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Departments of 1 Biochemistry and 2 Physiology, The Hebrew University, Hadassah Medical School, Jerusalem 91120, Israel
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Heat acclimation of rats has been shown to enhance endurance of rat hearts to ischemic insult and acute heat stress. Common protective features have been shown to be operative during both these stress-inducing conditions. To explore the role of membrane lipid composition in the adaptive response, we analyzed two major parameters that impact membrane dynamics and order, the nonesterified cholesterol levels and the acyl chain composition of phospholipids, in rat heart and salivary glands, both major thermoregulatory organs, in short- and long-term heat-acclimated rats. Before exposure to heat, control salivary gland tissue has a higher cholesterol-to-phospholipid mole ratio (0.32 ± 0.02) than heart (0.14 ± 0.01), and the acyl chains of its phospholipids are 50% more saturated. The remodeling strategies of the tissues after exposure to heat differed. Heart cholesterol levels increased after short-term heat acclimation (~50%), whereas salivary gland cholesterol levels decreased in acute heat stress and long-term heat acclimation (~32%). Remodeling of phospholipid acyl chains, particularly an increase in docosahexaenoic acid, was a protective strategy in both tissues (57% in heart and >100% in salivary glands). Modifying membrane lipid composition by treating rats with liposomes composed of egg phosphatidylcholine (PC) before exposure to heat resulted in a 38% increase in endurance to thermal stress. The density and affinity of muscarinic receptors of submaxillary salivary glands, involved in the acclimation response, were measured in control and PC liposome-treated rats, and then both groups were subjected to short-term heat acclimation. After PC treatment the well-established compensatory upregulation of the muscarinic receptors and concomitant decrease in their affinity was blunted. The substantial increase in the thermal endurance of heat-challenged intact rats after treatment with PC liposomes (600 vs. 200 min) suggests that membrane lipid composition plays a role in the ability of these tissues to respond to heat stress.
heat endurance; docosahexaenoic acid; liposomes; phosphatidylcholine; cholesterol; muscarinic receptors; submaxillary glands; heart
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE IDEA that the adaptive response to heat stress, or heat tolerance, is somehow affected by the stability of cellular lipids has its origin in 1924 in the work of Heilbrunn (26). The responsiveness of membrane lipid composition to changes in the environment is an established phenomenon (3). The response to temperature variation, one of the most studied environmental factors, has been described in terms of homeoviscous adaptation (12, 61). Detailed studies of this have demonstrated rapid adaptation by modifying acyl chain composition (53) and have focused on the response mechanism at the enzymatic level (14). In recent years it has been shown that the level of unsaturated fatty acyl chains of membrane phospholipids, in both animals and plants, depends on ambient temperature. Among poikilotherms, which are subject to a wide range of changes in their seasonal body temperatures, cold acclimation leads to an increased proportion of acyl chains containing unsaturated bonds. These changes underlie homeoviscous adaptation, allowing the maintenance of membrane fluidity within a certain range and competence in the face of a wide range of changes in body temperature (25, 53, 61). In contrast to the poikilotherms, mammalian acclimation is limited to a very narrow range of changes in body temperature, and little is known about lipid content remodeling under acclimation conditions. Marked changes in the affinity of various G protein-coupled receptors and Na-K-ATPase activity during the course of heat acclimation in mammals (30, 36) imply the possibility of changes in membrane lipid composition since changes in the function of these proteins have been shown to be membrane lipid dependent.
Many studies in recent years have focused on the relationship between membrane lipid composition and lateral organization of the membrane. Small changes in the level of unsaturation of membrane phospholipids have been shown to affect bulk membrane properties defined by fluidity, lateral phase separation, and a parameter referred to as "free volume." Free volume represents the free space generated in a bilayer to accommodate protein conformational transitions, aggregation, and motion (45, 46). These changes have been shown to affect specific modulation of various membrane integral or associated proteins (41). Cholesterol levels have also been shown to have major impact on the fluidity, packing (free volume), and lateral organization of membrane components (3, 45, 46). Lipid acyl chain composition and cholesterol levels have been shown to affect membrane permeability at the structural level and via interaction with specific proteins and enzymes (Ref. 22 and references therein). Lipid remodeling is thus likely to be involved in the cellular heat acclimation response. The purpose of this study was twofold: 1) to examine whether heat acclimation in rats induces changes in the lipid acyl chain composition and nonesterified cholesterol level in membranes of organs known to respond to heat acclimation, and 2) to determine whether manipulation of membrane lipid composition can affect heat endurance and properties of muscarinic receptors associated with water secretion for evaporative cooling.
