Intramembrane protein distribution in cell cultures is affected by 50 Hz pulsed magnetic fields

Intramembrane proteins (IMP) represent a class of proteins located in the lipid bilayer of the cell membrane which function as ion channels, enzymes or receptors. Since it has been argued that biological effects of extremely low frequency (ELF) electromagnetic fields are mediated by plasma membrane, this work was designed to study the possible effects of 50 Hz pulsed magnetic fields (PMF) of the type used to stimulate bone repair, on the distribution of IMP in the plasma membrane of Swiss NIH 3T3 fibroblasts. Evaluations were based on the calculation of a distribution factor, which allows discrimination between random, regular and clustered distribution of IMP, in electron microscope images of freeze-fractured membranes. The results indicate that cells exposed to PMF for more than two hours have a significant clustering of the IMP distribution compared to control unexposed cells. Bioelectromagnetics 18:463–469, 1997. © 1997 Wiley-Liss, Inc.     

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.     

Lipids and NADPH-dependent superoxide production in plasma membrane vesicles from roots of wheat grown under copper deficiency or excess

The effects of in vivo copper on the lipid composition of root plasma membrane and the activities of membrane-bound enzymes, such as NADPH-dependent oxidases and lipoxygenase, were studied. Plants were grown in hydroponic culture for 11 d without Cu supply or in the presence of 50 microM Cu. Control plants were supplied with 0.3 microM Cu. Growth of roots was severely affected in the 50 microM Cu-grown plants, whereas roots grown in Cu-deficient solution did not show any difference in comparison with the control. The 50 microM Cu concentration caused an increase in the leakage of K(+) ions as well. Excess metal supply resulted in a decrease in the total lipid content of plasma membrane, a higher phospholipid amount and a reduction of steryl lipids (free sterols, steryl glycosides and acylated steryl glycosides). Cu depletion in the growth solution had only a slight effect on the plasma membrane lipid composition. In comparison with the control, only the excess of Cu caused a decrease in the lipid to protein ratio as well as a change in the phospholipid composition, with a lower phosphatidylcholine to phosphatidylethanolamine ratio. The degree of unsaturation of root plasma membranes decreased following the 0 Cu treatment and even more after the 50 microM Cu supply. Plasma membranes of wheat grown under metal deficiency and excess showed increased NADPH-dependent superoxide-producing oxidase activities, whereas membrane-bound lipoxygenase was not increased or activated due to Cu treatments. The consequences of changes in plasma membrane lipid composition and activated oxygen production as a result of Cu treatments are discussed.     

Evidence that ADH-stimulated intramembrane particle aggregates are transferred from cytoplasmic to luminal membranes in toad bladder epithelial cells

In freeze-fracture (FF) preparations of ADH-stimulated toad urinary bladder, characteristic intramembrane particle (IMP) aggregates are seen on the protoplasmic (P) face of the luminal membrane of granular cells while complementary parallel grooves are found on the exoplasmic (E) face. These IMP aggregates specifically correlate with ADH-induced changes in water permeability. Tubular cytoplasmic structures whose membranes contain IMP aggregates which look identical to the IMP aggregates in the luminal membrane have also been described in granular cells from unstimulated and ADH-stimulated bladders. The diameter of these cytoplasmic structures (0.11 +/- 0.004 micrometers) corresponds to that of tubular invaginations of the luminal membrane seen in thin sections of ADH-treated bladders (0.13 +/- 0.005 micrometers). Continuity between the membranes of these cytoplasmic structures (which are not granules) and the luminal membrane has been directly observed in favorable cross-fractures. In FF preparations of the luminal membrane, these apparent fusion events are seen as round, ice-filled invaginations (0.13 +/- 0.01 micrometer Diam), of which about half have the characteristic ADH-associated aggregates near the point of membrane fusion. They are less numerous than, but linearly related to, the number of aggregates counted in the same preparations (n = 78, r = 0.71, P less than 0.01). These observations suggest that the IMP aggregates seen in luminal membrane after ADH stimulation are transferred preformed by fusion of cytoplasmic with luminal membrane.     

