The effect of oxidant gases on membrane fluidity and function in pulmonary endothelial cells

Free Radical Biology & Medicine, Vol. 4, pp. 121-134, 1988 Printed in the USA. All fights reserved.

÷

0891-5849/88 $3.00+ .00 © 1988 PergamonJournals Ltd.

Review Article THE EFFECT OF OXIDANT GASES ON MEMBRANE FLUIDITY AND FUNCTION IN PULMONARY ENDOTHELIAL CELLS

JAWAHARLAL M . PATEL a n d EDWARD R. BLOCK Department of Medicine, University of Florida and Veterans Administration Medical Center, Gainesville, FL

(Received and Accepted 30 July 1987)

Abstract--Free radicals and oxidant gases, such as oxygen (02) and nitrogen dioxide (NO2), are injurious to mammalian lung cells. One of the postulated mechanisms for the cellular injury associated with these gases and free radicals involves peroxidative cleavage of membrane lipids. We have hypothesized that oxidant-related alterations in membrane lipids may result in disordering of the plasma membrane lipid bilayer, leading to derangements in membrane-dependent functions. To test this hypothesis, we examined the effect of exposure to high partial pressures of 02 or NOz on the physical state and function of pulmonary endothelial cell plasma membranes. Both hyperoxia (95% 02 at 1 ATA) and NO2 exposure (5 ppm) caused early and significant decreases in fluidity in the hydrophobic interior of the plasma membrane lipid bilayer and subsequent depressions in plasma membranedependent transport of 5-hydroxytryptamine. Lipid domains at the surface of pulmonary endothelial cell plasma membranes are more susceptible to NO2-induced injury than to hyperoxic injury. Alterations in the fluidity of these more superficial domains are associated with derangements in surface dependent functions, such as receptorligand interaction. These results support our hypothesis and advance our understanding of how the chemical events of free radical injury associated with high 02 and NO2 tensions are translated into functional manifestations of 02 and NO2-induced cellular injury. Keywords--Oxidant, Membrane injury, Membrane fluidity

and their plasma membranes provide a semipermeable barrier that regulates transport of water, solutes, and particulate material between blood and tissue in the lung. ~-3 The pulmonary endothelium is also a metabolically active tissue that processes a variety of biologically active substances in the circulation either by carrier-mediated transmembrane transport, by receptor mediated internalization, or by way of enzymatic activity at the plasma membrane. 1,2 The structural and functional integrity of the pulmonary endothelial cell and its plasma membrane is, therefore, of major importance for normal cellular and organ function. The pulmonary endothelium is particularly susceptible to injury because it is vulnerable to noxious agents that are inhaled as well as to those delivered to the lung by way of the pulmonary circulation. Recent evidence indicates that a variety of exogenous and endogenous agents can cause structural derangement and loss of normal endothelial cell function. One of the better-studied models is normobaric oxygen (02) toxicity, where metabolic and physiologic studies indicate that injury to the pulmonary capillary endothelial cell, and in particular to its plasma membrane, leads to many

I. INTRODUCTION

Pulmonary endothelial cells form a continuous monolayer on the luminal surface of the lung vasculature, Correspondence and reprint requests should be sent to: E. R. Block, M.D., Research Service (151), Veterans Administration Medical Center, Gainesville, FL 32602. Jawaharlal M. Patel received a Ph.D. in Biochemistry from Marathwada University in India in 1973 and was a Visiting Fellow at the National Institute of Environmental Health Sciences in Research Triangle Park, NC from 1974-1977. Dr. Patel is currently an Assistant Research Scientist in the Pulmonary Medicine Division of the Department of Medicine at the University of Florida. Dr. Patel's primary research interests include drug metabolism, the biochemical toxicity of inhaled compounds, and the cell biology of the lung. Edward R. Block was awarded an M.D. by the Johns Hopkins School of Medicine in 1968. After completing internal medicine residency training at Johns Hopkins Hospital, he spent three years at the National Institute of Allergy and Infectious Diseases and then completed a fellowship in Pulmonary Medicine and Physiology at the University of Pennsylvania. Dr. Block is presently Associate Chief of Staff for Research at the Veterans Administration Medical Center in Galnesville and is a Professor of Medicine at the University of Florida, College of Medicine. He is interested in the cell biology of the pulmonary endothelium, and his current research activities focus on the effects of high and low partial pressures of oxygen on the structure and function of the plasma membrane of pulmonary endothelial cells. 121

122

J.M. PATELand E. R. BLOCK

of the pathophysiological sequelae of pulmonary 02 toxicity. 4-13 This is consistent with recent work indicating that the cytotoxic effects of 02 are mediated through 02 free radicals and that the plasma membrane is a critical site of free radical reactions. 14-16Since 02 free radicals can cause peroxidative cleavage of membrane lipids, it is plausible to hypothesize that exposure to high partial pressures of 02 alters the physical state of lipids in the plasma membranes of pulmonary endothelial cells and that these alterations in physical state mediate the derangements in plasma membrane function seen in 02 toxicity. To test this hypothesis, two issues must be addressed. First, it must be determined whether hyperoxia affects the physical state of the plasma membrane lipid bilayer of pulmonary endothelial cells. Second, a cause and effect relationship between hyperoxia-induced changes in membrane lipids and hyperoxia-induced alterations in membrane function must be established. Evidence relating to these two issues is presented in the following sections.

initial site and a critical site of interaction between 02free radicals and pulmonary endothelial cells. 15 The unsaturated bonds of membrane cholesteryl esters and fatty acids can readily react with molecular 02 and 02free radicals and undergo peroxidation, and Freeman et al. 23 have reported increased lipid peroxidation in cultured endothelial cells exposed to high 02 tensions under controlled conditions. Peroxidation of membrane lipids leads to the generation of short chain fatty acyl derivatives and the byproduct malonaldehyde, which can mediate a variety of cross-linking reactions. In addition, O2-free radicals can catalyze the oxidation of amino acids in membrane proteins and can cause protein-protein cross linking and protein strand scission (Fig. 1). The net result of these effects is a disordering of the molecular organization and of the physical state of the plasma membrane, leading to alterations in membrane function.

