AMER. ZOOL., 38:291-304 (1998)
Cholesterol Function in Plasma Membranes from Ectotherms:
Membrane-Specific Roles in Adaptation to Temperature1
ELIZABETH L. CROCKETT 2
Department of Biological Sciences, Ohio University, Athens, Ohio 45701
SYNOPSIS. Cholesterol is an essential component in the plasma membranes of animals with multiple effects on the physical properties of membranes including
membrane order (fluidity), phase behavior, thickness, and permeability. Cholesterol also affects functional attributes of cell membranes such as the activities of
various integral proteins. Because cholesterol provides rigidity to fluid phase membranes, it is a likely candidate to counter some of the temperature-induced perturbations in membrane order that would otherwise be experienced by animals
that live at varied body temperatures. If cholesterol contributes to homeoviscous
adaptation (HVA), more cholesterol is likely to be present in plasma membranes
from warm-bodied animals than from cold-bodied animals. This prediction is generally supported by studies examining cholesterol contents in membranes from
endothermic and ectothermic animals. Comparisons of cholesterol levels in temperature acclimated (or acclimatized) ectotherms reveal an increase in cholesterol
with temperature, no change in cholesterol content, or an increase in cholesterol
with a decrease in temperature. These different patterns largely represent tissue
and regional differences in the membranes (membrane domains). The membranespecific nature of the cholesterol response to temperature is likely to arise from
the multiplicity of the effects that cholesterol exerts on membranes, as well as the
heterogenous nature of plasma membranes. These factors also allow cholesterol to
perform more than a single role in temperature adaptation of plasma membranes
in animals.
INTRODUCTION
Ectothermic animals occupy a variety of
thermal environments, and as a result, body
temperatures of these animals vary appreciably. Many ectothermic organisms are
adapted to temperature variation allowing
physiological processes and biochemical reactions to proceed efficiently at different
temperatures. Changes in body temperature
threaten to compromise membrane integrity
and function because of alterations in the
physical properties of membranes. Virtually
all of the important functions of biological
membranes, including cell compartmentalization and the provision of specialized microenvironments for organizing protein-mediated events or metabolic pathways, require a membrane that performs as a fluid
1
From the Symposium The Biology of Lipids: Integration of Structure and Function presented at the
Annual Meeting of the Society for Integrative and
Comparative Biology, 26-30 December 1996, Albuquerque, New Mexico.
2
E-mail: [email protected]
matrix and compositionally represents a
mixture of asymmetrically distributed polar
and neutral lipids intermixed with proteins.
Many organisms adjust the compositions of
their membrane lipids to minimize effects
of temperature variation.
Cholesterol is a major constituent of the
plasma membranes in animals. Cholesterol
has pronounced effects on the physical
properties of membranes, and because of
this may be used to minimize effects of
temperature on membrane structure and
function. Much of the early work which examined changes in cholesterol content as a
function of body temperature focused on intracellular membranes (e.g., Wodtke, 1978;
Downer and Kallapur, 1981; Chang and
Roots, 1989). However, more recent studies
have demonstrated that membrane-associated cholesterol is largely confined to the
plasma membrane (e.g., Lange et al., 1989).
This review presents the rationale for why
cholesterol is likely to be important in thermal adaptation of plasma membranes of animals, discusses the evidence, and presents
291
292
ELIZABETH L. CROCKETT
a view of why cholesterol is involved in
thermal adaptation in a membrane-specific
manner.
\
A NATURAL HISTORY OF CHOLESTEROL
Cholesterol and its whereabouts
Sterols are found in the cell surface
membranes of virtually all eukaryotic organisms. Cholesterol is the most abundant
neutral lipid in the plasma membranes of
animals and is required for growth (Yeagle,
1988). Many animals (including most, if
not all, vertebrates) can synthesize cholesterol, although some invertebrates lack this
capacity (Bloch, 1991). Both the endoplasmic reticulum and the peroxisome are sites
of cholesterol biosynthesis in mammalian
hepatocytes (Keller et al, 1985; Thompson
et al, 1987; Thompson and Krisans, 1990).
Like polar lipids, cholesterol is amphipathic. The polar head of cholesterol is
small and consists of a 3(3-hydroxy group
(Fig. 1A). The rest of the cholesterol molecule is nonpolar and includes four fused
rings (the steroid nucleus) and a hydrocarbon (isooctyl) tail. The steroid nucleus is
planar with conformational restrictions. The
rigid character of this portion of the molecule increases order (decreases fluidity) in
the region of the bilayer with which it abuts
(Stockton and Smith, 1976). The hydrocarbon tail, on the other hand, is relatively
flexible and can undergo rotation (Duax et
al., 1988). Cholesterol intercalates in a
phospholipid bilayer in an orientation parallel with the polar lipids (Fig. IB) (Franks,
1976; Worcester and Franks, 1976; Mclntosh, 1978). The hydroxyl group of cholesterol may form hydrogen bonds with the
ester carbonyl (Huang, 1976) or the phosphate moiety (Sankaram and Thompson,
1990; Reinl et al, 1992) of the phospholipids. The specific position of the hydrogen
bond between cholesterol and a neighboring phosphopholipid may be temperaturedependent (Reinl et al, 1992).
Levels of cholesterol in biological membranes are species- and tissue-specific. Cholesterol is also asymmetrically distributed
within the cell. Cholesterol content (on a
molar basis) equals phospholipid content in
some plasma membranes (e.g., mammalian
B
FIG. 1. (A) Cholesterol. (B) The position of cholesterol in a phospholipid bilayer. Adapted from Franks
(1976).
erythrocytes, Nelson, 1967; mammalian intestinal epithelia, Meddings, 1989), and
cholesterol levels of plasma membranes
vary among tissues (Fig. 2). Cholesterol is
increasingly concentrated along the secretory pathway from its site of synthesis at
the endoplasmic reticulum to the Golgi apparatus and then to the plasma membrane
(Bretscher and Munro, 1993). Cholesterol
levels are extremely low in other intracellular membranes (Lange et al, 1989). Lateral heterogeneity of cholesterol (asymmetric distribution along the length of the
membrane) also exists within the plasma
membrane itself (El Yandouzi and Le Grimellec, 1992; Schroeder et al, 1995).
Membrane domains, which differ from the
rest of the membrane, include discrete cholesterol-rich and cholesterol-poor regions
(reviewed by Schroeder et al, 1991).
Properties of membrane cholesterol
The membrane localization of cholesterol
has chemical, physical, and functional consequences for the cell surfaces in which it
resides. Several key physical properties of
the membrane (discussed below) are governed by cholesterol. These physical properties require the precise molecular geometry that arises from the orientation of the
3(3-hydroxy group, the flat steroid nucleus.