The rat acclimation model was chosen for experimentation (27, 28). Previous studies have demonstrated that short- or long-term exposure of rats to mild heat stress results in enhanced endurance during acute heat challenge (28). Both rat heart and submaxillary salivary glands were studied because these two organs represent the major thermoregulatory effector organs of this species (27, 28). Changes in lipid composition were characterized in three groups of heat-treated rats, 1) after short-term (2 day) heat acclimation (AC); during which various adaptive responses are turned on; 2) after long-term acclimation (30-day AC), when acclimatory homeostasis manifested by a more rapid and efficient adaptive response is achieved (28); and 3) after acute heat stress.
To determine whether alterations in membrane lipid composition improve heat endurance of rats similarly to heat acclimation, membrane lipid composition was altered by repeated injection of liposomes (diameter 30-100 nm) composed of egg phosphatidylcholine (PC). In rats this treatment was shown previously to alter membrane lipid composition of heart and other tissues, mainly by reducing nonesterified cholesterol levels (4). Heat endurance, membrane acyl chain composition, and cholesterol levels were measured in parallel to M3 muscarinic receptor responses in the submaxillary gland. Activation of this receptor leads to water secretion and evaporative cooling. Changes in the affinity and density of this receptor during the different phases of heat acclimation underlie the acclimatory responses of this important effector organ receptor and serve as a sensitive marker of both heat stress and heat acclimation (30).
Our data demonstrate that heat acclimation induces changes in the lipid composition of both heart and salivary gland membranes. Changes in the cholesterol levels differed in the two organs; however, a marked increase in the level of highly unsaturated acyl chains, particularly docosahexaenoic acid (DHA), seems to be an important adaptive response in both organs, possibly helping to prolong maintenance of cellular functional integrity and thus improving heat endurance.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials
Egg PC (containing 95.6% PC, 3.1% sphingomyelin, 0.5% lysophosphatidylcholine, and 0.12% DL-
-tocopherol) was
purchased from Lipoid KG (Ludwigshafen, Germany); L--PC
(egg)(>99%) was purchased from Avanti Polar Lipids (Alabaster, AL),
and sterile nonpyrogenic saline was purchased from Travenol. The
Meth-Prep II kit for transmethylation of phospholipid acyl chains was
purchased from Alltech Associates (Deerfield, IL). All solvents used
were analytic or HPLC grade.
Animals
Male rats (Rattus norvegicus, Sabra strain, albino var., Harlan, Jerusalem) of initial weight 70-90 g were divided into four groups: normothermic controls (C), heat-acclimated (AC), heat-stressed, and normothermic groups treated with egg PC liposomes. All animals were kept in a 12:12-h light-dark cycle. Normothermic rats were kept at 24 ± 1°C. Heat acclimation was attained by continuous exposure to 34 ± 1°C and 30-40% relative humidity, for 2 days (2-day AC) or 30 days (30-day AC). Acute heat stress was attained by exposure to 40°C for 2 h. Membrane lipid compositions of rat heart and salivary glands were determined in all groups (see below). The endurance of rats subjected to heat stress was monitored in control and PC liposome-treated groups. In addition, muscarinic receptor density and affinity in salivary gland extracts were measured and compared with our database for control and acclimated rats (30). All experiments were performed in accordance with guidelines approved by the Hebrew University Committee for Animal Experimentation.Liposome Preparation, Characterization, and Administration
Small unilamellar liposomes composed of 10% PC in a size range of 30-100 nm were prepared in 0.9% saline, 200 µM desferal, 10 mM histidine buffer, pH 6.7, by shearing using a Polytron homogenizer, followed by extrusion through a two-stage high-pressure Gaulin LAB 60 homogenizer (APV Gaulin, Hilversum, The Netherlands) and sterilization by filtration through a Gelman 0.2-µm filter (Ann Arbor, MI). Liposome phospholipid concentration was determined by the modified Bartlett procedure (2), and their size distribution by photon correlation spectroscopy using a Coulter model N4 SD submicron analyzer (Coulter Electronics, Hialeah, FL). Sterility was assessed as described in Barenholz and Amselem (2).Rat Liposome Treatment
Two milliliters of PC liposomes (total of 800 mg/kg) was injected twice weekly for 2-3 wk. The plasma pharmacokinetics of the liposomes was followed using a Wako Choline Phospholipids B Kit (catalog no. 990-954009, Wako Chemicals) as described by Shmeeda et al. (58). One week after the completion of liposome treatment, rats were tested for heat endurance as described below or killed by cervical dislocation for lipid or receptor analysis.Heat Endurance