B. Measurement of membrane fluidity II. MEMBRANE STRUCTURE AND FLUIDITY

A. Theoretical considerations The basic structure of biological membranes can be represented by a phospholipid bilayer with embedded proteins. 17,18 It is widely accepted that the lipids regulate, to a large extent, the structural and functional integrity of biological membranes. 19,20According to the fluid mosaic model of membrane structure, membrane constituents are capable of rapid rotational and translational diffusion within the fluid matrix of the membrane. 19.21The mobility of the lipid bilayer is referred to as membrane fluidity and represents an inherent biophysical property of the membrane. 21 This membrane property is primarily influenced by the physical state and composition of the membrane lipids, but it can also be affected by membrane proteins and factors associated with the aqueous phase. 1g'21 Membrane perturbations that result in alterations in fluidity have been shown to interfere with a number of fundamental cellular functions, including cell cycling, differentiation, proliferation, and transmembrane signal transduction. ~8-22 In addition, numerous investigators have shown that the activity and kinetics of membranebound enzymes and carriers can be markedly affected by the membrane lipid composition and fluidity, 18-22 confirming that the physical state of the lipids surrounding various membrane proteins controls protein conformation and regulates protein function. It seems clear at the present time that optimal membrane function requires the fluidity of membrane lipids to be maintained within narrow limits. As noted earlier, the plasma membrane is both the

A number of physicochemical techniques have been developed to measure membrane physical parameters and to quantitate the fluidity of membrane constituents. 19'24'25 These include electron spin resonance spectroscopy (ESR), nuclear magnetic resonance spectroscopy (NMR), Raman spectroscopy, and fluorescence spectroscopy. Although each of these methods measures a different physical parameter, each can be useful for quantitating a particular characteristic of membrane lipid motion. For example, ESR utilizes spin-labeled molecular probes that partition into specific membrane lipid domains. An order parameter is calculated from spectral data derived from the probe, and this order parameter is a function of the rate of acyl chain motion in the lipid bilayer. 22'24 Similarly, dynamic details of the chemical and physical properties and the movement of atoms within bilayer lipid chains can be analyzed using proton or carbon NMR. 19,22,24,25 Raman spectroscopy, a relatively new and nonperturbing technique, yields information related to stretching vibrations, which occur in interatomic bonds within a molecule. Different bond types can be distinguished within a fatty acyl chain and inferences can be drawn concerning the relative types and extent of motion occurring about the various bond types. 22,24,25 Finally, fluorescence spectroscopy, which utilizes fluorescent probes that partition into specific membrane lipid domains, provides information on the rotational motion of these molecular probes within the lipid bilayer. 22'24 ESR, NMR, and fluorescence spectroscopic methods usually examine the rotational motion of acyl chains, although with ESR and with fluorescence recovery after photobleaching techniques, it is also pos-

Oxidant injury and membranefluidity

,4 ~ ~/~.~.

~

/

TaANSMEMaRANE GLYCOPROIEIN

~ •

M E M I R * ~ SUrfACE

DISULFIDE CROSSLINI{ING PIOTEIN STRAND \ LIPIO-PIK)'TEIN .z.. SClSSION \ CROSSLINKING

OA,,AO,, -

(1)o -,!? } ii i( i! i(

_c.3-s A,,,,NO'AO0 ~' OXIDATION

123

0

¢ ,~,

c,oss~..,.¢..,,o /

MALONDIALDEHYDE RELEASED FROM OXIDIZED FATTY ACIDS

FATTY ACID OJll DATION

Fig. 1. The structure of biologicalmembranesand the simplifieddepictionof the consequencesof free radical-inducedinjury. to the membranecomponents. [Reprintedwith permissionfrom Freeman,B. A.; Crapo, J. D. Biologyof disease: Free radicals and tissue injury. Lab. Invest. 47: 412-426; 1982]. sible to obtain a lateral diffusion constant for membrane lipids. In contrast, basic structural information, particularly in model membranes, is derived from Raman spectroscopy. However, this latter technique is limited by considerable interference between the phosphate and protein regions of biological membranes. In addition, Raman spectroscopy as well as ESR and NMR techniques require rather expensive apparatus. The advantage of ESR, NMR, and Raman spectroscopy over fluorescence techniques is that they do not require the use of optically pure and well-characterized probes. For a more detailed account of the advantages and disadvantages of each of these techniques as well as their applications, the reader is referred to several recent reviews. 19,22,24,25We have used fluorescence spectroscopy in the present studies for three reasons: (1) the inherent sensitivity of the technique, (2) the favorable time scale of the phenomenon of fluorescence, and (3) the availability of well-characterized fluorescent probes that partition into welldefined domains within membrane lipid bilayers. The principle of the fluorescent spectroscopic method is that polarization of the light emitted by a fluorescent probe incorporated in a membrane bilayer depends on its rotational motion which, in turn, is a function of the fluidity of the specific lipid domain in which the probe is partitioned. As the fluidity of the medium surrounding the probe decreases, the mobility of the probe is hindered, resulting in a change in its fluorescence polarization. This technique can be used to derive several fluidity parameters. For example, rotational relaxation time is a fluidity parameter that accurately reflects the rotational motion of fluorescent probes incorporated into the phospholipid bilayer of membranes. 21,22'24 In biophysical terms, it is the time required for a given probe molecule within the mem-

brane to rotate through an angle such that the cos 0 = e-l. This time varies inversely with the fluidity of the membrane surrounding the probe. Rotational relaxation time reflects both the rate of rotational motion of the probe as well as the angular range of rotational motion since it measures the time taken for a molecule to cover an angular distance. Fluorescence anisotropy, on the other hand, is a fluidity parameter that depends primarily on the molecular packing (i.e. the order) of the membrane lipids rather than the rotational rate of the fluorescence probe. 24'2~ An increase in anisotropy reflects an increase in lipid order, which is associated with a decrease in membrane fluidity. ~'26 Biological membranes represent a composite of many microenvironments. Structural evidence for heterogeneity of lateral organization of membrane lipids has been adduced in a variety of studies including xray diffraction, electron microscopy, lateral diffusion measurements, differential partitioning of lipid probes, and spin-label measurements. Since the lipid component of membranes is organized into specific domains, it is important in fluidity measurements to use fluorescent probes whose membrane localization as well as spectroscopic properties are well defined. In the studies described in Section III, we have used an SLM 4048s spectrofluorometer (SLM Instruments, Urbana, IL) to compare rotational relaxation times or fluorescence anisotropies of four well-characterized and widely-studied fluorescent probes incorporated into specific domains within the plasma membranes of control and oxidant-injured pulmonary endothelial cells. The four probes include 1,6-diphenyl- 1,3,5-hexatriene (DPH), trimethylamino-DPH (TMA-DPH), transparinaric acid (TPA), and fluorescamine. DPH is a fluorescent aromatic hydrocarbon with an all trans polyene system that partitions into the hydrophobic

124

J.M. PATELand E. R. BLOCK

region of the membrane. 27 Thus, DPH reports on the dynamics of the central acyl-side chain region of the phospholipid bilayer. TMA-DPH, a cationic analogue of DPH, has photophysical properties generally similar to those of DPH. 2s However, the cationic charge ensures that the TMA-DPH probe is anchored at the lipid-water interface with the DPH moiety intercalated between the upper portions of the fatty acyl chains. TMA-DPH reports on the physical state of the surface and/or glycerol-side chain region of the phospholipid bilayer. 29'3° TPA is a natural conjugated polyene fatty acid that orients in the membrane lipid phase parallel to the midregion of the fatty acyl-side chains of phospholipids) 1 TPA reports, therefore, on the mid acyl-chain region of the phospholipid bilayer. Finally, fluorescamine, a nonfluorescent reagent, reacts covalently with the external surface amino groups of phospholipids and proteins to form a highly fluorescent product. Fluorescamine reports on the hydrophilic surface region of the membrane. 32'33 Fluorescence microscopic studies, studies with isolated plasma membranes in normoxic and hyperoxic cells,34 and time-dependent studies of the distribution of these probes into the membranes of intracellular organelles of endothelial cells 35 indicate that these probes are localized to the plasma membrane of pulmonary endothelial ceils during the time course of the experiments to be described below. Phospholipids are major molecular components of biological membranes. Our probes reflect the dynamics of the polar head group and acyl chain moieties. Changes in fluidity thus reflect changes in the dynamic motion of the component parts of the phospholipids. III. OXIDANT-INDUCED ALTERATIONS IN MEMBRANE FLUIDITY AND FUNCTION