CHOLESTEROL FUNCTION IN THERMAL ADAPTATION
293
20°C-Acclimated Trout
0.75
•S
0.60-
Q. 0.45 -
O 0.30 -
£ 0.15 O 0.00
Liver
Gut
Kidney
Gill
FIG. 2. Cholesterol levels vary in plasma membranes from tissues of rainbow trout (Oncorhynchus tnykiss).
Molar ratios of cholesterol (expressed relative to phospholipid contents) were measured in plasma membranes
prepared from animals grown at 20°C. Error bars represent SEM (n a 4). Data for gut are from Crockett and
Hazel (1995) and those for other tissues are from Robertson and Hazel (1995).
and the eight carbon hydrophobic tail
(Suckling et al, 1979; Mattjus et al, 1995).
Cholesterol has multiple effects on membrane properties, so it is probably involved
with a variety of roles such as 1) stabilizing
the membrane, 2) reducing membrane permeability, 3) facilitating morphological
characteristics and interactions between
cells, 4) influencing phase transitions, and
5) providing a suitable microenvironment
(chemical and/or physical) for membraneassociated proteins. Other roles for cholesterol have been recently suggested. For example, cholesterol (along with a-tocopherol) may serve as a membrane antioxidant
(Parasassi et al, 1995). Cholesterol and atocopherol, a well-recognized antioxidant,
share many similar effects on membrane
physical and chemical properties (Stillwell
et al, 1996). The roles of cholesterol in biological membranes, however, are still not
clearly defined, or completely understood,
and the degree to which cholesterol exerts
its influence will depend on the cholesterol
content itself and on other molecular constituents of the membrane.
A widely accepted perspective is that
cholesterol stabilizes membranes {e.g.,
Bloom and Mouritsen, 1988; Bloch, 1991).
Cholesterol provides order to (rigidifies)
membranes that are in the fluid-phase (in
contrast with membranes that are in the gel
phase), and more so than its metabolic precursors (Dahl et al, 1980), by reducing the
number of trans-gauche isomerizations
within acyl chains of neighboring phospholipids (Yeagle, 1985). The ordering influence of cholesterol is particularly pronounced in membranes rich in saturated
phospholipids compared with those containing unsaturated phospholipids (Kusumi
et al, 1986; Merkle et al, 1987). The effect
of cholesterol on the motional freedom of
5-SASL (a phospholipid analogue) in phosphatidylcholine vesicles is dramatically reduced with unsaturation (inclusion of double bonds) of the fatty acyl chains. Furthermore, constants for lateral diffusion
(diffusion within the plane of the bilayer)
are reduced 4-fold by introduction of 30
mole % cholesterol in vesicles consisting of
saturated phospholipids, whereas only a
15% decrease in diffusion constants is observed with cholesterol addition to vesicles
of unsaturated composition (Kusumi et al,
1986).
Increased packing density of the hydrophobic tails of phospholipids in the presence of cholesterol has structural as well as
functional consequences. First, cholesterol
has a condensing effect on membranes and
as a result reduces the cross-sectional area
294
ELIZABETH L. CROCKETT
occupied by phospholipids in the bilayer
(Stockton and Smith, 1976; Ipsen et al,
1990). This, in turn, decreases membrane
permeability to ions and polar nonelectrolytes (Kroes and Ostwald, 1971; de Kruyff
et al., 1973). A 50% increase in cholesterol
content of guinea pig erythrocytes corresponds to a 2-fold decrease in permeability
of erythritol, a polar nonelectrolyte (Kroes
and Ostwald, 1971). Molecules small
enough to diffuse through the hydrophobic
barrier take advantage of defects in packing
to cross the otherwise impermeable membrane. Because cholesterol creates tighter
packing in the membrane, it reduces the
passage of permeants (Kroes and Ostwald,
1971). Incorporation of cholesterol into a
lipid bilayer also affects the dipole potential
at the membrane interface thus decreasing
the permeability for cationic particles (de
Gier, 1992). The ordering effect of cholesterol also increases membrane thickness
(Stockton and Smith, 1976), although the
nature of this depends on what phase the
membrane is in and the number of carbon
atoms in the acyl chains of phospholipids
(Mclntosh, 1978). For example, at 25°C,
addition of 30 mole % cholesterol to phosphatidylcholine vesicles (with one saturated
and one unsaturated acyl chain) increases
the thickness of the hydrophobic region
from 25.8 A to 29.9 A (Nezil and Bloom,
1992).
Cholesterol appears to be responsible for
the structural integrity and function of certain specialized regions of the plasma membrane. Caveolae (nonclathrin-coated invaginations in the plasma membrane) are particularly enriched with cholesterol. The
function and characteristic morphology of
caveolae both require cholesterol (Rothberg
et al., 1990; Chang et al., 1992). Cholesterol also promotes assemby of gap junctions in Novikoff cells and enhances communication via gap junctions between
smooth muscle cells (Meyer et al., 1990;
Zwijsen et al., 1992). Experiments using
cholesterol supplementation have shown
that rapid formation of gap junctions is correlated with increased levels of cellular cholesterol (Meyer et al., 1990).
In addition to the effects of cholesterol
on membrane order and related properties
(e.g., permeability, thickness), cholesterol
influences thermotropic phase transitions of
model membranes (liposomes). At sufficiently high cholesterol contents, the main phase
transition between gel and fluid (liquid
crystalline) phases is abolished in disaturated phosphatidylcholine
vesicles
(McMullen et al., 1993). At cholesterol
concentrations of less than 20 mole %, this
transition includes both sharp and broad
components as indicated by "high-sensitivity" differential scanning calorimetry
(DSC). Cholesterol affects the transition
temperature, enthalpy, and cooperativity of
the broad component (and is specific for
phospholipid acyl chain length). However,
only a slight effect from cholesterol is observed for the transition temperature and
cooperativity associated with the sharp
component (McMullen et al., 1993; McMullen and McElhaney, 1995). As has been
observed with membrane order, influences
of cholesterol on phase behavior/transitions
are more pronounced in saturated compared
with unsaturated membranes (Kariel et al.,
1991; Hernandez-Borrell and Keough,
1993). Cholesterol exerts little influence on
the gel to fluid phase transition in phophatidylcholine vesicles with two polyunsaturated fatty acids (Kariel et al., 1991).
