Hagen aerator. The Isc was monitored with non-polarizable Ag/AgCl electrodes placed at a distance of 15 mm from the epithelium and connected to a voltageclamp circuit (G. Me´traux Electronique, Crissier, Switzerland) set to maintain the PD across the skin at zero mV. The PD was measured with calomel-agar electrodes for 4 s at intervals of 2 min. Both parameters were monitored on a two-channel Cole-Parmer recorder. Experiments were started 60 min after the bioelectric parameters of the skin had reached a steady level. AlCl3 was applied to the outer and inner surface of the skin in the final concentrations specified below. The experiments were carried out at room temperature (18 –22°C). Statistical treatment was performed by means of Student’s t-test for paired data.
X-ray Diffraction Analysis of Phospholipid Multilayers Synthetic DMPC (lot 80H-8371 A grade MW 677.9) and DMPE (lot 68F-8350 A grade MW 635.9) from Sigma (St. Louis, MO, USA) were used without further purification. About 1 mg of each phospholipid was introduced into special glass capillaries (1 mm in diameter; Glas Technik & Konstruktion, Berlin, Germany), which were then filled with 200 l of distilled water or aqueous solutions of AlCl3 (10⫺2 mM to 400 mM). The specimens were x-ray diffracted 2 days after preparation in flat plate cameras provided with rotating devices. Specimen-to-film distances were 8 and 14 cm, standardized by sprinkling calcite powder on the capillary surface. Ni-filtered CuK␣ radiation from a Philips PW 1140 x-ray generator (Eindhoven, The Netherlands) was used. The relative reflection intensities on films were measured by peakintegration using a Bio-Rad GS-700 (Hercules, CA, USA) microdensitometer and Bio-Rad Molecular Analyst/PC image software; no correction factors were applied. The experiments were performed at 17 ⫾ 2°C, which is below the main phase transition temperature of both DMPC and DMPE. Each experiment was repeated three times and in case of doubts additional experiments were carried out. RESULTS Study of Human Erythrocytes Phase contrast and scanning electron microscopy observations revealed that human red blood cells incubated with 0.1 mM AlCl3 lost their normal discoid shapes. Figure 1 shows that AlCl3 induced echinocytosis, an alteration characterized by the development of blebs and/or protuberances on the red cell surfaces. Virtually all the cells underwent this shape change but with different intensities, i.e., some of them showed marked shape alterations, while such changes were minimal in other cells. Electrophysiological Measurements of Isolated Toad Skin The electrical response of the toad skin to AlCl3 (outer bathing solution) was represented by a decrease in the electrical parameters in respect to control values (Fig. 2A). For the maximal concentration used (0.1 mM) such decrease was 32 ⫾ 3% and 16 ⫾ 4% for Isc and PD, respectively, reaching a trough in 50 ⫾ 5 min (n ⫽ 7). The decrease was only partially reversible after washout of the compound. Furthermore, skin resistance increased by 20 ⫾ 5% (p ⬍ 0.01). The addition of 0.1 mM AlCl3 to the inner bathing solution decreased slightly, although significantly, the electrical parameters (Fig. 2B). In five out of seven experiments, after incubation with 0.1 mM AlCl3 applied to both surfaces of the skin, it was found that the decrease in the electrical parameters was preceded by a transient (22 ⫾ 3 min) and significant (p ⬍ 0.05) increase in Isc and PD (15 ⫾ 2%) (Fig. 2C). X-ray Diffraction Analysis of Phospholipid Multilayers The molecular interactions of AlCl3 with multilayers of the phospholipids DMPC and DMPE were investigated in an aqueous media. Figure 3A shows a comparison of the diffraction pattern of DMPC alone and that of DMPC incubated with AlCl3 in a 10⫺2 mM to 400 mM concentration range. As expected, pure water altered the structure of DMPC: its bilayer width increased from about 55 Å in its dry crystalline form [29] to 63 Å when immersed in water, and its reflections were reduced to only the first three orders of the bilayer width. On the other hand, a new and strong reflection of 4.2 Å appeared indicative of the fluid state reached by DMPC bilayers, and corresponded to the average distance between the fully extended acyl chains organized with rotational disorder in
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FIG. 1. Effect of AlCl3 on morphology of human erythrocytes. Images (obtained by scanning electron microscopy) of untreated erythrocytes (A), and erythrocytes incubated with 0.1 mM AlCl3 (B). Scale bar in (A): 3.8 m, in (B): 3.5 m.