A. Role of lipid peroxidation Peroxidation of membrane lipids has been implicated in the mechanism of cell injury due to free radicals generated secondary to high partial pressures of 02, ionizing radiation, aging, and a host of chemical agents and drugs. 36-3s Abstraction of a hydrogen atom from an unsaturated fatty acid is the initial step in lipid peroxidation, which leads to the formation of lipid radicals, peroxy radicals, hydroperoxides, and a variety of lipid fragments. 16'36-3s The preferential involvement of unsaturated fatty acids in the free radical initiated peroxidation of membrane lipids is particularly significant because of the potential impact on the physical state of the fatty acyl side chains in the bilayer. For example, peroxidative cleavage of membrane lipids can lead to alterations in cholesterol/phospholipid ratio, unsaturation index, fatty acyl chain length, and

the percentage distribution of fatty acids. Recent evidence reveals that the peroxidation of lipids in model and biological membrane systems is indeed responsible for alterations in the fluidity of these membranes. 39-43 For example, Dobretsov et a1.,39 using fluorescence spectroscopic methods, demonstrated an increase in bilayer rigidity in phospholipid vesicles after in vitro lipid peroxidation, and Ohyashiki et al. 41 reported that the lipid peroxidation of the porcine intestinal brushborder membrane results in a marked reduction of the lipid fluidity and causes structural changes in the lipid organization of the membrane surface. These authors were able to establish a correlation between the magnitude of the modifications in the physical state of the membrane and the extent of functional loss in the membrane. Similarly, Rosen et a1.,41 using electron paramagnetic resonance spectrometry, found a decrease in membrane fluidity in resealed ghosts of human erythrocytes exposed to reactive Oz species, and Girotti and Thomas, 43 using the same system, found an increase in lipid peroxidation and membrane permeability. Finally, Curtis et al.4° reported that peroxidation of hepatic microsomal membrane lipids resulted in an increase in the order parameter (i.e., a decrease in fluidity) evaluated by ESR using three stearic acid spin probes.

B. Oxidant gases 1. Effect of hyperoxia on plasma membrane fluidity of endothelial cells. To evaluate the effect of exposure to high partial pressures of O2 on plasma membrane fluidity, second- to fifth-passage porcine pulmonary artery endothelial cells in confluent monolayer were exposed at 37°C to 20% 02-75% N2-5% CO2 (control) or 95% 02-5% CO2 (hyperoxic) at 1 ATA for 3-42 h in an airtight 30.5 × 30.5 cm stainless steel chamber housed inside a COz incubator. Immediately after exposure, cells were washed, scraped, and suspended in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.15 M KC1 (KC1 buffer). Suspensions of cells were mixed with equal volumes of 20-/tM DPH or 12-/tM TPA. After a 30 min incubation, the ceils were washed free of unincorporated probe, resuspended, and used for spectroscopic measurements. There were no differences in the amount of probe incorporated into the plasma membrane, in the kinetics of the incorporation, or in the spectroscopic properties of the incorporated probe between control and hyperoxic c e l l s . 34 Exposure to 95% O2 for 4 h caused significant (p < 0.003) increases in the rotational relaxation times of TPA in pulmonary artery endothelial ceils. More prolonged exposure to O2 for 18 or 42 h caused further increases in rotational relaxation time (Fig. 2). Ex-

Oxidant injury and membrane fluidity

=E

60 5O

F

I

~!30

~

125

0

0

3.1 ~2 3.3 3.4 3.5 3.6

I

3.1 3.2 :~3 3.4 3.5 3.6 I/TEMPERATURE

3.1 3.2 33 ~.4 35 3.6

(°K) x 10 3

Fig. 2. Effect of 02 exposure on rotational relaxation times for TPA in pulmonary artery endothelial cells in culture. Cells were exposed to 20% 02 (0) or 95% O: (A) at 1 ATA for 4, 18, and 42 h. Each point represents the mean of at least 9 dishes. S.E. are too small to be represented. Differences between control and 95% 02 exposed curves are significant at p < 0.003, p < 0.0003, and p < 0.0001 for 4-, 18-, and 42-h exposures, respectively. [Reprinted with permission from Block, E. R.; Patel, J. M.; Angelides, K. J.; Sherighan, N. P.; Garg, L. C. Hyperoxia reduces plasma membrane fluidity: A mechanism for endothelial cell dysfunction. J. Appl. Physiol. 60: 826-835; 1986].

reported to interfere dramatically with a number of fundamental membrane functions. 18.2o,22 Endothelial cells were incubated with 10 pM TMADPH and then studied to determine whether more superficial lipid domains within the plasma membranes of endothelial cells were affected by hyperoxia, control, and hyperoxic pulmonary artery. As with TPA and DPH, exposure to 95% 02 at 1 ATA had no effect on the rate or amount of the probe incorporated into the plasma membrane or on the spectroscopic properties of the TMA-DPH probe. However, exposure to

posure to 95% 02 caused similar progressive increases in the rotational relaxation times of DPH in pulmonary artery endothelial cells (Fig. 3). These results indicate that normobaric hyperoxia decreases fluidity in the mid and central acyl side chain regions of the plasma membranes of pulmonary endothelial cells, and the magnitude of the alteration in fluidity is a function of the duration of hyperoxic exposure. In addition to being statistically significant, the differences illustrated in Figures 2 and 3 are biologically significant, since alterations in fluidity of a similar magnitude have been W

~=

70

60

40 ~ ~ 30

20 I0 o rr

0

. 5.1

. . . . . 5.2 3.3 3.4 3.5 3.6

I

i

.

i

i

I

3.1 3.2 3.3 3.4 3.5 3.6 I/TEMPERATURE

I

I

I

I

I

3.1 3.2 3.3 3.4 35

I

3.6

(°K) x 103

Fig. 3. Effect of 02 exposure on rotational relaxation times for DPH in pulmonary artery endothelial ceils in culture. Cells were exposed to 20% 02 ( 0 ) or 95% 02 (&) at 1 ATA for 4, 18, 42 h. Each point represents the mean of at least 9 dishes. S.E. are too small to be represented. Differences between control and 95% 02 exposed curves are significant at p < 0.003, p < 0.0003, and p < 0.0001 for 4-, 18-, and 42-h exposures, respectively. [Reprinted with permission from Block, E. R.; Patel, J. M.; Angelides, K. J.; Sherighan, N. P.; Garg, L. C. Hyperoxia reduces plasma membrane fluidity: A mechanism for endothelial cell dysfunction. J. Appl. Physiol. 60: 826-835; 1986].