Cholesterol also can modulate activity of
various integral proteins. The influence of
cholesterol on the behavior of membrane
proteins may arise via physical or direct
chemical means. The ordering effect of
cholesterol may limit the free volume in the
membrane required for conformational
changes. For example, elevated levels of
cholesterol reduce the rate of formation of
metarhodopsin II in phosphatidylcholine
vesicles (Mitchell et al., 1990). Keq for reactions involving metarhodopsin I and II is
directly related to fractional volume, a measured variable representing free volume
available for molecular mobility (Mitchell
et al., 1990). Fractional volume is also inversely correlated with cholesterol content
(Mitchell et al., 1990). Supraphysiological
levels of cholesterol reduce renal (Na+,K+)ATPase, again suggesting that cholesterol
ordering inhibits protein dynamics (Yeagle
et al., 1988). Cholesterol may also influence
activity of integral proteins via its contri-
CHOLESTEROL FUNCTION IN THERMAL ADAPTATION
bution to membrane thickness. Results of
numerous studies demonstrate a relationship between membrane thickness and activity of integral proteins (e.g., Johannsson
et al., 1981; In't Veld et al., 1991; Cornea
and Thomas, 1994).
A direct chemical interaction between
cholesterol and membrane proteins is suggested by results of several studies using
reconstituted transporters (e.g., Vetnuri and
Philipson, 1989; Shouffani and Kanner,
1990). Activity of several of these proteins
(sarcolemmal Na + -Ca 2+ exchanger and
Na+,K+-ATPase, but not sarcoplasmic reticular Ca2+-ATPase), and brain -y-aminobutyric acid transport require cholesterol; in
most cases cholesterol analogues, which
mimic physical properties of cholesterol, do
not restore activity (Vemuri and Philipson,
1989; Shouffani and Kanner, 1990). These
results 1) demonstrate that cholesterol is a
chemical requisite for certain transport proteins, and 2) provide a possible explanation
for variations in protein activities in the absence of any change in the physical properties of the membrane (i.e., membrane order and thickness, phase behavior).
POTENTIAL ROLES FOR CHOLESTEROL IN
THERMAL ADAPTATION
Two properties of biological membranes
that are particularly influenced by temperature are membrane order (increasing temperatures fluidize membranes; decreasing
temperatures order membranes) and membrane phase (Hazel and Williams, 1990).
Deviations from either an appropriate membrane fluidity and/or phase may compromise
cell viability. Two homeostatic paradigms
explain, at least in part, how ectothermic organisms cope with variable or extreme body
temperatures. Homeoviscous adaptation
(HVA) maintains membrane order (Sinensky, 1974) while homeophasic adaptation
(HPA) preserves the liquid crystalline phase
for membrane functionality (McElhaney,
1984; Hazel, 1995). Despite variation in
temperature regimes, HVA ensures a structurally sound membrane and also an optimal
physical environment for integral proteins.
HVA also should minimize changes in other
physical properties of membranes that like
fluidity are influenced by temperature; i.e.,
295
membrane permeability and membrane
thickness. HPA, on the other hand, prevents
membranes from undergoing a phase transition that would compromise their structural
integrity (e.g., Drobnis et al., 1993). HPA
also guarantees minimal disruption in membrane function by ensuring that the majority
of membrane lipids will occur in the liquid
crystalline phase.
Both of these adaptational responses may
be achieved by remodelling the molecular
constituents that make up the lipid matrix
of biological membranes (membrane restructuring) (Hazel and Williams, 1990). A
variety of taxononic groups (including representatives from several kingdoms) adjust
the compositions of their membranes to accommodate changes in temperature. This
restructuring of lipid compositions provides
the membrane properties necessary for
structural integrity and function in the face
of varying environmental temperatures.
Much attention has been given to the importance of phospholipid restructuring (e.g.,
Hazel and Williams, 1990). Less attention
has been given to cholesterol which may
also play significant roles in adaptation to
temperature.
Because cholesterol influences many of
the same physical properties of membranes
that temperature affects (i.e., membrane order, phase, permeability, thickness), cholesterol modulation is likely to counter temperature-related changes in the physical
properties of membranes. Adjustments in
cholesterol level could reverse, or at least
ameliorate, temperature-induced perturbations in the physical properties of membranes. Hyperfluidization and a reduction in
membrane thickness, brought on by an
acute rise in body temperature, could be
offset by increased levels of cholesterol.
Similarly, hypofluidity and an increase in
membrane thickness, triggered by a decline
in body temperature, could be countered by
a decrease in membrane cholesterol. In addition, poikilothermic organisms whose
plasma membranes are particularly enriched
with cholesterol are less likely to experience a phase transition during a sudden
change in body temperature than organisms
whose membranes have little cholesterol.
Modulating cholesterol levels could guar-
296
ELIZABETH L. CROCKETT
antee either some degree of HVA, HPA, or
else be a mechanism for maintainence of
membrane thickness.
Further support for the potential contribution of cholesterol to thermal adaptation
of plasma membranes is evinced from studies that demonstrate the contributions of
cholesterol to 1) thermal stabilities of integral proteins and 2) cell survival upon heat
stress. In general, membrane function is
more resilient to heat stress when lipid bilayers are fortified with cholesterol. Cholesterol has also been implicated in the thermal stability of various membrane proteins
(Artigues et al, 1989; Rotenberg and Zakim, 1991; Ortega et al, 1996). Loss of activity (thermal inactivation) and unfolding
(thermal denaturation) of Ca2+/Mg2+-ATPase in cardiac microsomes occurs at higher
temperatures when membranes are enriched
with cholesterol (Ortega et al, 1996). Heat
sensitivity among mammalian cell lines is
inversely correlated with cholesterol content normalized to membrane protein (Cress
and Grener, 1980). A similar relationship,
however, is not apparent when cholesterol
is normalized to phospholipid.
Cholesterol may be good choice for membrane restructuring during temperature adaptation because of its accessibility (Bloch,
1991). First, cholesterol modulation may be
rapid. Cholesterol can easily move from intracellular sites of biosynthesis or storage (as
cholesterol esters) to the plasma membrane
(Trotter and Voelker, 1994). In addition, cholesterol moves easily from one leaflet to another (Lange, 1992). Second, the use of cholesterol as a modulator may not require any
expenditure of ATP or reduced cofactors.
Circulating lipoproteins in carnivorous animals contain exogenous cholesterol. Third,
if necessary, cholesterol can be synthesized
de novo. Reliance on endogenous cholesterol could limit the role of cholesterol in thermal adaptation to selected animals because
not all animals appear capable of synthesizing cholesterol (Bloch, 1991).