hexagonal packing. The response to 10⫺2 mM AlCl3 consisted of a marked decrease in the phospholipid reflection intensities, all of which almost disappeared after exposure to 1 mM and 10 mM AlCl3. These results imply that the salt induced a strong molecular disorder of the DMPC bilayer in a concentration-dependent manner. However, this effect was reversed at 100 mM AlCl3 as the intensities increased to values somewhat similar to those shown by DMPC in pure water. The finding suggests that this concentration of the salt induced a molecular reordering of DMPC. The intensity of the 4.2 Å reflection was unchanged with higher AlCl3 concentration; however, a marked decrease of the remaining intensities was observed. This result implies that only the polar head region was structurally affected. The results of an x-ray diffraction analysis of DMPE incubated with water and AlCl3 is shown in Fig. 3B. As reported elsewhere [30], water did not significantly affect the bilayer structure of DMPE. However, 10⫺2 mM AlCl3 produced a considerable decrease of the reflection intensities. They remained almost unchanged with higher concentrations of the salt, although an increase of the low angle reflection intensity was observed when it reached a 400-mM concentration. DISCUSSION The present results indicate that Al(III) ions interact with the human erythrocyte membrane changing its normal discoid shape to an echinocytic form, characterized by the formation of blebs and/or protuberances over the cell surface. According to the bilayer couple hypothesis [21], shape changes induced in erythrocytes by foreign molecules are due to a differential expansion of the two monolayers of the membrane. Thus, the spiculated shape (echinocyte) arises when the added compound locates in the outer monolayer, whereas a cup shape (stomatocyte) is induced when the compound is inserted in the inner monolayer. The fact that 0.1 mM AlCl3 induced the formation of echinocytes is an indication that Al(III) ions accumulated in the outer moiety of the red cell membrane. This conclusion is supported by x-ray diffraction of bilayers
composed of DMPC and DMPE. They represent phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively [7]. These phospholipids differ only in their terminal amino groups, these being ⫹N(CH3)3 in DMPC and ⫹NH3 in DMPE. Moreover, both molecular conformations are very similar in their dry crystalline phases [29,30] with the hydrocarbon chains mostly parallel and extended, and the polar groups lying perpendicularly to them. However, the gradual hydration of DMPC results in water filling the highly polar interbilayer spaces. Thus, its bilayer width increases from 54.5 Å (when dry) up to about 63 Å (when fully hydrated). This phenomenon allows the incorporation of Al(III) ions into DMPC bilayers producing its structural perturbation at a 10⫺2 mM concentration and complete destruction when it is 1 mM. On the other hand, DMPE molecules pack tighter than those of DMPC due to their smaller polar group and higher effective charge, resulting in a very stable bilayer system that is not significantly affected by water [29,30] or by a number of compounds [23–26]. This organization did not prevent a low concentration of Al(III) (10⫺2 mM) from interacting with and perturbing its structure. However, these effects were much milder than those observed in DMPC bilayers. At relatively high Al(III) concentrations (100 mM in DMPC and 400 mM in DMPE), a reordering effect takes place in both phospholipid bilayers. Although these ordering-disordering effects might seem surprising, we have reported similar effects with Al(acac)3 [22] and other cations such as Zn(II) [27], Cu(II) [28], and Hg(II) [31]. The structural fluctuations observed with Al(III) ions can be explained as follows: as described previously, the lipid polar head groups lie perpendicularly to the extended acyl chains; contacts between monolayers to form bilayers occur via the acyl chain terminal amino groups. This arrangement is stabilized through hydrophobic interactions among the acyl chains and electrostatic interactions between the negatively charged phosphates and positively charged amino groups of neighboring polar heads [29,30]. At low AlCl3 concentrations Al(III) ions bind electrostatically to a