126

J . M . PATEL and E. R. BLOCK

24 hr

30hr

42 hr

45 W

_z F-

40

Y

i:f Z

:!f 2.

. 3.3

. 3.4 .

. 3.5.

3.6

32

,

. 3.4

I / TEMPERATURE

. ('K)

. 35

. 3.6.

3.2

33

3'.,

, 35

3,6

X 103

Fig. 4. Effect of 02 exposure on rotational relaxation times for TMA-DPH in pulmonary artery endothelial cells in culture. Cells were exposed to 20% O2 ( 0 ) or 95% 02 (O) at 1 ATA for 24, 30, or 42 h. Each point represents the mean of at least 6 dishes. Differences between control and hyperoxic curves are significant at p < 0.05 (24 h), p < 0.025 (30 h), and p < 0.05 (42 h).

95% 02 for 24 to 42 h did significantly increase the rotational relaxation times of TMA-DPH in pulmonary artery endothelial cells (Fig. 4). Exposure to 95% 02 for shorter periods, e.g. for 12 or 18 h, has no significant effect on the dynamics of this probe. These results indicate that hyperoxia decreased the fluidity of the lipid-water interface involving the glycerol-side chain region of the plasma membrane of pulmonary endothelial cells. The decrease in fluidity in this surface lipid domain required a 24-h exposure to 95% 02 at 1 ATA and did not progress with more prolonged exposure, whereas decreases in fluidity in the hydrophobic core of the lipid bilayer were detected after a 4-h exposure to 95% 02 and increased with more prolonged 02 exposure (Figs. 2 and 3). The later onset of O~ injury tc the hydrophilic head group region of the plasma membranes of pulmonary artery endothelial cells suggests that the unsaturated fatty acyl chains of the membrane phospholipids are more susceptible to free radical attack than the more ordered polar head group region. The results illustrated in Figures 2-4 demonstrate that O2-free radical-induced injury decreases fluidity in at least three distinct lipid domains within the plasma membrane lipid bilayer of pulmonary artery endothelial cells. These results also suggest that the decreases in plasma membrane fluidity are early manifestations of endothelial cell 02 toxicity and, at least in the hydrophobic core region of the membrane, progress with continued 02 exposure. The next issue to be addressed is whether these alterations in fluidity are responsible for derangements in plasma membrane function in these cells.

2. Temporal relationship between changes in plasma membrane fluidity and function in hyperoxic endothelial cells. 5-Hydroxytryptamine is a biologically active amine that is taken up by pulmonary endothelial cells by way of a specific, carrier mediated plasma membrane-dependent transport process. Since inhibition of the transmembrane transport of 5-HT is a sensitive and early index of hyperoxic injury to the plasma membrane of the pulmonary endothelial c e l l , 4'5'44 w e examined the relationship between hyperoxic alterations in plasma membrane fluidity and 5-HT transport in pulmonary endothelial cells. If the decreases in plasma membrane fluidity noted above are responsible for the decrease in 5-HT transport by hyperoxic pulmonary endothelial cells, then a compatible temporal relationship must exist between the two phenomena. In Figure 5, the effect of exposure to 95% 02 on 5HT uptake by pulmonary artery endothelial cells is compared to the effect of 95% 02 exposure on plasma membrane fluidity. The hyperoxic decrease in fluidity in the mid and central acyl-side chain regions of the plasma membrane, reflected in measurements of TPA and DPH, antedates the hyperoxic depression of 5-HT uptake, but the magnitudes of these reductions in fluidity and 5-HT uptake increase in a proportionate manner with continued 02 exposure. For example, although the rotational relaxation times for DPH and TPA are increased after a 4-h exposure and the decrease in 5HT uptake requires a 18-h exposure, all three change by comparable amounts (e.g. 13 to 15%) between 18and 42-h exposures to 95% 02. That the detection of alterations in plasma membrane fluidity precedes the

Oxidant injury and membrane fluidity detection of alterations in 5-HT uptake is not surprising because the sensitivity of the fluorescence spectroscopic method is much greater than the sensitivity of the method for measuring 5-HT uptake. In addition, it is not unexpected that alterations in the physical state of the plasma membrane lipids would precede derangements in plasma membrane function. In contrast to the results with DPH and TPA, the hyperoxic decrease in fluidity in the hydrophilic head group region of the plasma membrane, reflected in measurements at the rotational relaxation times of TMA-DPH, does not occur until a 24-h exposure to 95% Oz (Fig. 4), which is after the hyperoxic decrease in 5-HT uptake is observed (Fig. 5). In addition, there are no further increases in the rotational relaxation times of TMA-DPH between 24 and 42 h exposures to 95% 02. The relationship between the reversibility of the 02induced alterations in membrane fluidity and the reversibility of the O2-induced decrease in 5-HT uptake by pulmonary endothelial cells is illustrated in Figure 6. Rotational relaxation times of DPH and 5-HT uptakes were measured immediately (0 h), and 18, 24, and 48 h after an 18-h exposure to 95% 02 at 1 ATA. The results indicate that after 48 h there was no significant recovery from either the O2-induced decrease in fluidity in the hydrophobic core region of the plasma

5-HT ['TUPTAKE

14(

127

membrane or from the hyperoxic depression of 5-HT uptake.

3. Effect of NO2 exposure on plasma membrane fluidity of endothelial cells. If the alterations in plasma membrane fluidity observed in hyperoxic endothelial cells are responsible for the depression in transmembrane transport of 5-HT by these cells, then perturbations that cause similar alterations in the same lipid domains within the endothelial cell plasma membrane lipid bilayer should cause similar alterations in 5-HT transport. To test this, we measured plasma membrane fluidity in pulmonary artery endothelial cells exposed tO NO2. NO2 is a common indoor and outdoor environmental oxidant. Exposure to relatively high concentrations of NOz produces morphologic alterations in pulmonary endothelial cells similar to those described with O2 toxicity. 45'46 Moreover, like Oz, NO2 is known to promote lipid peroxidation in mammalian lungs in vivo 47-49 and in vitro. 5°-53 We considered that NOz may have an effect on the physical state of endothelial cell plasma membrane lipids similar to that of hyperoxia. In addition, NO2, unlike 02, is itself a free radical. 5°'54 Therefore, it is possible that NO 2 will also react directly with surface lipids in the plasma membranes of endothelial cells, resulting in alterations in the physical state of surface lipid domains that, in -X--X--X-

[]

pDPH

[ ] pTPA [ ]

pTMA-DPH

4(-

~

-l

4(-4(-4(-

I

13( .J

120

o

n.

IZ

110;

0

0 I1.

o i-

z

_[_

10© i

90l

ill

o

n. ill o.