CHOLESTEROL AS EFFECTOR IN THERMAL
ADAPTATION
Comparisons of ectotherms and
endotherms
As a rule, ectothermic animals have lower body temperatures than endotherms. If
cholesterol is involved in thermal adaptation, cholesterol levels in membranes from
homologous tissues should be different (and
consistently so) for warm- and cold-bodied
organisms. In most membranes that have
been surveyed, cholesterol contents are
higher in endothermic animals than in ectothermic animals adapted (or acclimated
to) to low body temperatures (Robertson
and Hazel, 1996). One interpretation of
these data is that cholesterol levels in the
membrane rise with body temperature to
stabilize membranes. At lower body temperatures, reduced levels of cholesterol
probably help to maintain a more fluid state
than would be possible with a membrane
more endowed with cholesterol. This explanation assumes some level of homeoviscous
adaptation (HVA) and is fully plausible given that HVA has been demonstrated in taxonomically diverse vertebrate animals {e.g.,
Behan-Martin et al, 1993).
Comparisons of temperature-acclimated/
acclimatized ectotherms
Cholesterol modulation in plasma membranes during temperature acclimation/acclimatization is variable. Every possible response to temperature has been observed in
the various studies that have been conducted. Cholesterol levels have been shown to
rise with temperature, stay the same, or decrease with an increase in acclimation temperature depending on the organism, tissue,
or plasma membrane domain (discussed below). If cholesterol modulation is involved
in HVA, then cholesterol contents should
increase with a rise in temperature. If cholesterol contents do not change with temperature, then HVA may be absent, or alternatively, homeoviscous efficacy is
achieved via restructuring of phospholipid
composition. Finally, an increase in cholesterol may be observed at low body temperatures. This may be accompanied by a lack
of HVA, and or an inverse compensation in
membrane order with respect to temperature. A homeoviscous response, in this case,
would have to arise from changes in the
composition of membrane constituents other than cholesterol and would have to be
large enough to offset both the effects of
CHOLESTEROL FUNCTION IN THERMAL ADAPTATION
temperature and adjustments in cholesterol
level.
Cholesterol levels increase with temperature.—Cholesterol levels rise with acclimation temperature in the plasma membranes of skeletal muscle from two species
of crabs (Fig. 3). In this study, changes in
cholesterol content were significant when
crabs were collected and acclimated to different temperatures in autumn; no significant differences were observed in spring.
Cholesterol levels in various tissues of rainbow trout (Oncorhynchus mykiss) rise with
acclimation temperature (Robertson and
Hazel, 1995). Molar ratios of cholesterolto-phospholipid are significantly higher in
plasma membranes from liver (31%
change), kidney (18% change), and gill (9%
change) in warm-acclimated (20°C) trout
than in cold-acclimated (5°C) animals.
No change in cholesterol content.—Cholesterol contents do not change with acclimation temperature in plasma membranes
from trout sperm when animals are fed either a corn or cod liver oil diet (Fig. 4).
Cholesterol levels in the plasma membranes
of teleost erythrocytes do not change
(Grove S0renson, 1990; Robertson and Hazel, 1995), increase (Grove S0renson,
1993), or decrease (Gabbianelli etal, 1996)
with growth temperature. Molar ratios of
cholesterol/phospholipid from lipid extracts
of skeletal muscle from crayfish do not
change with acclimation temperature (Cossins, 1976). Cholesterol levels also do not
vary with acclimation temperature in the
basolateral membranes (BLM) from intestinal epithelia of trout (Fig. 5, left side), although perfect homeoviscous adaptation
(explained by phospholipid restructuring) is
observed (Crockett and Hazel, 1995).
Cholesterol levels are elevated at low
temperatures.—Cholesterol levels are 1.3fold higher in brush border membranes
(BBM) from intestinal epithelia of cold-acclimated trout than warm-acclimated fish
(Fig. 5, right side). In addition, these apically-located regions of the plasma membrane have physical properties representing
inverse compensation with respect to temperature (i.e., at any given temperature
membranes from cold-acclimated animals
are more ordered than membranes from
297
warm-acclimated animals) (Crockett and
Hazel, 1995). Microsomal membranes from
the digestive gland of the Antarctic scallop
Adamussium colbecki have higher (1.3fold) cholesterol-to-phospholipid weight ratios than the temperate zone (Mediterranean) scallop Pecten jacobaeus (Viarengo
etal, 1994).
EXPLANATIONS FOR MEMBRANE-SPECIFIC
CHOLESTEROL RESPONSES
Several reasons may account for why individual membranes respond differently to
temperature both in the degree to which
cholesterol modulation occurs and/or the direction of cholesterol change. Two likely
explanations for the membrane-specific responses are discussed below.
Cholesterol has multiple effects on
membranes
A change in body temperature of an organism has immediate consequences for its
membranes. Some properties of the membrane are particularly important to preserve.
Membrane order, phase, thickness, and permeability properties may all be critical features, and some membrane restructuring
during temperature fluctuations is required
to maintain these. As pointed out earlier,
cholesterol is a convenient choice for remodelling the membrane. Because of the
multiple influences of cholesterol on physical/chemical/functional properties of membranes, any adjustment in cholesterol level
will, however, have more than a single effect. For example, at low body temperature
an animal may reduce its cholesterol content in the plasma membrane to prevent the
membrane from becoming overly rigid. If,
however, the activity of a particular membrane protein requires a direct chemical interaction with cholesterol, then depletion of
cholesterol could compromise some aspect
of membrane functionality.
Membranes are heterogenous
All membranes are not alike and may respond differently to varying levels of cholesterol. Differences among tissues and
membrane domains within an individual
cell may include chemical and physical
properties as well as differences in mem-
298
ELIZABETH L. CROCKETT
SKELETAL MUSCLE
0.75
o
Cancer
"o °
60
E,
Carcinus
-
!& 0.45 -
"5
Q.
O 0.30 -
«J 0.15 en
"5
O 0.00
FIG. 3. Cholesterol levels increase with acclimation temperature in plasma membranes from skeletal muscle of
crabs (Cancer pagurus and Carcinus maenas). Animals were collected in autumn and acclimated for 3 weeks
to either 5° or 22°C. Bars represent mean cholesterol/phospholipid molar ratios with error bars representing SEM
(n a 4). Significance (P < 0.05) is indicated by asterisk. Data are from Cuculescu et al. (1995).
brane function. One example of membrane
heterogeneity is the varying degrees of
membrane unsaturation (quantity of double
bonds within fatty acyl chains of polar lipids). Basolateral membranes (BLM) from
rainbow trout intestine have a higher degree
of membrane unsaturation (more double
bonds) than brush border membranes
(BBM) from intestine at either cold or
warm acclimation temperatures (Fig. 6).