The conscious rats, lightly restrained, were instrumented with colonic thermistor (YSI 702, Yellow Springs, OH), connected to a data-acquisition system (Codas, Dataq, OH) inserted 6 cm beyond the anal sphincter. The rats were then kept at an ambient temperature of 24 ± 1°C for colonic temperature (Tc) stabilization. Immediately thereafter, the rats were placed in a temperature-controlled heat stress chamber under heat stress of 40°C. Heat endurance was determined as the time taken to attain a Tc of 41.5°C.Membrane Lipid Analysis
The hearts and salivary glands were removed from killed rats, rinsed with cold saline, kept on ice, dried on absorbent paper, weighed, and homogenized in ice-cold saline (1.0-1.5 ml/g), using a Polytron homogenizer (Kinematica) for 3 min at setting 5 and then using a Dounce homogenizer (30 strokes). Homogenized samples were extracted for total lipids using the Bligh and Dyer procedure (5) as described in detail elsewhere (59). A 50-µl aliquot of the Bligh and Dyer lower phase was used to quantify phospholipids based on organic phosphorus by the modified Bartlett procedure (2). A 100-µl aliquot was evaporated under N2 and processed for gas chromatographic analysis of lipid acyl chains by transmethylation in 50 µl of toluene and 20 µl of Meth-Prep II for 30 min at 25°C and injected into a Perkin Elmer Autosystem gas chromatograph and Autosampler using a 6FT 10% Silar 10C column (Alltech), dry N2 as the carrier gas, and flame ionization for detection. Samples were prepared with pentadecanoic acid (1 mg/ml) as a marker (2, 55). To enable quantitative calculation of remodeling of acyl chain composition, their level was calculated in mole percent rather than weight percent.Calculation of the mole-weighted (average) acyl chain melting temperature (MP) was based on the mole percent of each acyl chain component multiplied by the melting point temperature of that component (43). This average MP value is a measure of membrane phospholipid average fluidity; the higher the average MP is, the lower the fluidity (53). Fluidity is a complex expression, including both structural elements such as free volume and packing and dynamic parameters. Within the context of this study, we are using average MP without resolution of its components, to express the bulk implications of phospholipid acyl chain composition (53).
A 1.5-ml portion of the Bligh and Dyer lower phase was evaporated and used for determination of nonesterified cholesterol (59). We will use "cholesterol" to denote nonesterified cholesterol (esterified cholesterol was not determined). Samples were analyzed on HPLC using a Kontron type 400 solvent delivery system, equipped with a Kontron autosampler MSI 660 (Kontron, AG, Zurich, Switzerland) using a normal phase silica column (Alltech, Deerfield, IL) of length 5 cm, diameter 4 mm, particle size of 3 µm. Samples were resolved in a mobile phase composed of hexane:isopropanol (500:6) at a flow rate of 1 ml/min based on Smith et al. (62). Detection was performed at 212 nm using an ultraviolet-visible spectrophotometer coupled to a Kontron data analyzer.
Muscarinic Receptor Binding
Measurements were performed on a particulate fraction of gland homogenate. Submaxillary glands were extirpated, weighed, pooled, and homogenized in 0.32 M sucrose (10% wt/vol). Membranes were prepared as described by Pimoule et al. (51) and Kloog et al. (37). The supernatant from the centrifugation at 1,000 g for 10 min was then centrifuged for 20 min at 20,000 g. The resulting pellet was resuspended in 50 mM sodium phosphate buffer (pH 7.4) and recentrifuged as before. The final pellet was resuspended in phosphate buffer to yield a membrane preparation containing 2-6 mg protein/ml. In the binding assay, 10-µl samples were used. The assay was performed as described previously (30), using various concentrations (0.1-20 nM) of N-[3H]methylscopolamine (NMS). Nonspecific binding was determined in the presence of 50 µM atropine. After incubation (60 min, 37°C), the reaction was terminated by the addition of cold buffer and filtration using GF/C glass microfiber filters (Whatman) to separate bound from free ligand. Radioactivity was then determined using Quicksafe cocktail A (Zinsser Analytic). Protein was assayed according to Bradford (7). Muscarinic receptor densities and dissociation constants for the ligand were calculated from Scatchard plots, using a curve-fitting routine for one and two binding sites. To verify the homogeneity of receptor population, Hill coefficients were calculated.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of Various Heat Treatments on Acyl Chain Composition of Rat Heart and Salivary Glands
The total phospholipid acyl chain composition of heart left ventricles and salivary glands, before and after exposure to a variety of heat treatments, was examined (Tables 1 and 2, respectively). Next, the acyl chain composition of lipids was examined in liposome-treated rats before and after 2-day AC.