80 I

't

6

4

18

42

t4

DURATION OF 02 EXPOSURE (hours) Fig. 5. Relationship between the effect of exposure to 95% 02 on the rotational relaxation times for DPH (pDPH), TPA (pTPA), and TMA-DPH (pTMA-DPH) and on the uptake of 5-HT at 37°C in pulmonary artery endothelial cells. 5-HT uptake, pDPH, pTPA, and pTMA-DPH were measured after 4-, 18-, and 42-h exposures to 95% O2 at 1 ATA. The results are presented as mean percent (+ S.E.) of control cells that were simultaneouslyexposed to 20% 02 at 1 ATA. pDPH, pTPA, and pTMA-DPH are inversely related to fluidity and are presented as percent of control averaged over temperature range 5°C to 40°C. *p < 0.01, **p < 0.05, ***p < 0.005, vs. control.

128

J . M . PATEL and E. R. BLOCK

D-Hr

130

u rA E

~pDPH

120 •W.,W. I10 ,_~

I00

T

._I 0r~ 9O Z

80

8

70

O I-- 6O z W U

50.

tlJ O_

40: 30

20 I0 0

24

48

HOURS AFTER EXPOSURE Fig. 6. Reversibility of the hyperoxic alterations in plasma membrane fluidity and 5-HT uptake in pulmonary artery endothelial cells. 5-HT uptakes and rotational relaxation times of DPH (pDPH) were measured immediately (0 h), 24 h after, and 48 h after an 18-h exposure to 95% 02 at 1 ATA. The results are mean percent ( + S.E.) of control cells that were simultaneously exposed to 20% 02 at 1 ATA. pDPH, which is inversely related to fluidity, is represented as percent of control averaged over the temperature range 5°C to 40°C. *p < 0.01 vs. control. **p < 0.05 vs. control.

turn, lead to derangements in surface membrane-dependent functions, e.g. receptor-ligand interactions. To assess the effect of NO2 exposure on fluidity in the hydrophobic core region of the plasma membrane that is affected by hyperoxia, we measured the rotational relaxation times of DPH in pulmonary artery endothelial cells exposed to 5 ppm NO2 in air or exposed to air alone (control). As with hyperoxia, NOz had no effect on the rate of probe incorporation, the amount of probe incorporated, or the spectroscopic properties of the incorporated probe, 53 but did cause significant increases in DPH rotational relaxation times in pulmonary artery endothelial cells. The magnitude of the changes in the rotational relaxation times were directly proportional to the duration of exposure to NO2 (Fig. 7). Since the decreases in fluidity in the hydrophobic core of the plasma membrane of endothelial cells exposed to NO2 are similar to those observed in O2-exposed endothelial ceils, we next examined the effect of NO2 exposure on 5-HT uptake and whether the time course of the NO2 effect is compatible with the time

course of the alterations in fluidity. Exposure to 5 ppm N O 2 for 3 to 12 h did not alter the uptake of 5-HT. In contrast, exposure to 5 ppm NO: for 24 h significantly reduced 5-HT uptake in pulmonary artery endothelial cells (Fig. 8). Therefore, N O 2 , like 02 exposure, causes a decrease in 5-HT uptake that is preceded by a decrease in fluidity in the hydrophobic core region of the plasma membrane of pulmonary artery endothelial cells. To test whether more superficial lipid domains within the plasma membrane of endothelial cells were affected by NO2, control and NOE-exposed pulmonary artery endothelial cells were incubated with 10-~M TMA-DPH or 100-#M fluorescamine and then studied. As with DPH, NO2 had no effect on the rate or amount of the probes incorporated into the plasma membrane or on the spectroscopic properties of the probes but did significantly increase anisotropy values of TMA-DPH in cells exposed to 5 ppm NO2 (Fig. 9). Indentical results were obtained using fluorescamine. Because exposure to NOz also caused significant and early decreases in the fluidity of hydrophilic surface

Oxidant injury and membrane fluidity

plasma membrane-dependent transport of amines, we examined the effect of enrichment of plasma membrane cholesterol content on plasma membrane fluidity and 5-HT uptake. Incorporation of cholesterol into biological membranes has been used as an experimental tool to examine the effect of decreases in fluidity in the hydrophobic region on a variety of membrane functions.IS,22 We incubated confluent monolayers of pulmonary artery endothelial cells for 3 h with 0.1 mM cholesterol or vehicle (control) and then measured 5HT uptakes and rotational relaxation times of DPH and TMA-DPH. Cholesterol contents in the plasma membranes of treated cells were significantlyincreased compared to control cells (0.307 --- 0.012 vs. 0.219 --0.10 #g cholesterol/#g protein, p < 0.001). Rotational relaxation times of DPH and TMA-DPH were also significantly increased in the cholesterol-treated cells, similar to those in hyperoxic and NO2-exposed cells, whereas 5-HT uptakes were significantly decreased (Table 1).

regions of the plasma membrane (measured by TMADPH and fluorescamine), we evaluated the possible relationship between these changes in fluidity and alterations in surface functions of these cells, such as the binding of 125I-insulin to the insulin receptor on the plasma membrane of pulmonary artery endothelial cells. ~5's6 Our studies35 indicate that insulin receptor binding to cells exposed to 5 ppm NO2 for 12 or 24 h was significantly less (p < 0.05) than binding to control cells (Fig. 10). Scatchard analysis of the binding of 125I-insulin to pulmonary artery endothelial cells suggests that there are fewer binding sites on the NO2exposed cells compared to controls, primarily due to the reduction in receptor number in NO2-exposed cells) 5 As with hyperoxia, the time courses for the recovery of the fluidity changes and for the recovery of the alterations in insulin receptor binding were nearly identical, providing additional indirect support for a causal relationship between alterations in fluidity and specific derangements in membrane function.

C. Effect of modulation of membrane lipid composition on fluidity and function

IV. C O N C L U S I O N S AND F U T U R E D I R E C T I O N S

The results summarized in this review demonstrate that the oxidant gases O2 and NO2 decrease fluidity in physically distinct lipid domains within the plasma membrane of pulmonary endothelial cells. In cells ex-

To further strengthen the cause and effect relationship between decreases in fluidity in pulmonary endothelial cell plasma membranes and derangements in

80

129

A.

B.

A

q-

/

7.O

/ / /

LIJ

/

60

/

/

/

Z

O

v-

5.o

~o

4.0

////

.d

z o

50

1.0

i

3.2

i

i

i

i

3.3

3.4

3.5

36

I/TEMPERATURE

i

32

i

I

i

I

3.3

3.4

3.5

3.6.

(°K) x I0 3

Fig. 7. Effect of NO2 exposure on rotational relaxation times for DPH in pulmonary artery endothelial cells in culture. Cells were exposed to air (control) (O) or 5 ppm NO: ( 0 ) for 3 h (panel A) or 24 h (panel B). Each point represents the mean -S.E. of 4 to 8 experiments. Differences between control and NOz curves are significant at p < 0.05 (panel A) and p < 0.001 (panel B). Printed with permission from Patel and Block (1986).

130

J . M . PATEL and E. R. BLOCK

45O r-

i

T

400-

T

550 ~% 3o0

,.X-

250

2oo ~

150 i

,oo: 50O[

N=9

N-6

3

HOURS

24 HOURS DURATION OF EXPOSURE

Fig. 8. Effect of NO2 exposure on 5-HT uptake in pulmonary artery endothelial cells. Cells were exposed to air (control) (open bar) or 5 ppm NO2 (hatched bar) for 3 to 24 h. Results are expressed as mean -+ S.E., and the number of experiments is shown at the bottom of each bar. *p < 0.05 vs. control.