Assuming the influence of cholesterol on
membrane physical properties is greatest in
more saturated membranes, as has been
shown for model membranes, variations in
cholesterol level in the BBM will have
more pronounced effects than similar
changes in cholesterol in the BLM. Cholesterol levels do not change with acclimation
temperature in the BLM (Fig. 5). For cholesterol to affect the physical properties of
membranes in this highly unsaturated membrane, cholesterol modulation would prob-
TROUT SPERM
0.60
corn oil
18°C
cod liver oil
§ 0.45 -|
•Q.
en
° 30 "
o
0.
"2 0.15 "o
O O.00
FIG. 4. Cholesterol levels do not change with acclimation temperature in plasma membranes from trout sperm
(Oncorhynchus mykiss). Animals were acclimated for nine months to either 8° or 18°C and fed an isoenergetic
diet of corn oil or cod liver oil. Bars represent mean cholesterol/phospholipid molar ratios with error bars
representing SEM (n a 6). No statistically significant difference in cholesterol levels is apparent. Data are from
Labbe et al. (1995).
CHOLESTEROL FUNCTION IN THERMAL ADAPTATION
299
TROUT INTESTINAL EPITHELIA
o 0.30 BLM
5°C
20°C
BBM
•5
al Polar L ipic
0.24 -
6
2
0.18 0.12 0.06 -
a)
0.00
O
FIG. 5. Cholesterol levels stay the same in basolateral membranes (BLM) but increase at low acclimation
temperatures in brush border membranes (BBM) from trout intestinal epithelia. Animals were acclimated for 4
weeks to either 5° or 20°C. Bars represent mean cholesterol/total polar lipid molar ratios with error bars representing SEM (n = 4). Total polar lipids (phospholipids and glycolipids) were used to normalize cholesterol
levels. Total polar lipids were used because of the likelihood that glycolipids in trout membranes represent a
significant membrane constituent as has been shown in plasma membranes of mammals (Simons and van Meer,
1988). Asterisk indicates significance (P < 0.05). Data are from Crockett and Hazel (1995).
ably have to be relatively large. Any major
deviation in cholesterol level could easily
have negative consequences for (Na+,K+)ATPase activity. Like the mammalian enzyme, teleost (Na+,K+)-ATPase activity is
particularly sensitive to cholesterol level
(Raynard and Cossins, 1991; Crockett and
Hazel, 1997).
A noteworthy result of the comparison
for brush border and basolateral membranes
is that in BBM unsaturation index (a measure of the average number of double bonds
per fatty acyl chain) is unaffected by acclimation temperature, while the BLM show
a typical response of an increase in membrane unsaturation at low temperature (Fig.
6). These results are in accord with those
obtained earlier by Lee and Cossins (1990)
whose work indicates that the greatest
changes in membrane unsaturation in temperature-acclimated carp is in BLM and not
in BBM. HVA is achieved in trout BLM by
adjustments in membrane unsaturation rather than modulating cholesterol (Crockett
and Hazel, 1995). On the other hand, in
BBM, the increase in cholesterol at low acclimation temperatures explains inverse
compensation in membrane order (Crockett
and Hazel, 1995). Membranes from cold-
acclimated animals are more ordered than
membranes from warm-acclimated animals.
Temperature-induced differences in the microenvironment which bathes the brush
border membranes {e.g., bile) may require
a more stable (ordered) membrane at low
body temperature.
Another feature that varies among membranes is their cholesterol contents. Some
membranes are naturally more fortified with
cholesterol than others (Fig. 2). Cholesterol
levels in trout tissues vary; plasma membranes from liver and intestine have lower
levels of cholesterol while plasma membranes from kidney and gill have relatively
high cholesterol levels. Modulating cholesterol contents in membranes highly enriched in cholesterol (e.g., kidney) has less
of an effect on membrane order than doing
so in membranes less endowed with cholesterol (e.g., intestine) (Crockett and Hazel, 1997). While these results could reflect
differences in acyl chain unsaturation between renal and intestinal BLM, other observations suggest that differences in cholesterol content may be the important factor.
In the same study renal BLM that have
been experimentally enriched with cholesterol are significantly more ordered than na-
300
ELIZABETH L. CROCKETT
TROUT INTESTINAL EPITHELIA
FIG. 6. Unsaturation indices (a measure of the average number of double bonds per fatty acyl chain) for
basolateral membranes (BLM) and brush border membranes (BBM) from intestinal epithelia of trout acclimated
to either low (5°C) or high (20°C) temperatures. Animals were acclimated for 4 weeks. Bars represent mean
unsaturation indices with error bars representing SEM (n = 4, except BBM at 20°C n = 3). Asterisk indicates
significance (P < 0.05). Data are from Crockett and Hazel (1995).
tive membranes when the membranes are
prepared from cold-acclimated fish. A similar comparison for BLM prepared from
warm-acclimated animals is not significantly different. Because a higher degree of unsaturation in membranes from cold-acclimated animals should diminish the effect of
cholesterol enrichment, the relevant distinguishing feature of renal membranes from
the two acclimation groups is cholesterol
content (cholesterol levels are higher in
warm-acclimated trout). Additional cholesterol, experimentally added, has a more significant impact on membrane order in the
membrane with native cholesterol contents
that are lower (BLM from the cold-acclimated group).
The observation that renal BLM with
varying cholesterol levels (either as a result
of acclimation temperature or experimental
cholesterol enrichment) have membrane orders with different thermal sensitivities is
related to effects of cholesterol restructuring in heterogenous membranes (Crockett
and Hazel, 1997). The order of membranes
with relatively high levels of cholesterol is
less affected by acute temperature change
than membranes with less cholesterol. This
result agrees with studies using model
membranes (liposomes) which show reduced temperature coefficients for segmen-
tal order parameter (Smol) in the presence of
50 mole % cholesterol compared with
membranes which lack cholesterol (Sankaram and Thompson, 1990).
How do these results relate to cholesterol
modulation (or lack thereof) in plasma
membranes from ectotherms? First, animals
or tissues whose membranes are naturally
more enriched with cholesterol may suffer
less perturbation of their physical properties
from a modest change in temperature than
those animals or tissues whose membranes
have lower endogenous cholesterol. Eurythermal animals, particularly those which
undergo temperature change on a regular
basis (e.g., diurnal cycles of temperature),
may benefit from having membranes with
higher cholesterol levels. Comparison of
cholesterol levels in a stenothermal crab
(Cancer pagurus) with a eurythermal crab
(Carcinus maenas) supports this prediction
(Fig. 3), although other factors such as average environmental temperature or phylogeny could also explain this difference. Second, because cholesterol has its greatest effect on membrane order in membranes with
relatively low levels of cholesterol, changes
in cholesterol level in tissues with already
high cholesterol contents (e.g., trout kidney
and gill), may have greater impact on some
other membrane property besides mem-
CHOLESTEROL FUNCTION IN THERMAL ADAPTATION
brane order. The increase in cholesterol
content in plasma membranes of gill from
warm-acclimated trout may reflect requirements for minimizing changes in the distance from physiological body temperatures
to phase transition temperatures (an aspect
of HPA discussed by Hazel, 1995). High
cholesterol levels in gill may be a major
contributor to the permeability properties of
this epithelium (Robertson and Hazel,
1995), and a change in cholesterol content
with temperature may reflect compensatory
adjustments to maintain permeabilities
within relatively narrow limits. This may be
particularly critical in a tissue, such as gill,
that represents a key interface between organism and environment. Finally, changes
in cholesterol content in plasma membranes
with already high levels could be related to
minimizing the effects of temperature on
membrane thickness.