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Acyl chain composition of control heart and salivary glands. The acyl chain composition of control heart tissue and salivary glands is displayed in Tables 1 and 2. Heart tissue contains substantially more 20:4, 22:5, and 22:6 compared with salivary tissue, which contains a smaller proportion of polyunsaturated fatty acids (PUFA) but a greater variety of PUFA species (18:3, 20:3, 20:5, 22:4, and 22:6). The ratios of unsaturated fatty acids (UFA), monounsaturated fatty acids (MUFA), and PUFA to saturated fatty acids (SFA) are twofold higher in heart compared with salivary glands. The average MP of salivary gland acyl chains is 27.8°C, which is much higher than that of heart tissue, 8.9°C. The higher average MP of salivary glands is due to the predominance of SFAs and indicates a generally less "fluid" phospholipid membrane environment.
Effects of heat treatments on heart tissue (Table 1). Hearts of rats acclimated for 2 days displayed a 42% reduction in the levels of saturated acyl chains composition (16:0, 18:0) and a net increase in PUFA, resulting in an increased ratio of PUFA to SFA, from 1.29 to 1.9. The major changes in the UFA composition were found in 22:5 and 22:6; the mole ratio of 22:5 decreased 40%, and that of 22:6 increased 53%, suggesting metabolic conversion of 22:5 to 22:6. Arachidonic acid (20:4), a significant component (12.7%) of the control heart tissue acyl chain profile, increased 15%. As a result of these changes, the ratio of UFA to SFA increased considerably, from 1.94 in untreated rat hearts to 2.70 in hearts of 2-day AC rats. The ratio of MUFA to SFA increased in this treatment in contrast to all others.
The metabolic pathways of long-chain fatty acids relevant to our discussion are shown in Fig. 1, where the pathways for generation of long-chain PUFA (n-6, n-3) are compared with the major pathway for synthesis of long-chain MUFA (n-9) (11). We have used the ratio of the level of DHA (22:6), of the n-3 pathway, divided by that of
-arachidonic acid
(20:4), the major n-6 pathway metabolite, to reflect the relative flux
through these two pathways (n-3/n-6) (6, 24). This ratio
is an underestimation because the n-3 components should include
eicosapentaenoic acid (EPA), which may be diverted to prostanoids (see
DISCUSSION), and the n-6 component is actually less because
20:4 includes -arachidonic acid, an n-3 metabolic intermediate. This
ratio increased in 2-day AC heart tissue from 0.84 to 1.12. The average
MP of the acyl chains in 2-day AC hearts decreased from 9.0 to 1.43, indicating a small increase in the average fluidity of the membrane
phospholipids.
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Effect of heat treatments in salivary glands. The lipid acyl composition of salivary glands was significantly different from heart (Table 2), in particular the relative amounts of SFA and PUFA. Keeping the lower levels of PUFA in mind, some of the relative changes in their acyl chain profile after the various heat treatments still appear to be significant, but the changes in absolute terms are small. After 2-day AC there was a major decrease in 18:3, 20:4, and 20:5 and a parallel increase in the C22 series. DHA increased in particular (608%). The ratio of UFA to SFA decreased and the ratio of PUFA to SFA increased only slightly due to the smaller amount of PUFA in this tissue. The ratio of the n-3/n-6 pathways increased substantially. Average MP was not affected in any of the heat treatments.
In 30-day AC rats, SFA changed <10%, 20:3 and 22:5 decreased
50%,
and 22:6 increased 92%, but not nearly as strikingly as after
2-day AC (or heat stress). The ratios of UFA, PUFA, and MUFA to SFA did
not change compared with the controls. The n-3/n-6 ratio increased slightly.
Acute heat stress led to insignificant changes in SFA, 18:1 and 18:2.