.40

3 HR

24HR "I

Or) rr"

>13.

.36

.32

0

rr. I--

0

6"--.~.5 " ~ . $ .28

O0

z < .24

-

.20 0

, 10

, 20

, 30

l

40

0

z

J

10

20

=

30

J 40

TEMP ° C Fig. 9. Effect of NO2 exposure on steady-state fluorescence anisotropies (r,) for TMA-DPH in pulmonary artery endothelial ceils in culture. Cells were exposed to air (control) (O) or 5 ppm NOz (Q) for 3-24 h. Each point represents the mean -+ S.E. of 4 experiments. Differences between control and NO2 curves are significant at p < 0.05 (3 h) and p < 0.001 (24 h). Exposure to 5 ppm NO2 for 6 h or 12 h caused increases in r, for TMA-DPH similar to those observed after 3 h NO2 exposure (not shown).

Oxidant injury and membrane fluidity

__1 c~ p.zQ

131

CONTROL 100

~

!

m

Ido x v

75 -

z ¢¢1 z ._J --n co

50

I

25

00 3

6

12

24

NO2 EXPOSURE (HR) Fig. 10. Effect of NO2 exposure on ~251-insulin binding to pulmonary artery endothelial cells. I m m e d i a t e l y after exposure to 5 ppm NO2, cells were incubated with ~251-insulin for 2 h prior to measuring binding. Data are expressed as percent of control (mean -+ S.E. f o r N = 4). Specific binding in controls is 1.65 -+ 0.13% b o u n d / m g protein. *p < 0.05 vs. control.

posed to high 02 tensions, the earliest alterations are observed in the central and mid acyl-chain regions of the plasma membrane lipid bilayer. After more prolonged exposure to 02, alterations in the fluidity of the polar head group region of the plasma membrane are observed. Similar alterations in fluidity are observed in the plasma membranes of endothelial cells exposed to NO2. However, the time course is slightly different in the NO2-exposed cells in that the onset of the changes in the hydrophilic polar head group region coincide with the onset of the changes in the fluidity of the hydrophobic core of the plasma membrane. The reason for this difference is not known. Since NOz itself is a free radical, it may react directly with the membrane lipids upon contact at the cell surface. In contrast, hyperoxic injury to monolayer cultures of endothelial cells requires the generation of intracellular free radical intermediates. Several lines of evidence indicate that the hyperoxic and NO2-induced decreases in plasma membrane fluidity may be directly responsible for alterations in plasma membrane function in pulmonary artery endothelial cells. First, the time courses of 02- and NO2induced decreases in fluidity and the time courses of the alterations in 5-HT uptake are compatible with a causal relationship. In addition to the similar time

course for the development of the hyperoxic decrease in plasma membrane fluidity and the reduction of 5HT uptake, the time courses of post-hyperoxic recovery appeared comparable as well. Concordance of recovery patterns was also observed after NO2 exposure. Finally, incorporation of cholesterol, a nonoxidant agent and a natural membrane rigidizer, into the plasma membranes of pulmonary artery endothelial cells mimicked the perturbations in plasma membrane fluidity and 5-HT uptake observed after exposures to 95% 02 or 5 ppm NO2. Table 1. Effect of Cholesterol Incorporation on Rotational Relaxation Times of DPH and T M A - D P H and on 5-HT Uptake in Pulmonary Artery Endothelial Cells in Culture Rotational Relaxation Time in nsec

Control Cholesterol

DPH

TMA-DPH

5-HT Uptake p m o l e / h / / x g protein

29.47 --- 1.57 35.21 - 1.94"

24.11 --- 0.80 30.95 --- 1 . 3 1 t

2.50 --- 0,16 2.18 --- 0.09:~

Note: Confluent monolayers of endothelial cells were incubated for 3 h with culture medium containing 0. I mM cholesterol in 0.5% ethanol or with culture medium containing 0.5% ethanol (control). Immediately after the 3 h incubation, rotational relaxation times of DPH and TAM-DPH and 5-HT uptakes were measured at 37°C. Data shown are mean -+ SE. *p < 0.01. tp < 0.00l. Sp < 0.025.

132

J . M . PATELand E. R. BLOCK

This evidence supports our original hypothesis that alterations in plasma membrane fluidity may mediate derangements in plasma membrane-dependent function that are prominent manifestations of free radical-induced pulmonary endothelial cell injury. Uptake of 5HT represents the first example of an endothelial cell plasma membrane function that may be directly affected by alterations in the fluidity of the hydrophobic region of the plasma membrane. Our preliminary data with NO2 indicate that oxidant injury to more superficial regions of the plasma membrane affects the physical state of surface lipid domains that may be responsible for derangements in surface membranedependent functions, e.g. receptor ligand interactions. Whether a similar relationship exists for other plasma membrane functions in cells injured by free radical mechanisms is not known. Whether the manifestations of non-free radical induced injury to pulmonary endothelial cells are mediated via alterations in membrane fluidity is also not known. Further studies are needed to answer these questions. Whereas the lung is the organ in which functional and structural alterations are most prominent during exposure to high 02 tensions under normobaric conditions and the capillary endothelial cell is the critical target cell in the lung, 12other organs, such as the brain and kidney, are susceptible to 02 toxicity. Since the chemical events responsible for injury in these nonpulmonary tissues are identical to those occurring in lung endothelial cells, it is possible that similar alterations in the physical state of the membrane lipids occur in the constituent cells from these nonpulmonary tissues. Indeed, recent evidence indicates that hyperoxia decreases fluidity in the central acyl-side chain region of the plasma membrane of human newborn foreskin fibroblasts but does not affect fluidity in the mid acyl-side chain or polar head group regions of the plasma membrane of these cells. 34These observations, coupled with the results in pulmonary endothelial cells, suggest that a decrease in fluidity in the central acylside chain region of the plasma membrane may be a universal manifestation of cellular 02 toxicity. For example, this alteration in the physical state of plasma membrane lipids (or other as yet unidentified alterations in physical state) could explain the increase in osmotic fragility of red blood cells 57,5sor the inhibition of plasma membrane-dependent migration, phagocytosis, and pinocytosis by alveolar macrophages 59,6°exposed to high 02 tensions. Similarly, altered plasma membrane fluidity could account for the block in nerve conduction and the changes in brain electrolyte distribution observed following exposure of animals to high partial pressures of 02 .61'62 Since the exact nature of the hyperoxic alterations in membrane fluidity will de-

pend upon the composition and distribution of lipids within the membrane bilayer, it is even possible that differences in the susceptibility to and the manifestations of hyperoxic injury observed in different cell types may be explained, at least in part, by differences in the physical state of the membrane lipids. Thus, cells with membranes in which the fatty acyl chains are more resistant to the disordering effects of high 02 tensions may prove to be more resistant to O2-induced injury and vice versa. Clearly, further studies are needed to verify these intriguing speculations. The data presented and the studies reviewed herein demonstrate that hyperoxia, as well as exposure to NO2, affects the physical state of the plasma membrane lipid bilayer of pulmonary endothelial cells and provide intriguing, albeit indirect, evidence that these alterations in physical state are responsible for derangements in membrane function. Future studies should strive to develop more direct evidence in support of the causal relationship between the changes in fluidity and function. For example, the effects of high partial pressures of O2 on specific plasma membrane lipids and on the 5-HT transporter itself, both within its natural lipid environment and in reconstituted systems, need to be defined. Similarly, lipid depletion and repletion studies will help clarify the role of specific membrane lipids. The results of these and other studies may provide a missing link between the well-documented chemical events of 02 toxicity, that is, the generation of highlyreactive, partially reduced O2 species, and NO2 toxicity and the less well understood functional and clinical manifestations of 02 and NO2 cytotoxicity. REFERENCES