I have attempted to provide the most
likely (but probably not the only) explanations for the membrane-specific response of
cholesterol to temperature variation in ectotherms. Interactions of cholesterol with
plasma membranes are complex, and the
membranes themselves are physically,
chemically, and functionally heterogenous.
These factors allow cholesterol to play a variety of roles in temperature adaptation of
plasma membranes from ectotherms. When
cholesterol levels rise with temperature,
cholesterol is likely to contribute to homeoviscous adaptation and possibly to conservation of membrane thickness. In membranes which experience no change in cholesterol level, activities of (Na+,K+)-ATPase
(and perhaps other membrane-associated
proteins that are influenced by direct chemical interaction with cholesterol) are less
likely to be affected. In these cases, levels
of protein activity may be conserved, if in
addition to cholesterostasis, membrane
physical properties are maintained by compositional restructuring among the polar lipids. An increase in cholesterol with reduced
temperatures may ensure the necessary stability in an otherwise perturbed membrane
microenvironment (intestinal lumen). Tissues with high native levels of cholesterol
are unlikely to experience phase transitions
or significant changes in membrane order
301
when body temperatures deviate. These
scenarios are all likely to be adaptive, but
further study is required to identify, more
precisely, the elements which drive the
membrane-specific cholesterol responses.
ACKNOWLEDGMENTS
I thank Jeffrey R. Hazel for advice and
insight on research that I conducted under
his mentorship. I also thank two anonymous reviewers and Gary C. Packard for
comments on the manuscript. Much of my
original research on cholesterol was supported by a Maytag Postodoctoral Fellowship from Arizona State University and by
NSF IBN 9205234. Ohio University has
provided support for this paper and my participation in the symposium.
REFERENCES
Artigues, A., M. T. Villar, A. M. Fernandez, J. A. Ferragut, and J. M. Gonzalez-Roz. 1989. Cholesterol
stabilizes the structure of the nicotinic acetylcholine receptor reconstituted in lipid vesicles. Biochim. Biophys. Acta 985:325-330.
Behan-Martin, M. K., G. R. Jones, K. Bowler, and A.
R. Cossins. 1993. A near perfect temperature adaptation of bilayer order in vertebrate brain membranes. Biochim. Biophys. Acta 1151:216-222.
Bloch, K. 1991. Cholesterol: Evolution of structure
and function. In D. E. Vance and J. Vance (eds.),
Biochemistry of lipids, lipoproteins and membranes, pp. 363-381. Elsevier, New York.
Bloom, M. and O. G. Mouritsen. 1988. The evolution
of membranes. Can. J. Chem. 66:706-712.
Bretscher, M. S. and S. Munro. 1993. Cholesterol and
the Golgi apparatus. Science 261:1280-1281.
Chang, M. C. J. and B. I. Roots. 1989. The lipid composition of mitochondrial outer and inner membranes from the brains of goldfish acclimated at 5
and 30°C. J. Therm. Biol. 14:191-194.
Chang, W-J., K. G. Rothberg, B. A. Kamen, and R. G.
W. Anderson. 1992. Lowering the cholesterol content of MA 104 cells inhibits receptor mediated
transport of folate. J. Cell Biol. 118:63-69.
Cornea, R. L. and D. D. Thomas. 1994. Effects of
membrane thickness on the molecular dynamics
and enzymatic activity of reconstituted Ca-ATPase. Biochemistry 33:2912-2920.
Cossins, A. R. 1976. Changes in muscle lipid composition and resistance adaptation to temperature
in the freshwater crayfish, Austropotamobius pallipes. Lipids 11:307-316.
Crockett, E. L. and J. R. Hazel. 1995. Cholesterol levels explain inverse compensation of membrane order in brush border but not homeoviscous adaptation in basolateral membranes from the intestinal
epithelia of rainbow trout. J. Exp. Biol. 198:11051113.
Crockett, E. L. and J. R. Hazel. 1997. Cholesterol af-
302
ELIZABETH L. CROCKETT
fects physical properties and (Na+,K+)-ATPase in
basolateral membranes of renal and intestinal epithelia from thermally acclimated rainbow trout. J.
Comp. Physiol B. 167:344-351.
Cuculescu, M., D. Hyde, and K. Bowler. 1995. Temperature acclimation of marine crabs: Changes in
plasma membrane fluidity and lipid composition.
J. Therm. Biol. 20:207-222.
Dahl, C. E., J. S. Dahl, and K. Bloch. 1980. Effect of
alkyl-substituted precursors of cholesterol on artificial and natural membranes and on the viability
of Mycoplasma capricolum. Biochemistry 19:
1462-1467.
de Gier, J. 1992. Permeability barriers formed by membrane lipids. Bioelect. Bioenerg. 27:1-10.
de Kruyff, B., W. J. de Greef, R. V. W. van Eyk, R.
A. Demel, and L. L. M. van Deenen. 1973. The
effect of different fatty acid and sterol compositions on the erythritol flux through the cell membrane of Acholeplasma laidlawii. Biochim. Biophys. Acta 298:479-499.
Downer, R. G. H. and V. L. Kallapur. 1981. Temperature-induced changes in lipid composition and
transition temperature of flight muscle mitochondria of Schistocerca gregaria. J. Therm. Biol. 6:
189-194.
Drobnis, E. Z., L. M. Crowe, T. Berger, T. J. Anchordoguy, J. W. Overstreet, and J. H. Crowe. 1993.
Cold shock damage is due to lipid phase transitions in cell membranes: A demonstration using
sperm as a model. J. Exp. Zool. 265:432-437.
Duax, W. L., Z. Wawrzak, J. F. Griffin, and C. Cheer.
1988. Sterol conformation and molecular properties. In P. L. Yeagle (ed.), Biology of cholesterol,
pp. 1-18. CRC Press, Boca Raton.
El Yandouzi, E. H. and C. Le Grimellec. 1992. Cholesterol heterogeneity in the plasma membranes of
epithelial cells. Biochemistry 31:547—551.