We found that 18:3 and 20:3 decreased substantially as in the salivary
glands of 2-day AC, a 17% increase in 20:5, and substantial decreases
in 22:4 and 22:5. The increase in 22:6 by 158% was more than for
30-day AC (92%) but much less than in 2-day AC (608%). The decline in
20:4 under all heat treatments in salivary glands differs from
its response in heart. The ratio of n-3/n-6 increased only slightly as
in 30-day AC.
If we look at the ratio of MUFA and PUFA to the SFA in salivary glands,
we find much smaller changes after heat challenge compared with those
in heart tissue in both groups of fatty acids. The most pronounced
change was observed in tissue of rats acclimated for 2 days. Although
the ratio of UFA to SFA decreased in all treatments, the average MP
values in the various treatments do not change significantly; thus
homeoviscous adaptation seems to be operative here as in heart tissue.
The salivary glands of liposome-treated rats demonstrated increased
18:3, decreased 20:3 and 20:5, and substantially increased 22:6 in
contrast to its lack of effect in heart. In PC liposome-treated 2-day
AC salivary glands, the C18 and C20 series were
similar to that found in 2-day AC salivary gland not exposed to
liposomes, including the decrease in 20:4. In contrast to 2-day AC
salivary gland, 22:4 decreased, with higher levels of 22:5 and
22:6. Liposome treatment generated an acyl chain pattern
similar to the 2-day AC profile (except for 18:2 and 22:4).
Effects of Heat Treatments on Cholesterol Levels in Heart and Salivary Glands
The level of cholesterol and total phospholipids was measured in tissues of untreated (control) rats and of rats after heat acclimation and PC liposome treatment. The results (Table 1 and 2) are expressed as cholesterol-to-phospholipid mole ratio (Chol/PL) which is related to membrane fluidity as well as to the membrane phase structure (47, 48). The partitioning of cholesterol in phospholipids is affected by the level of phospholipid acyl chain saturation (45, 46). Chol/PL in control rat heart was 0.14 ± 0.01. In 2-day AC hearts, this ratio doubled to 0.26 ± 0.02. In contrast, 30-day AC did not result in a sustained increase of cholesterol levels. PC liposome treatment, which has been shown to cause nonesterified cholesterol depletion from membranes (4, 59, 66), reduced Chol/PL levels by 22%. In hearts of 2-day AC liposome-treated rats, the absolute rise in Chol/PL was less than found in hearts not injected with liposomes (0.18 ± 0.01 vs. 0.26 ± 0.02); thus liposome treatment reduced the 2-day AC-induced cholesterol increase, but it still represents a moderate change (0.26/0.14 vs. 0.18/0.11).Salivary gland cholesterol level did not change in 2-day AC; however, it decreased ~35% after 30-day AC and heat stress. This parallels a reduced response in the PUFA acyl chain composition and suggests a significant role for nonesterified cholesterol in 30-day AC and heat stress. Liposome treatment reduced cholesterol levels by 22%. Two days of AC led to no further changes in Chol/PL.
Heat Endurance
Upon subjection to heat stress, rectal temperature rises, up to establishment of the hyperthermic plateau, at which the heat-stressed body temperature is regulated (32). This temperature is physiologically determined by the onset of salivation, which clamps body temperature at this hyperthermic level. Failure of temperature regulation coincides with depressed salivation and is marked by the upward shift of body temperature beyond the regulated range. It is evident from the data presented in Fig. 2 that while the untreated rats maintained their regulated temperature for ~200 min, the liposome-treated rats maintained temperature regulation for a significantly longer period of at least 600 min. This prolonged endurance resembled that observed for 30-day AC rats.
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Muscarinic Receptor Studies
Muscarinic receptor density and affinity were measured in submaxillary salivary glands of rats treated with PC liposomes and compared with untreated rats (Fig. 3). Two subtype receptor populations, high affinity (HA) and low affinity (LA), are present. After PC liposome treatment, marked receptor upregulation was measured with no significant change in the binding affinity and in the LA-to-HA receptor subtype population ratio, suggesting no change in their coupling to the G protein.