1. Fishman, A. P. Endothelium: A distributed organ of diverse capabilities. Ann N.Y. Acad. Sci. 401: 1-8; 1982. 2. Ryan, J. W.; Ryan, U. S. Pulmonary endothelial cells. Fed. Proc. 36: 2683-2691; 1977. 3. Fishman, A. P.; Pietra, G. G. Handling of vasoactive material by the lung. N. Engl. J. Med. 291: 884-890,953-959; 1974. 4. Dobuler, K. J.; Catravas, J. D.; Gillis, C. N. Early detection of oxygen-induced lung injury in conscious rabbits. Am. Rev. Respir. Dis. 126: 534-539; 1982. 5. Block, E. R.; Fisher, A. B. Depression of serotonin clearance by rat lungs during oxygen exposure. J. Appl. Physiol. : Respirat. Environ. Exercise Physiol. 42: 33-38; 1977. 6. Krulewitz, A. H.; Fanburg, B. L. The effect of oxygen tension on the in vitro production and release of angiotensin converting enzyme by bovine pulmonary artery endothelial cells. Am. Rev. Respir. Dis. 130: 866-869; 1984. 7. Barry, B. E.; Crapo, J. D. Patterns of accumulation of platelets and neutrophils in rat lungs during exposure to 100% and 85% oxygen. Am. Rev. Respir. Dis. 132: 548-555; 1985. 8. Boogaerts, M. D.; Yamada, O.; Jacob, H. S.; Moldow, C. F. Enhancement of granulocyte endothelial cell adherence and granulocyte-induced cytotoxicity by platelet release products. Proc. Natl. Acad. Sci. U.S.A. 79: 7019-7023; 1982. 9. Repine, J. E.; Bowman, C. M.; Tate, P. M. Neutrophils and lung edema. Chest 815: 47-50; 1982.

Oxidant injury and membrane fluidity 10. Valimaki, M.; Kivisaan, J.; Niinikoski, J. Permeability of alveolar-capillary membrane in oxygen poisoning. Aerospace Med. 45: 370-374; 1974. 11. Newman, J. H.; Loyd, J. E.; English, D. K.; Ogletree, M. L.; Fulkerson, W. J.; Brigham, K. L. Effect of 100% oxygen on lung vascular function in awake sheep. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 54: 1379-1386; 1983. 12. Erdmann, A. J. III; Huttemeier, P. C.; Landolt, C.; Zapol, W. M. Pure oxygen breathing increases sheep lung microvascular permeability. Anesthesiology 58: 153-158; 1983. 13. Shasby, D. M.; Lind, S. E.; Shasby, S. S.; Goldsmith, J. C.; Hunninghake, G. W. Reversible oxidant induced increases in permeability of cultured endothelium: alterations in cell shape and calcium homeostasis. Blood 65: 605-614; 1985. 14. Deneke, S. M.; Fanburg, B. L. Oxygen toxicity of the lung: an update. Br. J. Anaesth. 54: 737-749; 1982. 15. Freeman, B. A.; Crapo, J. D. Biology of disease: free radicals and tissue injury. Lab. Invest. 47: 412-426; 1982. 16. Weiss, S. J. Oxygen, ischemia and inflamation. Acta. Physiol. Scand. 548: 9-37; 1986. 17. Singer, S. J.; Nicolson, G. L. The fluid mosaic model of the structure of cell membranes. Science 175: 720-731; 1972. 18. Houslay, M. D.; Stanley, K. K. Dynamics of Biological Membranes. New York: John Wiley and Sons; 1983. 19. Stubbs, C. D. Membrane fluidity: structure and dynamics of membrane lipids. Essays in Biochemistry 19: 1-39; 1983. 20. Stubbs, C. D.; Smith, A. D. The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim. Biophys. Acta 779: 89-137; 1984. 21. Quinn, P. J.; Chapman, D. The dynamics of membrane structure. CRC Crit. Rev. Biochem. 8:1-117; 1980. 22. Shinitzky, M. Membrane fluidity and cellular functions. In: Shinitzky, M. editor. Physiology of Membrane Fluidity. Vol. I. Boca Raton, FL: CRC Press; 1984; 1-51. 23. Freeman, B. A.; Young, S. L,; Crapo, J. D. Liposome-mediated augmentation of superoxide dismutase in endothelial cells prevents oxygen injury. J. Biol. Chem. 258: 12534-12542; 1983. 24. Barchi, R. L. Physical probes of biological membranes in studies of the muscular dystrophies. Muscle Nerve 3: 82-97; 1980. 25. Andersen, H. C. Probes of membrane structure. Ann. Rev. Biochem. 47: 359-383; 1978. 26. van Blitterswijk, W. J.; van Hoeven, R. P.; van Der Meer, B. W. Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements. Biochim. Biophys. Acta 644: 323-332; 1981. 27. Pessin, J. E.; Salters, D. W.; Glaser, M. Use of a fluorescent probe to compare the plasma membrane properties in normal and transformed cells: Evaluation of the interference by triacylcerols and alkyldiacylglycerols. Biochemistry 17, 19972004; 1978. 28. Prendergast, F. G.; Haugland, R. P.; Callahan, P. J. 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene: synthesis, fluorescence properties, and use as a fluorescence probe of lipid bilayers. Biochemistry 20: 7333-7338; 1981. 29. Kuhry, J. G.; Fronteneau, P.; Duportail, G.; Maechling, C.; Laustriat, G. TMA-DPH: a suitable flurorescence polarization probe for specific plasma membrane fluidity studies in intact living cells. Cell Biophys. 5: 129-140; 1983. 30. Kuhry, J. G.; Duportail, G,; Bronner, C.; Laustriat, G. Plasma membrane fluidity measurement on whole living cells by fluorescence anisotropy of trimethylaminodiphenylhexatriene. Biochim. Biophys. Acta 845: 60-67; 1985. 31. Schroeder, E; Holland, J. E; Vagelos, P. R. Use of 13-parinaric acid, a novel fluorometric probe, to determine characteristic temperatures of membranes and membrane lipids from cultured animals cells. J. Biol. Chem. 251: 6739-6746; 1976. 32. Udenfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.; Leimgruber, W.; Weigele, M. Fluorescamine: A reagent for assay of amino acids, peptides, proteins, and primary amines in the picomolar range. Science 178: 871-872; 1972.

133

33. Hawkes, S. P.; Meehan, T. D.; Bissell, M. J. The use of fluorescamine as a probe for labeling the outer surface of plasma membrane. Biochem. Biophys. Res. Commun. 32: 1033-1042; 1976. 34. Block, E. R.; Patel, J. M.; Angelides, K. J.; Sheridan, N. P.; Garg, L. C. Hyperoxia reduces plasma membrane fluidity: a mechanism for endothelial cell dysfunction. J. Appl. Physiol. 60: 826-835; 1986. 35. Patel, J. M.; Edwards, D. A.; Block, E. R.; Raizada, M. K. Nitrogen dioxide decreases surface membrane fluidity and insulin receptor binding of pulmonary endothelial cells. Biochem. Pharmacol. in press; 1988. 36. Pryor, W. A.; Lightsey, J. W.; Prier, D. G. The production of free radicals in vivo from the action of xenobiotics: the initiation of autoxidation of polyunsaturated fatty acids by nitrogen dioxide and ozone. In: Yagi, K. editor. Lipid Peroxides in Biology and Medicine. New York: Academic Press; 1982: 1-22. 37. Bus, J. S.; Gibson, J. E. Lipid peroxidation and its role in toxicology. In: Hodgson, E.; Bend, J. R.; Philpot, R. M., editors. Reviews in Biochemical Toxicology. North Holland: Elsevier; 1979: 125-149. 38. Proctor, P. H.; Reynolds, E. S. Free radicals and disease in man. Physiol. Chem. Phys. Med. NMR 16: 175-195; 1984. 39. Dobretsov, G. E.; Borschevskaya, T. A.; Petrov, V. A.; Vladimirov, Y. A. The increase of phospholipid bilayer rigidity after lipid peroxidation. FEBS Lett. 84: 125-128; 1977. 40. Curtis, M. T.; Gilfor, D.; Farber, J. L. Lipid peroxidation increases the molecular order of microsomal membranes. Arch. Biochem. Biophys. 235: 644-649; 1984. 41. Ohyashiki, T; Ohtsuka, T.; Mohri, T. A change in the lipid fluidity of the procine intestinal brush-border membranes by lipid peroxidation. Studies using pyrene and fluorescent stearic acid derivatives. Biochim. Biophys. Acta 861: 311-318; 1986. 42. Rosen, G. M.; Barber, M. J.; Rauckman, E. J. Disruption of erythrocyte membranal organization by superoxide. J. Biol. Chem. 258: 2225-2228; 1983. 43. Girotti, A. W.; Thomas, J. P. Damaging effects of oxygen radicals on resealed erythrocyte ghosts. J. Biol. Chem. 259: 17441752; 1984. 44. Block, E. R.; Patel, J. M.; Sheridan, N. P. Effect of oxygen and endotoxin on lactate dehydrogenase release, 5-hydroxytryptamine uptake, and antioxidant enzyme activities in endothelial cells. J. Cell. Physiol. 122: 240-248; 1985. 45. Guidotti, T. L. Toxic inhalation of nitrogen dioxide: Morphologic and functional changes. Exp. Molec. Pathol. 33: 90-103; 1980. 46. Brown, R. E R.; Clifford, W. E.; Marrs, T. C.; Cox, R. A. The histopathology of rat lungs following short term exposures to mixed oxides of nitrogen (NOx). Br. J. Exp. Pathol. 64: 579593; 1983. 47. Thomas, H. V.; Mueller, P. K.; Lyman, R. L. Lipoperoxidation of lung lipids in rats exposed to nitrogen dioxide. Science 159: 532-534; 1968. 48. Sevanian, A.; Mead, J. E; Stein, R. A. Epoxides as a product of lipid autoxidation in rat lungs. Lipids 14: 634-643; 1979. 49. Sugai, M.; Ichinose, T.; Kubota, K. Studies on the biochemical effects of nitrogen dioxide. IV. Relation between the changes in lipid peroxidation and the antioxidant protective system in rat lungs upon life span exposure to low levels of NO:. Toxicol. Appl. Pharmacol. 73: 444-456; 1984. 50. Pryor, W. A.; Lightsey, J. W. Mechanism of nitrogen dioxide reactions: initiation of lipid peroxidation and the production of nitrous acid. Science 214: 435-437; 1981. 51. Mustafa, M. G.; Tierney, D. E Biochemical and metabolic changes in lung with oxygen, ozone and nitrogen dioxide toxicity. Am. Rev. Respir. Dis. 118: 1061-1090; 1978. 52. Guidotti, T. L. The higher oxides of nitrogen: inhalation toxicology. Environ. Res. 15: 443-472; 1978. 53. Patel, J. M.; Block, E. R. Nitrogen dioxide-induced changes in cell membrane fluidity and function. Am. Rev. Respir. Dis. 134: 1196-1202; 1986. 54. Brand, J. C. D.; Stevens, I. D. R. Mechanism and stereochem-

134

J . M . PATEL and E. R. BLOCK

istry of the addition of nitrogen dioxide to olefins. J. Chem. Soc. 1958: 629-638; 1958. 55. Bar, R. S.; Dolash, S.; Spector, A. A.; Kaduce, T. L.; Figard, P. H. Effect of membrane lipid unsaturation on the interactions of insulin and multiplication stimulating activity with endothelial cells. Biochim. Biophys. Acta 804: 466-473; 1984. 56. Bar, R. S.; Goldsmith, J. C.; Rechler, M. M.; Peacock, M. L.; Nissley, S. P. Interactions of multiplication-stimulating activity with bovine endothelium: comparative studies in primary, passaged, and cloned cultures from the pulmonary and systemic circulations. Endocrinology 110: 990-996; 1982. 57. Mengel, M. E.; Kann, H. E. Jr. Effect of hyperoxia on erythrocytes. III. In vitro peroxidation of erythrocyte lipids. J. Clin. Invest. 45: 1150-1158; 1966.

58. Goldstein, J. R.; Mengel, M. E. Hemolysis in mice exposed to varying levels of hyperoxia. Aerospace Med. 411: 12-13; 1969. 59. Simon, L. M.; Axline, S. G.; Robin, E. D. The effect of hyperoxia on phagocytosis and pinocytosis in isolated pulmonary macrophages. Lab. Invest. 39: 541-546; 1978. 60. Bowles, A. L.; Dauber, J. H.; Daniele, R. P. The effect of hyperoxia on migration of alveolar macrophages in vitro. Am. Rev. Respir. Dis. 120: 541-545; 1979. 61. Kaplan, S. A.; Stein, S. N. Effect of oxygen at high pressure on the transport of potassium, sodium and glutamate in guinea pig brain cortex. Am. J. Physiol. 190: 157-162; 1957. 62. Cymerman, A.; Gottlieb, S. F. Effects of increased oxygen tensions on bioelectric properties of frog sciatic nerve. Aerospace Med. 41: 36-39; 1970.