Franks, N. P. 1976. Structural analysis of hydrated egg
lecithin and cholesterol bilayers. I. X-ray diffraction. J. Mol. Biol. 100:345-358.
Gabbianelli, R., G. Falcioni, L. Mazzanti, E. Bertoli,
and G. Zolese. 1996. Seasonal variations of physical and biochemical membrane properties in trout
erythrocytes (Salmo irideus). Comp. Biochem.
Physiol. B 114:275-279.
Grove-S0renson, P. 1990. Phospholipids and fatty acid
esters from flounder (Platichthys flesus L.) erythrocyte plasma membrane and changes of the lipids
from the membrane as a result of long term temperature acclimation. Comp. Biochem. Physiol. B
96:571-576.
Grove-S0renson, P. 1993. Changes of the composition
of phospholipids, fatty acids and cholesterol from
the erythrocyte plasma membrane from flounders
(Platichthys flesus L.) which were acclimated to
high and low temperatures in aquaria. Comp. Biochem. Physiol. B 106:907-912.
Hazel, J. R. 1995. Thermal adaptation in biological
membranes: Is homeoviscous adaptation the explanation? A. Rev. Physiol. 57:19-42.
Hazel, J. R. and E. E. Williams. 1990. The role of
alterations in membrane lipid composition in enabling physiological adaptation of organisms to
their physical environment. Prog. Lipid Res. 29:
167-227.
Hernandez-Borrell, J. and K. M. W. Keough. 1993.
Heteroacid phosphatidylcholines with different
amounts of unsaturation respond differently to
cholesterol. Biochim. Biophys. Acta 1153:277282.
Huang, C-H. 1976. Roles of carbonyl oxygens at the
bilayer interface in phospholipid-sterol interation.
Nature 259:242-244.
In't Veld, G., A. J. M. Driessen, J. A. F. Op den Kamp,
and W. N. Konings. 1991. Hydrophobic membrane thickness and lipid-protein interactions of
the leucine transport system of Lactococcus lactis.
Biochim. Biophys. Acta 1065:203-212.
Ipsen, J. H., O. G. Mouritsen, and M. Bloom. 1990.
Relationships between lipid membrane area, hydrophobic thickness, and acyl-chain orientational
order. Biophys. J. 57:405-412.
Johannsson, A., C. A. Keightley, G. A. Smith, C. D.
Richards, T. R. Hesketh, and J. C. Metcalfe. 1981.
The effect of bilayer thickness and n-alkanes on
the activity of the (Ca2+ + Mg2+)-dependent ATPase of sarcoplasmic reticulum. J. Biol. Chem. 256:
1643-1650.
Kariel, N., E. Davidson, and K. M. W. Keough. 1991.
Cholesterol does not remove the gel-liquid crystalline phase transition of phosphatidylcholine
containing two polyenoic acyl chains. Biochim.
Biophys. Acta 1062:70-76.
Keller, G. A., M. C. Barton, D. J. Shapiro, S. J. Singer.
1985. 3-Hydroxy-3-methylglutaryl-coenzyme A
reductase is present in peroxisomes in normal rat
liver cells. Proc. Natl. Acad. Sci. U.S.A. 82:770774.
Kroes, J. and R. Ostwald, 1971. Erythrocyte membranes—effect of increased cholesterol content on
permeability. Biochim. Biophys. Acta 249:647650.
Kusumi, A., W. K. Subczynski, M. Pasenkiewicz-Gierula, J. S. Hyde, and H. Merkle. 1986. Spin-label
studies on phosphatidylcholine-cholesterol membranes: Effects of alkyl chain length and unsaturation in the fluid phase. Biochim. Biophys. Acta
854:307-317.
Labbe, C, G. Maisse, K. Miiller, A. Zachowski, S.
Kaushik, and M. Loir. 1995. Thermal acclimation
and dietary lipids alter the composition, but not
fluidity, of trout sperm plasma membrane. Lipids
30:23-33.
Lange, Y. 1992. Tracking cell cholesterol with cholesterol oxidase. J. Lipid Res. 33:315-321.
Lange, Y, M. H. Swaisgood, B. V. Ramos, and T. L.
Steck. 1989. Plasma membranes contain half the
phospholipid and 90% of the cholesterol and
sphingomyelin in cultured human fibroblasts. J.
Biol. Chem. 264:3786-3793.
Lee, J. A. C. and A. R. Cossins. 1990. Temperature
adaptation of biological membranes: Differential
homeoviscous responses in brush-border and basolateral membranes of carp intestinal mucosa.
Biochim. Biophys. Acta 1026:195-203.
Mattjus, P., R. Bittman, C. Vilcheze, and J. R Slotte.
1995. Lateral domain formation in cholesterol/
CHOLESTEROL FUNCTION IN THERMAL ADAPTATION
phospholipid monolayers as affected by the sterol
side chain conformation. Biochim. Biophys. Acta
1240:237-247.
McElhaney, R. N. 1984. The structure and function of
the Acholesplasma laidlawii plasma membrane.
Biochim. Biophys. Acta 779:1-42.
Mclntosh, T. J. 1978. The effect of cholesterol on the
structure of phosphatidylcholine bilayers. Biochim. Biophys. Acta 513:43-58.
McMullen, T. P. W. and R. N. McElhaney. 1995. New
aspects of the interaction of cholesterol with dipalmitoylphosphatidylcholine bilayers as revealed
by high-sensitivity differential scanning calorimetry. Biochim. Biophys. Acta 1234:90-98.
McMullen, T. P. W., R. N. A. H. Lewis, and R. N.
McElhaney. 1993. Differential scanning calorimetric study of the effect of cholesterol on the
thermotropic phase behavior of a homologous series of linear saturated phosphatidylcholines. Biochemistry 32:516-522.
Meddings, J. B. 1989. Lipid permeability of the intestinal microvillus membrane may be modulated by
membrane fluidity in the rat. Biochim. Biophys.
Acta 984:158-166.
Merkle, H., W. K. Subczynski, and A. Kusumi. 1987.
Dynamic fluorescence quenching studies on lipid
mobilities in phosphatidylcholine-cholesterol
membranes. Biochim. Biophys. Acta 897:238248.
Meyer, R., B. Malewicz, W. J. Baumann, and R. G.
Johnson. 1990. Increased gap junction assembly
between cultured cells upon cholesterol supplementation. J. Cell Sci. 96:231-238.
Mitchell, D. C, M. Straume, J. L. Miller, and B. J.
Litman. 1990. Modulation of metarhodopsin formation by cholesterol-induced ordering of bilayer
lipids. Biochemistry 29:9143-9149.