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SUWALSKY ET AL. few lipid polar groups and as a consequence their stabilizing inter-head group interactions are disrupted and some head groups and acyl chains change their orientations. This type of structural perturbation would result in the fainting of the phospholipid reflection intensities. However, as the AlCl3 concentration increases, more Al(III) ions link to neighboring polar groups leading to a cooperative ordering of the lipid molecules. Experiments performed on isolated toad skin showed a moderate, although significant decrease in the Isc and in the PD after application of AlCl3, which could indicate inhibition of the active transport of ions. The effect, especially evident when the compound was applied to the outer surface, was far more pronounced than that of aluminum acetylcetonate, which at 0.15 mM concentration (outer surface) induced a 3% decrease in Isc [22], whereas 0.1 mM AlCl3 applied in the current study was followed by a 32% decline in Isc. The fact that AlCl3 was less active at the inner surface of the skin suggests that the main site of interaction is the outward facing epithelial membrane. It has been recently shown that one of the primary targets of aluminum is the plasma membrane [32], which is affected by the metal from the cell exterior. The initial transient but significant rise in the electric parameters found in several experiments on application of AlCl3 to both surfaces might be partly understood by the fact that 0.1 mM Al(III) is able to open a metal ion-activated channel [17] and that micromolar concentrations of AlCl3 were found to markedly enhance the voltage-activated sodium currents in the neurons of a pond snail [6]. Furthermore, Homma and Harris [10] found transient stimulation of Na⫹/K⫹/Cl⫺ co-transport in rat glomerular mesangial cells. On the other hand, these authors [10] also showed that the increase in co-transport was followed by a progressive decrease of its baseline activity, and Missiaen et al. [15] showed that AlF4⫺ inhibits the activity of cation-transport ATPases. Advances in the understanding of epithelial ion channels [9] argue for several putative mechanisms of action for metal toxicity, such as (a) disturbance of lipid bilayer integrity; (b) alterations in lipid structure which lead to membrane protein changes; (c) direct interaction with receptor proteins, enzymes or ion channels, which could involve specific receptor sites for metal ions. Mechanism (a) can be ruled out because it involves decreased resistance across the bilayer, whereas the maximal Al(III) concentration applied to the outer surface of the skin induced a significant increase in resistance. Evidence for mechanism (b) was found by x-ray diffraction which showed molecular disorder of DMPC after exposure to Al(III), thus altering protein activity, which could then affect apical Na⫹ channels. Micromolar concentrations of AlCl3 decreased the conductance of single outer membrane channels from rat brain mitochondria [14]. Taken together, these findings could link the biological activity of aluminum to changes in channel protein structure and function which lead to the impairment of ion fluxes; the metal ion is a potent specific blocker of calcium channel currents in mammalian neurons [3]. Such actions may be limited at higher AlCl3 concentrations due to a reordering effect of the phospholipid bilayers, which for the permeable toad skin may take place at far lower Al(III) concentrations than for a relatively rigid model membrane. Further evidence for mechanism (b) is provided by x-ray diffraction studies which revealed a structural perturba-
FIG. 2. (A) and (B) represent the effects of increasing concentration of AlCl3 on the electrical properties of the isolated toad skin. Results are expressed as percentage decrease of control values. Each point represents the mean ⫾ standard error of the mean; n ⫽ 7. Abbreviations: PD, potential difference; Isc, short-circuit current. Values for untreated skins were: PD,
35.8 ⫾ 3.4 mV, and Isc 45.5 ⫾ 4.9 A/cm2. (A) Outer surface, AlCl3 0.01, 0.03 and 0.1 mM. (B) Inner surface, AlCl3 0.03, 0.067 and 0.1 mM. In contrast with control values, *p ⬍ 0.05, **p ⬍ 0.01, ***p ⬍ 0.001; NS ⫽ not significant. (C) illustrates the tracing representative of five experimental runs showing the effect of 0.1 mM AlCl3 on the bioelectric parameters of the skin.
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FIG. 3. (A) Microdensitograms from x-ray diffraction patterns of dimyristoylphosphatidylcholine (DMPC) in water and aqueous solutions of AlCl3. (B) Microdensitograms from x-ray diffraction patterns of and dimyristoylphosphatidylethanolamine (DMPE) in water and aqueous solutions of AlCl3. In both (A) and (B), specimen-to-film distance: (a) 14 cm, (b) 8 cm.
tion of DMPE molecules after interaction with Al(III), a process which could alter membrane proteins or enzymes and affect ion transport at the inner facing membrane. The minimum Al(III) concentrations that in the present work induced structural and functional perturbations ranged from 10 M to 100 M. These concentrations are relevant since a level of 14 – 40 M aluminum was found in the plasma of uremic patients [16] and much higher levels in serum and brain of such patients [13]. ACKNOWLEDGEMENTS
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The authors thank Sylvia Marchant, Fernando Neira, and Jose´ Morales for technical assistance. This work was supported by grants from FONDECYT (1990289), DIUC (95.24.09-1) and the CONICYT (Chile)-CNR (Italy) International Program of Scientific Cooperation (99091).
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