Alpha-adrenergically mediated changes in membrane lipid fluidity and Ca2/ binding in isolated rat liver plasma membranes

Noradrenaline (0.1-5 microM, in the presence of 5 microM propranolol to block beta-receptors), ATP (100 microM) and angiotensin II (0.1 microM), which are thought to increase cytosolic Ca2+ concentration by mobilizing Ca2+ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the alpha-antagonists phenoxybenzamine (50 microM) and phentolamine (1 microM). The response to a maximal dose of noradrenaline (5 microM) and that to ATP (100 microM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 microM) along with the foreign amphiphile Na+-oleate (1-30 microM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg2+, reducing free Ca2+ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca2+ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 microM) and angiotensin II (0.1 microM) also promoted a small release of 45Ca2+ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the alpha-antagonist phentolamine (5 microM). It is proposed that noradrenaline, via alpha-adrenergic receptors and other Ca2+ -mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca2+ bound to phospholipids, removing thus the mechanical constraints brought about by this ion.     

Brain glucose bisphosphatase requires inosine monophosphate

Glucose bisphosphate phosphatase has been partially purified from the cytosol of mouse brain. Enzyme activity required Mg2+ and a heat-stable cofactor. The activator was present in boiled extracts of mouse brain mitochondrial-nuclear fraction, of red blood cells, or of rabbit muscle. The chemical properties of the activator are consistent with its identification as inosine monophosphate (IMP), including its mobility in a high pressure liquid chromatography (HPLC) system capable of resolving all of the biologically important mononucleotides. A large number of other biologically important compounds were not effective, including AMP, cAMP, cGMP, and UMP, GMP, purified by HPLC, (50 or 74 microM), gave a rate about 35% of that obtained with IMP (5 microM). The enzyme was separated completely from phosphoglucomutase and significantly from glucose bisphosphate synthase. The products of the reaction are glucose-P and Pi. Fructose bisphosphate at 500 microM inhibited only 40% in the presence of 20 microM glucose bisphosphate. The activation by IMP follows hyperbolic kinetics with an apparent Ka of 5 microM in the presence of 12 microM glucose bisphosphate. The apparent Km of glucose bisphosphate was 10 microM in the presence of 50 microM IMP. There was no inhibition by 5 or 50 microM AMP or ADP. The possible regulatory importance of glucose bisphosphate in carbohydrate metabolism and the significance of the regulation of the phosphatase by the nucleotide are discussed.     

Freeze-fracture cytochemistry of sympathetic ganglia. Distribution of filipin and tomatin induced membrane deformations in neurons and satellite cells

Application of filipin to sympathetic ganglia results in membrane deformations in both the neurons and the satellite cells. The plasma membranes of the principal ganglion cells show a non-homogeneous distribution of filipin induced deformations with fewer deformations in the perikaryal plasma membrane than in the nerve fiber membrane. The filipin induced membrane lesions are correlated to the number of IMPs of the neuronal membrane i.e. a high density of intramembrane particles (IMP) gives fewer deformations and vice versa. The membrane of the satellite cells contain a higher density of probe induced lesions than the neuronal membrane. The filipin induced deformations in the satellite cells are not correlated to the number of IMPs or to the number of orthogonal arrays of small particles (OAP). Specialized membrane areas such as the gap junction is always devoided of filipin induced lesions. A similar distribution of membrane lesions was found when tomatin was used instead of filipin. These results indicate a possible difference in lipid content between various parts of the neurons and between the neuronal and satellite cell plasma membrane in guinea pig sympathetic ganglia.     

Ultrastructure of reconstituted rat liver microsomal membranes and cytochrome b5- or P-450-containing proteoliposomes

Thin sectioning and freeze-fracture electron microscopy have been used to show that it is possible to obtain topologically closed vesicles by means of reconstitution of rat liver microsomal membrane "ghosts." The reconstitution by 15 hr dialysis resulted in the formation of vesicles with intramembrane particles (IMP) while after 40 hr dialysis no IMP were observed in the membranes. The protein/lipid ratio and functional activity of NADPH- and NADH-linked enzyme systems were similar in both cases. Cytochrome P-450 (LM2) was incorporated into liposomes of different composition (protein: lipid ratio--1:200). IMP were observed only when the incorporation of cytochrome P-450 was performed in the presence of detergent Emulgen 913 as specific additive to the initial protein-lipid-sodium cholate mixture or in the course of incubation of proteoliposomal suspensions at 37 degrees C. After the incorporation of cytochrome b5 into azolectin liposomes vesicular membranes contain IMP if the incorporated membrane protein: lipid ratio is at least 1:50. Pronase-induced splitting off of a 11 kDa heme-containing fragment of cytochrome b5 did not affect IMP content. The conditions of IMP formation in reconstituted membranes and in microsomal ghosts are discussed.     