Nelson, G. J. 1967. Lipid composition of erythrocytes
in various mammalian species. Biochim. Biophys.
Acta 144:221-232.
Nezil, F. A. and M. Bloom. 1992. Combined influence
of cholesterol and synthetic amphiphilic peptides
upon bilayer thickness in model membranes. Biophys. J. 61:1176-1183.
Ortega, A., J. Santiago-Garcfa, J. Mas-Oliva, and J. R.
Lepock. 1996. Cholesterol increases the thermal
stability of the Ca2+/Mg2+-ATPase of cardiac microsomes. Biochim. Biophys. Acta 1283:45—50.
Parasassi, T, A. M. Giusti, M. Raimondi, G. Ravagnan, O. Sapora, and E. Gratton. 1995. Cholesterol
protects the phospholipid bilayer from oxidative
damage. Free Rad. Biol. Med. 19:511-516.
Raynard, R. S. and A. R. Cossins. 1991. Homeovisous
adaptation and thermal compensation of sodium
pump of trout erythrocytes. Am. J. Physiol. 260:
R916-R924.
Reinl, H., T. Brumm, and T. J. Bayerl. 1992. Changes
in the physical properties of the liquid-ordered
phase with temperature in binary mixtures of
DPPC with cholesterol. Biophys. J. 61:10251035.
Robertson, J. C. and J. R. Hazel. 1995. Cholesterol
content of trout plasma membranes varies with ac-
303
climation temperature. Am. J. Physiol. 269:
R1113-R1119.
Robertson, J. C. and J. R. Hazel. 1996. Membrane constraints to physiological function at different temperatures: Does cholesterol stabilize membranes at
elevated temperatures? In C. M. Woods and D. G.
McDonald (eds.), Society for experimental biology
seminar series 61: Global warming: Implications
for freshwater and marine fish, pp. 25—49. Cambridge University Press, Cambridge.
Rotenberg, M. and D. Zakim. 1991. Effects of cholesterol on the function and thermotropic properties
of pure UDP-glucuronosyltransferase. J. Biol.
Chem. 266:4159-4161.
Rothberg, K. G., Y-S. Ying, B. A. Kamen, and R. G.
W. Anderson. 1990. Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J.
Cell Biol. 111:2931-2938.
Sankaram, M. B. and T. E. Thompson. 1990. Modulation of phospholipid acyl chain order by cholesterol. A solid-state 2H nuclear magnetic resonance
study. Biochemistry 29:10676-10684.
Schroeder, E, J. R. Jefferson, A. B. Kier, J. Knittel, T.
J. Scallen, W. G. Wood, and I. Hapala. 1991.
Membrane cholesterol dynamics: Cholesterol domains and kinetic pools. Proc. Soc. Exp. Biol.
Med. 196:235-252.
Schroeder, E, J. K. Woodford, J. Kavecansky, W. G.
Wood, and C. Joiner. 1995. Cholesterol domains
in biological membranes. Mol. Membrane Biol.
12:113-119.
Shouffani, A. and B. I. Kanner. 1990. Cholesterol is
required for the reconsitution of the sodium-and
chloride-coupled, -y-aminobutyric acid transporter
from rat brain. J. Biol. Chem. 265:6002-6008.
Simons, K. and G. van Meer. 1988. Lipid sorting in
epithelial cells. Biochemistry 27:6197—6202.
Sinensky, M. 1974. Homeoviscous adaptation—a
homeostatic process that regulates viscosity of
membrane lipids in Escherichia coli. Proc. Natl.
Acad. Sci. U.S.A. 71:522-525.
Stillwell, W, T. Dallman, A. C. Dumaual, E T. Crump,
and L. J. Jenski. 1996. Cholesterol versus a-tocopherol: Effects on properties of bilayers made
from heteroacid phosphatidylcholines. Biochemistry 35:13353-13362.
Stockton, G. W. and I. C. P. Smith. 1976. A deuterium
nuclear magnetic resonance study of the condensing effects of cholesterol on egg phosphatidylcholine bilayer membranes. I. Perdeuterated fatty acid
probes. Chem. Phys. Lipids 17:251-263.
Suckling, K. E., H. A. F. Blair, G. S. Boyd, I. F. Craig,
and B. R. Malcolm. 1979. The importance of the
phospholipid bilayer and the length of the cholesterol molecule in membrane structure. Biochim.
Biophys. Acta 551:10-21.
Thompson, S. L., R. Burrows, R. J. Laub, S. K. Krisans. 1987. Cholesterol synthesis in rat liver peroxisomes. J. Biol. Chem. 262:17420-17425.
Thompson, S. L. and S. K. Krisans. 1990. Rat liver
peroxisomes catalyze the initial step in cholesterol
synthesis. J. Biol. Chem. 265:5731-5735.
Trotter, P. J. and D. R. Voelker. 1994. Lipid transport
304
ELIZABETH L. CROCKETT
processes in eukaryotic cells. Biochim. Biophys.
Acta 1213:241-262.
Vemuri, R. and K. D. Philipson. 1989. Influence of
sterols and phospholipids on sarcolemmal and sarcoplasmic reticular cations transporters. J. Biol.
Chem. 264:8680-8685.
Viarengo, A., R. Accomando, G. Roma, U. Benatti, G.
Damonte, and M. Orunesu. 1994. Differences in
lipid composition of cell membranes from Antarctic and Mediterranean scallops. Comp. Biochem.
Physiol. B 109:579-584.
Wodtke, E. 1978. Lipid adaptation in liver mitochondrial membranes of carp acclimated to different
environmental temperatures: Phospholipid composition, fatty acid pattern, and cholesterol content. Biochim. Biophys. Acta 529:280-291.
Worcester, D. L. and N. P. Franks. 1976. Structural
analysis of hydrated egg lecithin and cholesterol
bilayers. II. Neutron diffraction. J. Mol. Biol. 100:
359-378.
Yeagle, P. L. 1985. Cholesterol and the cell membrane.
Biochim. Biophys. Acta 822:267-287.
Yeagle, P. L. 1988. Cholesterol and the cell membrane.
In P. L. Yeagle (ed.), Biology of cholesterol, pp.
121-145. CRC Press, Boca Raton.
Yeagle, P. L., J. Young, and D. Rice. 1988. Effects of
cholesterol on (Na+,K+)-ATPase ATP hydrolyzing
activity in bovine kidney. Biochemistry 27:64496452.
Zwijsen, R. M. L., I. M. J. Oudenhoven, and L. H. J.
de Haan. 1992. Effects of cholesterol and oxysterols on gap junctional communication between
human smooth muscle cells. Eur. J. Pharm. 228:
115-120.
Corresponding Editor: Gary C. Packard