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To further study the effect of liposome treatment on membrane receptors, we measured receptor density in liposome-treated 2-day AC rats. Glands of 2-day AC rats upregulate their muscarinic receptors concomitantly with a decrease in their affinity (30). In the 2-day AC liposome-treated rats, there were no further changes in receptor profile compared with the preacclimation group. In addition, the ratio of HA/LA in this group is 0.39 compared with 1.0 in both liposome-treated and 2-day AC glands. Concomitantly, no significant affinity changes were found in these receptors.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Remodeling of lipid composition in membranes occurs in response to changes in ambient temperature. Such remodeling may result either in changes in the physical state of the membrane, or in specific lipid interactions with proteins (45). In this investigation, the response of membrane lipid composition to heat acclimation (chronic heat) and acute heat stress was compared in two mammalian thermoregulatory effector organs: the heart and the submaxillary gland, the latter, the major evaporative cooling organ of the rat. Both heat acclimation and acute heat stress led to dynamic lipid remodeling, resulting in maintenance of homeoviscosity. A common feature of the response to both stressors was an increase in PUFA, particularly DHA, whereas the Chol/PL ratio varied. An additional important finding was that intravenous injection of high doses of PC liposomes provided protection during acute heat stress. This was accompanied by upregulation of the density of submaxillary gland muscarinic receptors, although with decreased affinity, suggesting that receptor capacity is maintained.
Relationship Between Changes in Chol/PL and Fatty Acyl Chain Unsaturation in Control Rat Organs
Major differences were found, as expected, in the composition and physical state of the membranes of the two tissues (Tables 1 and 2). Hearts have significantly higher UFA/SFA and PUFA/SFA and lower MP and Chol/PL than salivary glands. Higher levels of unsaturation lead to less cohesive acyl chain packing, increased fluidity and compressibility, reducing the energy needed for elastic deformation (49, 38). Heart tissue is thus inherently more "fluid" than salivary glands. The UFA-to-SFA ratio in salivary glands is <1.0 in all conditions observed, whereas in heart it is >1.5 (Tables 1 and 2). This suggests that salivary gland membranes consist mainly of phospholipids with a saturated acyl chain in position 1 and an unsaturated acyl chain at position 2 (1-SFA, 2-UFA), whereas heart has a rather large population of biunsaturated phospholipids (about 1/3 of total) and possibly no bisaturated phospholipids. The change in amount of bisaturated and biunsaturated phospholipids may be a major contributor to dynamics and organization of membranes (41). Cholesterol segregates preferentially with bisaturated phospholipids, thus affecting lateral phase separation or domain formation (45, 46).Studies in retinal rod segments have shown that phospholipids containing DHA packed less cohesively, resulting in higher membrane free volume and cholesterol segregation into DHA-poor domains, generating lateral phase separation (52). Membrane enrichment with DHA has been suggested to represent a strategy to protect integral and membrane-associated proteins by providing increased free volume or a less compressed membrane, while cholesterol apparently plays a complementary role by fine-tuning the free volume such that membrane stability is maintained (45, 46).
Relationship Between Changes in Chol/PL and Acyl Chain Unsaturation in Heat-Treated Rat Organs
In 2-day AC heart, both cholesterol and DHA increased. Although subcellular fractionation of the tissues to determine where these changes occur would provide more specific information, based on previously reported studies of the phospholipid composition of heart tissue, in which mitochondrial membranes dominate, and where the microsomal and mitochondrial compartments contain 70-80% PC and phosphatidylethanolamine (65), the extent of the PUFA changes suggests that all heart membranes will be affected. Thus, wherever the cholesterol levels rise and encounter DHA, lateral phase separation (microdomain formation) may be anticipated.In salivary glands where cholesterol levels are already high there is no further change after 2-day AC. Here the relative increase in DHA probably acts locally because only a few phospholipids contain this component in this tissue. The substantially reduced cholesterol in 30-day AC and heat stress (~32%) certainly reflects a more fluid situation overall, where small changes in DHA could have significant local membranal effects.
Increased DHA, like elevated temperature, results in increased disorder in membranes. The ability to maintain structural integrity while accommodating the disorder introduced by a temperature increase may be a unique property of DHA or a function of the interplay between DHA and cholesterol. Both DHA and cholesterol have been shown to affect membrane permeability properties that will directly impact stress endurance during heat challenge (Refs. 22 and 33 and references therein). Increased free volume generated by DHA could increase the water capacitance of the phospholipids. Recently, Haines (22) has proposed that cholesterol proportionally reduces passive water permeability through lipid bilayers by interacting with aliphatic chains of membrane lipids at the bilayer interphase. This could help prevent dehydration of the cells (22). Cholesterol has also been proposed to contribute to energy saving either by controlling proton leakage, which leads to energy dissipation during thermal/cold adaptation in mitochondrial membranes, by acting as a membrane insulator and energy buffer, or by buffering Na+ leakage through the cell membrane (22). In 30-day AC rats, the metabolic rate is decreased. In 2-day AC heart, increased cholesterol may reflect an early protective function, facilitating heat dissipation and acting to stabilize metabolic rate.