Plasma Membrane Domains and the Site of Sperm Entrance in Discoglossus pictus (Anura) Eggs

Freeze-fracture quantitative analysis reveals three different plasma membrane (PM) domains in the unfertilized egg of the anuran Discoglossus pictus . One of these is specific to the sperm entrance site (D1). where the plasma membrane shows a larger number of intramembranous particles (IMP) than the rest of the egg surface. Such an increment is due to a markedly higher number of the IMPs anchored to the P-face. The two other domains (D2 and D3) are characterized by a lower IMP density at the P-face with respect to D1. The IMP density decreased within 10 min after fertilization by about 33% in all domains observed, probably due to the insertion of new membrane through exocytosis. The possibility that the IMPs located in D1 may represent putative plasma membrane proteins playing a role in sperm-egg interaction and/or in egg activation is discussed.     

New potent inhibitors of aquaporins: silver and gold compounds inhibit aquaporins of plant and human origin

Silver and gold compounds were tested as potential inhibitors of aquaporins of plant- and human origin. Silver as AgNO(3) or silver sulfadiazine inhibited with high potency (EC(50) 1-10 microM) the water permeability of the peribacteroid membrane from soybean (containing Nodulin 26), the water permeability of plasma membrane from roots (containing plasma membrane integral proteins), and the water permeability of human red cells (containing aquaporin 1). Gold as HAuCl(4) was less effective but still inhibited peribacteroid membrane water permeability (EC(50)=10 microM). Silver and gold are more potent inhibitors of aquaporins than the presently widely used mercury containing compounds.     

Freeze-fracture observations on mammalian oocytes

Freeze-fracture studies on mammalian oocytes have been hampered by the relatively small numbers of cells available at a given time as well as by difficulties encountered in effectively freezing these large, watery cells. We have nevertheless pursued this area because of the benefits of visualizing membrane faces involved in various fusion reactions by the freeze-fracture method. Our observations indicate no overall change in intramembranous particle (IMP) distribution before and after sperm penetration, although the question of possible alterations of these structures at the precise locus of sperm attachment remains open. Preliminary statistical analysis indicates that there is a much higher IMP density on the P face than on the E face of the plasma membrane and that the microvillar membranes bear more IMPs than those of the intermicrovillus regions. Probes of lipid subclasses were used to determine the distribution of cholesterol and anionic lipid in the egg plasma membrane. Filipin and tomatin showed extensive complex formation in microvillus as well as nonmicrovillus regions, whereas anionic lipids (using polymyxin B) have been difficult to detect on the oocyte surface. These results are discussed relative to current views of membrane fusion mechanisms.     

Phosphorylation of lysophosphatidylinositol by carrot membranes.

sn-1 Palmitoyl lysophosphatidylinositol is found in carrot suspension culture cells and can be phosphorylated to [32P]lysophosphatidylinositol monophosphate (LPIP) when [gamma 32P]ATP is added to isolated membranes. Based on in vivo labeling studies, [3H]inositol sn-1 palmitoyl LPIP was found predominantly in the plasma membrane-rich fraction or upper phase isolated by aqueous two-phase partitioning and LPI was found in the intracellular membrane-rich fraction or lower phase (Wheeler and Boss, Plant Physiol. 85, 389-392, 1987). While both membrane fractions phosphorylated LPI in vitro, the apparent Km for LPI in the intracellular membrane fraction was 180 microM and for the plasma membrane was 580 microM. When cells were treated with the ionophore, monensin, the percentage of [3H]inositol LPIP increased in the whole cell lipid extract. However, the monensin treatment decreased the amount of [3H]inositol LPIP and PIP recovered in the plasma membrane fraction relative to the sum of the individual lipid, [3H]inositol LPIP or PIP, respectively, recovered in both membrane fractions.     

An intracellular (ATP + Mg2+)-dependent calcium pump within the N1E-115 neuronal cell line

An intracellular (ATP + Mg2+)-dependent Ca2+ pumping mechanism has been identified and characterized within the cultured clonal neuroblastoma cell line N1E-115. Using cell suspensions treated with 0.005% saponin which selectively permeabilizes the plasma membrane in 95-98% of the cells, it was possible to show clearly that the intracellular Ca2+ pump mechanism is of non-plasma membrane origin and therefore can be compared directly with the Ca2+ pump characterized in detail in synaptosomal membrane vesicles (Gill, D. L., Grollman, E. F., and Kohn, L. D. (1981) J. Biol. Chem. 256, 184-192; Gill, D. L., Chueh, S. H., and Whitlow, C. L. (1984) J. Biol. Chem. 259, 10807-10813) which was proven by flux reversal studies to be derived from the neural plasma membrane (Gill, D. L. (1982) J. Biol. Chem. 257, 10986-10990). The intracellular Ca2+ pump in N1E-115 cells is distinct from mitochondrial Ca2+ accumulation and is increased up to 8-fold higher as cells reach confluency. In similarity to the neural plasma membrane pump, the intracellular Ca2+ pump within N1E-115 cells has high affinity for Ca2+ (Km = 0.28 microM), is dependent on both ATP (Km = 26 microM) and either Mg2+ or Mn2+ which half-maximally activate Ca2+ pumping at 0.35 mM and 0.32 mM, respectively, and shows similar specificity for Sr2+ and Ba2+ which half-maximally inhibit Ca2+ transport at 50 microM and 1.5 mM, respectively. In contrast to the neural plasma membrane pump, the intracellular Ca2+ pump displays approximately 40-fold higher sensitivity to La3+ (IC50 = 5 microM) and an apparent 400-fold lower sensitivity to VO4(3-) (IC50 = 185 microM), although the inhibitory effectiveness of VO4(3-) is increased 37-fold by a 15-min preincubation of the permeabilized cells with VO4(3-) in the absence of ATP (apparent IC50 = 5 microM). In further contrast to the neural plasma membrane Ca2+ pump, the intracellular pump within N1E-115 cells is stimulated more than 20-fold by oxalate (giving prolonged linear Ca2+ accumulation), is resistant to low saponin concentrations, and is not modified by calmodulin even after extensive treatment with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and/or calmodulin antagonist drugs. However, calmidazolium is effective in inhibiting the intracellular Ca2+ pump with an IC50 of approximately 2 microM.     

Isoproterenol increases active lipoprotein lipase in adipocyte medium and in rat plasma

White adipose tissue (WAT) lipoprotein lipase (LPL) activity channels diet fat towards storage in adipocytes. Adrenaline (ADR) is accepted to reduce WAT or adipocyte LPL activity (LPLa), but available data are not clear-cut regarding long exposure to ADR in vitro or in vivo. We studied the effects of long exposures to ADR or beta-adrenergic agonist on LPL: in isolated rat adipocytes (3 h) and in rats (>1 day). Isoproterenol (ISO) (1 microM) did not alter LPLmRNA nor LPLa in adipocytes, but increased LPLa in medium more than twofold (3.58 +/- 0.35 vs. 1.32 +/- 0.35 mU/10(6) adipocytes, P < 0.001). Effect was time (not present at 1 h, clear at 2 h) and concentration dependent (high sensitivity from 10 to 100 nM, max at 1 microM). Adenylate cyclase activator or cyclic AMP (cAMP) analogue produced a similar increase. Thus in adipocytes ISO produced an increase in LPLa release and/or a decrease in extracellular LPLa degradation. ADR or ISO treated rats had a two to fourfold decrease in WAT LPLa vs. unchanged LPLmRNA. This decrease was 10-fold in WAT heparin-releasable LPLa (5.7 +/- 0.6 vs. 57.3 +/- 10.2 mU/g, P < 0.001), which represents peri/extracellular LPLa. Plasma LPLa was increased 11-fold by ADR (3.30 +/- 0.58 vs. 0.32 +/- 0.08 mU/ml, P < 0.001) whereas only threefold by ISO (P > 0.01). We suggest that in vivo ADR increased release of active LPL to plasma from endothelial cells of LPL-rich tissue(s)-WAT was probably one of these tissues releasing LPL since it lost 90% of its peri/extracellular LPLa-and/or decreased degradation of plasma active LPL. Since liver LPLa was not increased, plasma active LPL might be kept away from hepatic degradation by binding to stabilising entities in plasma (fatty acids (FA), lipoproteins or soluble heparan sulphates (HS)). In conclusion, we believe this is the first report stating that: (a) ISO increases LPLa in isolated adipocyte medium, and (b) ADR administration to rats decreases WAT extracellular active LPL and increases preheparin plasma active LPL.     