Changes in average MP of the membrane phospholipids are small under all conditions; however, there is actually extensive compensatory remodeling going on. In 30-day AC heart, there is little remodeling, and cholesterol levels returned to control values. Here we find the lowest average MP and the highest n-3/n-6 ratio (see below), contributing to enhanced endurance. Cell membrane protection may be further facilitated by protein-mediated strategies because the level of the 72-kDa heat shock protein (HSP72) in 30-day AC heart is increased by >200% (42), and there is a marked increase in muscarinic receptors in glandular cell membranes (30). The increased DHA and decreased cholesterol may help to accommodate the increased protein density.
In control salivary glands, the ratio of Chol/PL is significantly higher than in hearts (0.32 compared with 0.14). Two-day AC did not lead to a change in Chol/PL, as seen in heart tissue. There is no change in average MP despite the increased PUFA, which emphasizes the compensatory nature of acyl chain remodeling. In 30-day AC and heat stress, the changes in acyl chain composition and DHA in particular were less dramatic. In all treatments the DHA component increased significantly (>100%) and to a greater extent than most other changes; however, the absolute level of DHA in salivary glands is much smaller than in heart, so its relative increase is misleadingly large. No significant changes were found in average MP under any conditions because the saturated components are dominant in this tissue and the absolute amount of DHA remained small. On an absolute scale these changes are not as substantial as those noted in heart tissue. Small amounts of DHA may act locally, influencing membrane lateral organization and in-plane domain formation (47), or may have impact as a modulating or signaling component of the response to heat challenge. Low levels of DHA could specifically affect the activity of membrane proteins such as protein kinase C (16), the sodium channel (64) and its level of gene expression (34, 57), or, as recently suggested, DHA may act as a ligand (15).
In summary, after 2-day AC, 30-day AC, and heat stress, the particular changes described produce stability in the average fluidity, which is reflected by the lack of changes in average MP, and represent the achievement of homeoviscous adaptation. This does not preclude changes in microdomains such as in rafts or specific changes in phospholipid headgroup composition that may otherwise affect the lateral organization of the membrane or affect particular proteins. The adaptive capability or flexibility of the system is seen in the heat acclimation process where transient, possibly overprotective, changes occur upon 2-day AC and are stabilized upon 30-day AC, where acclimatory homeostasis is achieved (28), allowing an enhanced protective response on subsequent heat challenge. This preconditioned state, which has been demonstrated at the protein level, is now supported by changes in the lipid components of these membranes. The remodeling strategies of these membranes differ from those of bacteria and poikilotherms, where increasing temperature results in a rise in SFA with an overall increase in average MP, thereby decreasing the phospholipid fluidity (53).
Metabolic Implications
Heat acclimation and stress increased the flux through the n-3 pathway relative to that of n-6.-Arachidonic acid (20:4), the major
n-6 pathway metabolite, has been implicated in proinflammatory processes because it can be further metabolized to active products such
as prostaglandins (9, 44). The PUFAs of the n-3 pathway, EPA (22:5) and DHA (22:6), have both been shown to modulate the activity of cyclooxygenase and competitively inhibit the conversion of
arachidonic acid to prostaglandins (8-10), diverting
an oxidative (inflammatory) course to a course generating prostanoids
from EPA, which consume excess oxygen. Increased flux through the n-3 pathway results in preconditioning against various stressors that induce production of free radicals (17-19). Severe
heat stress induces free radicals in brain and in cell cultures
(23). Evidence of an intrinsic free radical trapping
ability in DHA-modified brain lipids (19, 20) and an
ability to stimulate the activity of antioxidant enzymes in endothelial
cells (13) further suggests that this molecule can
function as a structural and biochemical "stress absorber." The
ratio of the n-3/n-6 pathways, which can be approximated by dividing
the level of DHA by that of -arachidonic acid (22:6/20:4), may be
used to reflect the anti-inflammatory/inflammatory status of the
membrane acyl chain milieu. Heat acclimation and stress increased this
ratio in heart and salivary glands, thus suggesting a rise in
anti-inflammatory capacity.
Although the net ratio favors n-3, these treatments actually triggered both the n-6 and n-3 pathways. A combined response of both pathways has been noted in other studies where both the n-6 and n-3 pathway induction were important in prevention of coronary disease (6, 24). Recent studies in normal human subjects undergoing heat acclimation have demonstrated changes in serum prostaglandins: an initial upregulation (2 wk) was followed by a return to normal levels when stabilization was achieved (Y. Shani and E. Shohami, personal communication). This may indicate different magnitudes or temporally varying activation of the n-6 and n-3 pathways with progression of acclimation. In salivary glands, although both 22:6 and 20:4 levels are relatively low compared with heart, their ratio favors the n-3 pathway in all heat treatments, thus supporting a role for DHA in this tissue despite its low level.
PC Liposome Treatment: Effects on Lipid Parameters, Receptor Profile, and Heat Endurance
Various studies on the dynamics of heat acclimation have demonstrated changes in the density and affinity of the muscarinic receptors in the submaxillary gland, the evaporative cooling effector organ of the rat, as well as in the atrium of the heart and gastrointestinal tract (reviewed in 31). Two-day AC upregulates the muscarinic receptors and results in decreased binding affinity (30), decreased inositol 1,4,5-trisphosphate-sensitive endoplasmic Ca2+ pool size, and a decreased Ca2+ signal, collectively impairing salivary glandular function, which is compensated in 2-day AC by increased nervous excitability. (35). In 30-day AC, there is further upregulation of the muscarinic receptors and a return to their preacclimation affinity (30), concomitant with enhanced performance to subsequent challenge, despite maintenance of a lowered Ca2+ signal (35). Modulation of the lipid environment of these receptors, and other proteins, could provide an important means of protecting their integrity under heat stress. The fact that there were no general changes in average acyl chain fluidity (MP) does not exclude the possibility that local heterogeneities in membrane composition and organization occur that affect these receptors. Decreased receptor affinity upon 2-day AC could be related to increased DHA. Increased DHA led to decreased affinity of
-adrenergic receptors to their ligand in cardiomyocytes
(21), suggesting that in an attempt to maintain the same
level of responsiveness, more receptors are necessary. Studies of other
G protein-coupled signaling pathways have implicated important roles
for both cholesterol and DHA in receptor coupling. The kinetics of
coupling of rhodopsin receptors with G proteins were reduced by
cholesterol and increased by DHA, which buffered the adverse affects of
cholesterol (45). In this investigation we show that
selectively modulating cell membrane lipids by PC liposome treatment,
which reduces membrane cholesterol levels, upregulated receptor density
and tended to decrease their affinity, similar to the 2-day AC
response. Two-day AC after liposome treatment did not further affect
the receptor profile, suggesting that modulation of the lipid
environment of these receptors provided sufficient protection of the
membranes/protein. Lipid acyl chain and cholesterol analysis showed
1) a significant reduction in cholesterol levels compared
with untreated rats in both tissues (22%), 2) a significant
increase in 18:3 in both heart and salivary glands, which reflects a
metabolic response because 18:3 is very low in the injected PC
liposomal acyl chain composition, and 3) major acyl chain
remodeling in salivary glands, including an increase in DHA. In the PC
liposome-treated heart, DHA is not induced. Here increased 18:3, which
affects water permeability, may contribute to endurance, a role
alternatively played by DHA (33, 50).
Modulating cell membrane lipids by PC treatment increased the thermal endurance of the rats almost as effectively as heat acclimation (29). It is likely that enhanced thermal endurance in the liposome-treated rats is also associated with improved glandular capacity to secrete water, as suggested by the increased density of the receptors. Water is secreted due to changes in electrolyte pumps creating osmotic force, initiated by the activation of muscarinic receptors. In the heart PC treatment may provide an alternative means of providing protection where heat acclimation is not applicable. The efficacy of PC treatment on a short time scale has yet to be determined.
In summary, this study demonstrates that homeoviscous adaptation to thermal stress includes the elevation of PUFA levels, which presumably plays an important role in maintaining membrane integrity in heart, and probably serves a more exclusive signaling or modulatory role in salivary glands. The increase in DHA in both heart and salivary glands generates further evidence for a unique protective role for this fatty acid. Further investigation of the involvement of PUFA and DHA in particular in protective strategies will support its therapeutic application in a variety of disease processes.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Shmeeda, Dept. of Experimental Oncology, Shaarei Zedek Medical Center, PO Box 3235, Jerusalem 91031, Israel (E-mail: hilary{at}szmc.org.il).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpregu.00423.2001
Received 20 July 2001; accepted in final form 31 January 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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