A freeze-etch study of the effects of extracellular freezing on cellular membranes of wheat

Seedlings of Triticum aestivum L. cv. Lennox were grown in different environments to obtain different hardiness. Pieces of laminae and leaf bases were slowly cooled to sub-zero temperatures and the damage caused was assessed by an ion-leakage method. Comparable pieces of tissue were slowly cooled to temperatures between 2° and-14°C and were then freeze-fixed and freeze-etched. Membranes generally retained their lamellar structures indicated by the abundance of typical membrane fracture faces in all treatments, and some membrane fracture faces had patches which lacked the usual scattering of intramembranous particles (IMP). These IMP-free areas were present in the plasma membrane of tissues given a damaging freezing treatment, but were absent from the plasma membrane of room-temperature controls, of supercooled tissues, and of tissues given a non-damaging freezing treatment. The frequency of IMP-free areas and the proportion of the plasma membrane affected increased with increasing damage. In the most damaged tissue (79% damage; leaf bases exposed to-8°C), 20% of the plasma membrane was IMP-free. The frequencies of IMP at a distance from the IMP-free areas were unaffected by freezing treatments. There was a patchy distribution of IMP in other membranes (nuclear envelope, tonoplast, thylakoids, chloroplast envelope), but only in the nuclear envelope did it appear possible that their occurrence coincided with damage. The IMP-free areas of several membranes were sometimes associated together in stacks. Such membranes lay both to the outside and inside of the plasma membrane, indicating that at least some of the adjacent membrane fragments arose as a result of membrane reorganization induced by the damaging treatment. Occasional views of folded IMP-free plasma membrane tended to confirm this conclusion. The following hypothesis is advanced to explain the damage induced by extracellular freezing. Areas of plasma membrane become free of IMP, probably as a result of the freezing-induced cellular dehydration. The lipids in these IMP-free patches may be in the fluid rather than the gel phase. The formation of these IMP-free patches, especially in the plasma membrane, initiates or involves proliferation and possibly fusion of membranes, and during or following this process, the cells become leaky.Abbreviations EF exoplasmatic fracture face - IMP intramembranous particles - PF protoplasmatic fracture face     

Inhibition by linoleic acid hydroperoxide of alveolar macrophage superoxide production: effects upon mitochondrial and plasma membrane potentials

Linoleic acid hydroperoxide (LOOH) is a naturally occurring product of lipid peroxidation. Incubation of rat alveolar macrophages with LOOH produced alterations of membrane properties and function at concentrations of LOOH as low as 0.1 microM. These included phorbol myristate acetate (PMA)-stimulated superoxide production, mitochondrial membrane potential, and plasma membrane potentials. These effects were clearly separated from gross loss of structural integrity as measured by lactate dehydrogenase release, in terms of both time of incubation and concentration of LOOH. PMA-stimulated superoxide production measured 15 min after addition of 10 microM LOOH was inhibited approximately 50%; however, addition of this concentration of the hydroperoxide after PMA stimulation was without effect. Superoxide production was also measured in a cell-free system produced by incubation of alveolar macrophages with sodium dodecyl sulfate. Prior incubation of alveolar macrophages with LOOH, H2O2, or t-butyl hydroperoxide, under conditions that significantly inhibited superoxide production by the intact cells, did not produce inhibition of the NADPH-dependent superoxide generating system in the cell-free preparation. These results suggest that the effect of LOOH was upon signal transduction involved in the stimulation of superoxide production rather than on the NADPH oxidase itself. Measurements of membrane potential changes were made using the lipophilic ions, 3,3'-dipentyloxacarbocyanine (DiOC5(3] and bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethineoxonol (oxonol V). On the basis of their charge, DiOC5(3) fluorescence primarily reports mitochondrial potential and oxonol V absorbance reports plasma membrane potential. With 10 microM LOOH, depolarization of the plasma and mitochondrial membranes appeared to occur within seconds. As prior depolarization depresses superoxide production, these hydroperoxide-induced changes in membrane potential may be responsible for decreased PMA-stimulated superoxide production.     

Freeze-fracture studies of Cryptosporidium muris.

The attachment site of Cryptosporidium muris to host cells was investigated using the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with the host plasma membrane at the dense band, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was dramatically altered at the above two junctures. The outer parasitophorous membrane showed low IMP-density as compared to the host plasma membrane, although both membranes were continuous. The inner parasitophorous membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP. When the attachment sites of parasites and host cells were fractured, circular-shaped fractured faces were observed on both sites of the parasite and host cell. These exposed faces corresponded to the dense bands and were very similar in size in each parasite.     

Covalent incorporation of 3'-O-(4-benzoyl)benzoyl-ATP into a P2 purinoceptor in transformed mouse fibroblasts.

ATP, 3'-O-(4-benzoyl)benzoyl-ATP (BzATP), a photoaffinity analog of ATP, and several other ATP analogs induced an increase in plasma membrane permeability to monovalent ions and normally impermeant metabolites, including nucleotides, in transformed 3T6 mouse fibroblasts. The rank order of agonist potency for induction of nucleotide channels was BzATP (EC50 = 15 microM) greater than ATP (EC50 = 50 microM) approximately adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) greater than 2-methylthio-ATP (EC50 = 75 microM) approximately 3'-amino-3'-deoxy-ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) (EC50 = 175 microM). Long wavelength UV illumination of 3T6 cells in the presence of greater than or equal to 20 microM BzATP at 4 degrees C, a nonpermeabilizing temperature, followed by removal of unbound BzATP, resulted in the efflux of 86Rb+ and the release of a prelabeled pool of cytoplasmic nucleotides when the temperature was shifted to 37 degrees C. Photoincorporation of BzATP was inhibited by ATP, ATP alpha S, ATP gamma S, and other ATP analogs that induced an increase in plasma membrane permeability to nucleotides in 3T6 cells under nonphotoactivating conditions. GTP, ITP, UTP, adenosine, and ATP analogs that did not alter plasma membrane permeability to nucleotides under nonphotoactivating conditions also had no effect on BzATP photoincorporation. Photoincorporation of BzATP occurred optimally between pH 6.6 and pH 8.2 but was inhibited at pH 6.0. Photoincorporation of BzATP was also modulated by the osmolarity and the divalent cation concentration of the assay medium. The increase in plasma membrane permeability to nucleotides induced by photoincorporated BzATP occurred at the same rate and had the same temperature, pH, ionic strength, and divalent cation requirements as the increase in plasma membrane permeability to nucleotides induced by ATP and BzATP under nonphotoactivating conditions. These findings support the hypothesis that BzATP can be covalently incorporated into a P2 purinoceptor in 3T6 cells that is coupled to plasma membrane channels for ions and other metabolites.     

Freeze-Fracture Analysis of Plasma Membranes of CHO Cells Stably Expressing Aquaporins 1-5

Several studies suggest that aquaporin water channels can be identified in membranes by freeze-fracture electron microscopy. For this report, Chinese Hamster ovary cells were stably transfected with cDNAs encoding aquaporins 1–5. Measurement of the osmotic water permeability of the cells confirmed that functional protein was expressed and delivered to the plasma membrane. By freeze-fracture electron microscopy, a 20% increase in intramembrane particle (IMP) density was found in plasma membranes of cells expressing AQP2, 3 and 5, and a 100% increase was measured in AQP1-expressing cells, when compared to mock-transfected cells. On membranes of cells expressing AQP4, large aggregates of IMPs were organized into orthogonal arrays, which occupied 10–20% of the membrane surface. IMP aggregates were never seen in AQP2-transfected cells. Hexagonally packed IMP clusters were detected in ∼5% of the membranes from AQP3-expressing cells. Particle size-distribution analysis of rotary shadowed IMPs showed a significant shift from 13.5 (control cells) to 8.5 nm or less in AQP-expressing cells; size distribution analysis of unidirectionally shadowed IMPs also showed a significant change when compared to control. Some IMPs in AQP expressing cells had features consistent with the idea that aquaporins are assembled as tetramers. The results demonstrate that in transfected CHO cells, AQP transfection modifies the general appearance and number of IMPs on the plasma membrane, and show that only AQP4 assembles into well-defined IMP arrays. Received: 17 March 1998/Revised: 19 June 1998     

A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes

Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 +/- 0.6 microM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 +/- 150 to 130 +/- 30 microM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane.