`Vol. 91, pp. 8234-8238, August 1994
`Biochemistry
`
`Structural order of membranes and composition of phospholipids in
`fish brain cells during thermal acclimatization
`(temperature adaptation/membrane fluidity/fatty adds)
`CSABA BUDA*, INDRANIL DEY*, NANDOR BALOGHt, LASZLO I. HORVATHt, KATALIN MADERSPACH*,
`MIKLOS JUHASZt, YOUNG K. YEO*§, AND TIBOR FARKAS*$
`*Institute of Biochemistry and tInstitute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary; and $Institute of
`Biochemistry, J6zsef Attila University, Szeged, Hungary
`
`Contributed by Tibor Farkas, May 6, 1994
`
`ABSTRACT
`A comparison of the structural orders of
`membranes of a mixed brain-cell population isolated from
`Cyprinus carpio L. acclimated to either summer (23-250C) or
`winter (50C) revealed a high degree of compensation (80%) for
`temperature, as assayed by electron spin resonance spectros-
`copy. The cells rapidly forget their thermal history and adjust
`the physical properties of the membranes when shifted to the
`other extreme of temperature either in vivo or in vitro. Phos-
`pholipids separated from both types of animals exhibit only
`around 10% compensation. Arachidonic and docosahexaenoic
`acids are the major polyunsaturated fatty acids in the brains,
`but the fatty acid composition of the brain total phospholipids
`does not vary with adaptation to temperature. Separation of
`phosphatidylcholines and phosphatidylethanolamines into mo-
`lecular species revealed a 2- to 3-fold accumulation of 18:1/
`22:6, 18:1/20:4, and 18:1/18:1 species in the latter; 18:0/22:6
`showed an opposite tendency. Molecular species composition of
`phosphatidylcholines did not vary with the temperature. The
`same trends of changes were seen with brains offreshwater fish
`from subtropical (Coda cada L.) or boreal (Acerina cernua)
`regions. It is concluded that the gross amount of docosa-
`hexaenoic acid (22:6) plays only a minor role in adjusting the
`membrane physical properties to temperature. Factors other
`than lipids might be involved in the adaptation processes. Due
`to their specific molecular architecture, molecules such as
`18:1/22:6, 18:1/20:4, or 18:1/18:1 phosphatidylethanolamine
`might prevent the contraction of membranes in the cold and
`may provide an environment for some other components
`involved in the temperature regulation ofphysical properties of
`nerve cell membranes.
`
`Most poikilotherms respond to thermal changes by adapting
`the physical properties of their membranes to the new
`situation to preserve the functional and structural integrity of
`these structures, a phenomenon that Sinensky (1) termed
`"homeoviscous adaptation." The homeoviscous efficacy,
`the extent to which the cells compensate for temperature
`changes, varies among the tissues and membranes (2, 3).
`Adjustment of the physicochemical properties of the mem-
`branes to the temperature is expected to be rapid and
`reversible to ensure proper functioning under fluctuating
`thermal conditions in fish. Wodtke and Cossins (4) have
`shown that the fluidity of the mitochondria in fish liver
`follows changes in the environmental temperature. It has also
`been demonstrated that the plasma membrane of carp eryth-
`rocyte rapidly adjusts to temperature under both in vivo and
`in vitro conditions (5, 6). The functions of neural tissue are
`highly dependent on membrane processes. Adaptation of the
`physical state of the synaptic vesicles in fish brain (2, 7), of
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`8234
`
`the synaptic vesicles, mitochondria, and myelin fractions of
`an air-breathing, subtropical fish, Channa punctatus (8), and
`of the synaptosomal and myelin fraction of carp brain (9) has
`been described. Changes in environmental temperature have
`been shown to cause an alteration in conduction in axons of
`goldfish (10) and in the velocity of conduction in the vagus
`nerve of carp (9). All the above experiments were carried out
`with fish exposed to the experimental temperature for a
`prolonged time, and since adaptation of the membrane phys-
`ical state to temperature takes place quite rapidly in fish
`erythrocytes (5, 6) and the liver endoplasmic reticulum (4), it
`seemed interesting to test whether this response was also
`present in the brain cells. The freshwater fish Cyprinus carpio
`was selected for this study because of its eurythermic nature
`and the abundance of data concerning its lipid composition
`and metabolism. For comparison, brains of freshwater fish
`evolutionarily adapted to the two temperature extremes were
`also investigated.
`
`MATERIAULS AND METHODS
`Experimental Design. Carp (Cyprinus carpio L.) of 1.0-1.5
`kg were obtained from a local fish farm. They were main-
`tained in well-aerated, recirculated, and thermostatted
`aquaria at 250C or 50C. Warm temperature-acclimated
`("warm" -acclimated) fish were collected in the summers of
`1992 and 1993 at water temperature of 25TC. Cold-acclimated
`fish were collected in the winter of 1992-1993 at water
`temperature of 50C. Brains were collected also from Acerina
`cernua captured in Vaasa, Finland, at a water temperature of
`5PC and from Catla catla captured in West Bengal, India, at
`a water temperature of 250C.
`For warm-acclimated carp, the temperature was shifted
`down from 250C to 5PC in steps of -0.50C/hr, whereas for
`cold-acclimated carp, the temperature was shifted up from
`5PC to 250C with 0.50C/hr steps. In the in vitro experiments,
`brains of both warm- and cold-acclimated fish were used. In
`every case, either in vivo or in vitro, at least five fish were
`involved.
`Preparation and Incubation of Isolated Brain Cell Suspen-
`sions. Isolated brain cell suspensions were prepared by a
`sieving method (11). After dissection of the entire brain, the
`meninges were removed and the brains were finely chopped
`in Hanks' balanced salts solution (Ca2+- and Mg2+-free)
`(HBSS) at 250C for warm-acclimated and temperature down-
`shifted fish, and at 5PC for cold-acclimated and tempera-
`
`Abbreviations: DPH-PA, 3-[p-(6-phenyl-1,3,5-hexatrienyl)phenyl]-
`propionic acid; 14-PGSL, 1-palmitoyl-2-[14-(4,4-dimethyl-N-oxyl)-
`stearoyl]-sn-glycero-3-phosphoglycerol.
`§Visiting scientist from Kyung Pook National University, Taegu,
`Korea.
`ITo whom reprint requests should be addressed at: Institute of
`Biochemistry, Biological Research Center, H-6701 Szeged, Hun-
`gary.
`
`000001
`
`
`
`Biochemistry: Buda et A
`
`ture-up shifted fish. Cell viability was shown by trypan blue
`exclusion to be 95%.
`Brain cell suspensions obtained from warm- and cold-
`acclimated fish were incubated at the alternative temperature
`for 80 min, subsequently restored to their acclimatization
`temperature, and then kept at that temperature for another 80
`min. During this time, fluorescence anisotropy measure-
`ments were carried out on 100-1.l aliquots of cell suspension
`at intervals of 10 min throughout the duration of incubation.
`Steady-State Fluorescence Anisotropy Measurements. Cells
`were exposed to the opposite (experimental) temperature
`extremes for various times and then were labeled with 5 1.d of
`1 mM fluorescent dye 3-[p-(6-phenyl-1,3,5-hexatrienyl)phe-
`nyl]propionic acid (DPH-PA) (Molecular Probes) in situ and
`steady-state fluorescence anisotropy measurements were
`carried out at the incubation temperature by using a com-
`puter-controlled thermostatable spectrophotofluorimeter
`(Hitachi MPF-2A) according to Dey and Farkas (5). It has
`been found that DPH-PA labels the outer leaflet of plasma
`membrane (5, 6, 12). To correct the fluorescence intensity
`and anisotropy for light scattering, measurements were also
`made on an unlabeled sample under the same conditions as
`for the labeled samples. These measurements were based on
`Kuhry et al. (13). The error of these determinations was
`<0.005 unit.
`To study the effect of temperature on membrane structural
`order in the spectrophotofluorimeter cell itself, a different
`approach was followed. Concentrated cell suspensions from
`warm- and cold-acclimated fish were labeled in HBSS at 25°C
`and 5°C, respectively. They were then kept at their respective
`temperature for 20 min to ensure the labeling of the cells and
`equilibration of the fluorescent dye in the membrane. Next,
`they were transferred into the precooled (5°C) or preheated
`(25°C) chamber of the spectrophotofluorimeter in a concen-
`tration to give 0.05 OD unit, and the changes in the anisotropy
`were recorded at intervals of 2 min until the anisotropy
`assumed a constant value.
`Electron Spin Resomance (ESR) Spectroscopy. A cell sus-
`pension containing 1 mg of phospholipid was mixed with 10
`,ug (1 mg/ml in ethanol) of 1-palmitoyl-2-[14-(4,4-dimethyl-
`N-oxyl)stearoyl]-sn-glycero-3-phosphoglycerol (14-PGSL)
`in HBSS at room temperature for 15 min. Samples were
`contained in 1-mm (i.d.) capillary tubing. Phospholipid (5
`,uM) in chloroform was mixed with 15 nmol of 14-PGSL in
`ethanol at room temperature for 5 min. For the labeling of
`phospholipids, the method ofDey et al. (6) was followed. The
`
`Proc. Natl. Acad. Sci. USA 91 (1994)
`
`8235
`
`samples were transferred into the capillary and accommo-
`dated in a standard quartz ESR tube. Spectra were recorded
`between 5°C and 25°C with a computerized ECS-106 (Bruker,
`Billerica, MA) ESR spectrometer equipped with air-flow
`temperature regulation. The rate of motion of the spin label
`14-PGSL was quantitated with the effective rotational cor-
`relation time (R) according to Kivelson (14). Effective order
`parameters were calculated by the formula of Jost et al. (15).
`Lipid Extraction and Analysis. Total lipids were extracted
`from the brains or the washed cells according to Folch et al.
`(16). Phospholipids from the total lipids were separated by
`silicic acid column chromatography, using chloroform to
`remove the neutral lipids and methanol to elute the polar
`lipids. Phospholipid subclasses were separated by one-
`dimensional TLC according to Fine and Sprecher (17). Phos-
`phorus content was determined according to Rouser et al.
`(18). Methyl esters were separated on 10%o FFAP on 80- to
`100-mesh Supelcoport (Supelco) in a 2-m column (2-mm i.d.).
`A Hitachi model 263-80 gas chromatograph connected to a
`Hitachi model 263-80 data processor was used. Peaks were
`identified by comparison with authentic standards (6).
`Molecular species composition was determined according
`to Takamura et al. (19). The dinitrobenzoyl derivatives of
`diacylglycerols, obtained by phospholipase C hydrolysis
`(from Bacillus cereus for phosphatidylethanolamines and
`from Clostridium perfringens for phosphatidylcholines) (Sig-
`ma) of the phospholipids were separated by HPLC (Waters
`model 440) on a Nucleosil C18 column [5-,um particle size; 4
`mm (i.d.) x 250 mm] using acetonitrile/2-propanol (80:20,
`vol/vol) of HPLC grade (Carlo Erba, Milan) isocratically as
`the mobile phase (flow rate, 1.0 ml/min) and monitoring the
`eluent at 254 nm. The peaks were recorded and calculated by
`using a data processor (Hitachi model 263-80). Peaks were
`identified by 1,2-diacylglycerol derivatives of authentic stan-
`dards (6) and their relative elution times (20).
`
`RESULTS
`The carp brain total phospholipids proved to be rather rich in
`docosahexaenoic acid. Ethanolamine phosphoglycerides
`were richer in docosahexaenoic acid than choline phospho-
`glycerides, but the difference between cold-acclimated and
`warm-acclimated animals was not significant (Table 1). The
`ratio of saturated to unsaturated fatty acids varied with the
`temperature, being lower in the cold-adapted fish (Table 1).
`High levels ofdocosahexaenoic acid have been reported from
`
`Fatty acid composition of total phospholipids, phosphatidylcholines (PC), and phosphatidylethanolamines in
`Table 1.
`carp brain in relation to acclimation temperature
`
`Total
`
`250C
`50C
`Fatty acid
`13.4 ± 4.2
`16:0
`13.8 ± 3.2
`8.5 ± 1.5
`7.9 ± 3.1
`16:1
`18:0
`12.5 ± 1.3
`7.5 ± 1.9
`18:1(n - 9)
`20.5 ± 3.1
`17.2 ± 1.4
`18:2(n - 6)
`1.1 ± 0.6
`0.6 ± 0.5
`18:3(n - 3)
`1.0 ± 0.6
`2.4 ± 0.2
`20:4(n - 6)
`10.6 ± 2.0
`8.6 ± 0.6
`20:5(n - 3)
`0.3 ± 0.1
`0.2 ± 0.1
`22:4(n - 6)
`0.4 ± 0.2
`0.5 ± 0.1
`3.3 ± 0.5
`24:1(n - 9)
`4.7 ± 1.2
`22:5(n - 3)
`0.8 ± 0.3
`0.3 ± 0.1
`22:6(n - 3)
`30.5 ± 0.7
`25.5 ± 5.2
`Others
`4.9
`3.0
`(sat/unsat)*
`(0.26)
`(0.35)
`n = 8 for 5YC fish and n = 6 for 250C fish.
`*Saturated/unsaturated ratio.
`
`wt % (mean ± SD)
`PC
`
`PE
`
`50C
`17.1 ± 6.9
`7.5 ± 2.7
`11.2 ± 6.5
`20.7 ± 5.4
`0.8 ± 0.3
`2.5 ± 0.5
`9.7 ± 0.5
`0.5 ± 0.3
`0.3 ± 0.2
`4.7 ± 2.6
`0.8 ± 0.6
`21.8 ± 5.4
`2.4
`(0.33)
`
`250C
`19.4 ± 7.7
`6.8 ± 3.0
`15.4 ± 12.0
`18.6 ± 8.1
`0.5 ± 0.5
`1.4 ± 0.3
`8.1 ± 6.5
`0.2 ± 0.1
`0.3 ± 0.2
`3.8 ± 2.1
`0.7 ± 0.5
`18.9 ± 7.1
`5.9
`(0.53)
`
`50C
`8.1 ± 3.6
`7.6 ± 4.6
`5.3 ± 1.3
`16.8 ± 5.4
`1.4 ± 1.3
`2.6 ± 0.4
`12.9 ± 1.8
`0.7 ± 0.6
`0.5 ± 0.3
`1.3 ± 0.5
`1.3 ± 0.5
`34.1 ± 4.0
`7.1
`(0.15)
`
`250C
`8.5 ± 4.2
`9.4 ± 0.8
`9.9 ± 5.5
`16.5 ± 1.0
`0.1 ± 0.1
`1.5 ± 0.1
`8.5 ± 0.8
`0.6 ± 0.6
`0.6 ± 0.4
`1.2 ± 0.2
`0.4 ± 0.2
`31.7 ± 2.0
`11.5
`(0.22)
`
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`8236
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`Biochemistry: Buda et A
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`Proc. Natl. Acad. Sci. USA 91 (1994)
`
`other fish neural elements (9, 21, 22), but, as in our case, an
`effect of environmental temperature on docosahexaenoic
`acid has not been observed. Chang and Roots (21, 22) found
`that a reduction in the ratio of saturated to unsaturated fatty
`acids in the mitochondria and microsomes accompanied cold
`adaptation in goldfish (Carassius auraus) brains, but carp
`nerve phospholipids (9) and garfish (Lepisosteus osseus)
`axon phospholipids (22) did not show a similar response.
`There were characteristic differences with acclimation
`temperature in molecular species composition between phos-
`phatidylcholines and phosphatidylethanolamines of carps.
`Similar differences were seen also with fish evolutionarily
`adapted to the temperature (Tables 2 and 3). The 18:0/22:6,
`18:0/20:4, and 16:0/18:1 species were predominant in the
`phosphatidylcholines, with additionally 16:0/22:6 in the phos-
`phatidylethanolamines. However, 16:0/18:1 was only a mi-
`nor component in the phosphatidylethanolamine fraction.
`The level of 18:0/22:6 tended to be higher in the phosphati-
`dylcholines and phosphatidylethanolamines in the brains of
`warm-acclimated fish than those in cold-acclimated fish. In
`agreement with findings in the present study, the level of
`18:0/22:6 has been found to be low in the brains ofmarine and
`freshwater fish adapted to low environmental temperature
`(20, 23). A characteristic difference between the two phos-
`pholipids is observed in the level of 18:1/22:6, which was
`significantly higher in the phosphatidylethanolamines than in
`the phosphatidylcholines in both cold- and warm-adapted
`animals. Moreover, cold-acclimated fish accumulated 2-3
`times more of this species, along with 18:1/20:4 and 18:1/
`18:1, than the warm-acclimated fish, and its level also varied
`slightly with an upshift of the temperature (from 14.1 ± 1.8
`to 10.7 ± 0.7 wt % in the case of 18:1/22:6). Elevated levels
`of 18:1/22:6 phosphatidylethanolamine in the livers of fresh-
`water and marine fish adapted to low temperature have
`already been reported (23-25). The rotational correlation
`time of 14-PGSL embedded in phospholipid vesicles pre-
`pared from brains of cold-acclimated carp was less than for
`Molecular species composition of brain
`Table 2.
`phosphatidylethanolamines in fish seasonally or
`evolutionarily adapted to contrasting temperatures
`% total
`
`At 250C
`At 50C
`Acyl
`C. catla
`C. carpio
`C. carpio
`A. cernua
`(n = 7)
`(n = 8)
`(n = 5)
`(n = 6)
`groups
`2.7 ± 2.8
`22:6/22:6
`2.6 ± 1.2
`0.5 ± 0.3
`1.3 ± 0.7
`0.4 ± 0.3
`20:5/20:5
`0.3 ± 0.2
`0.7 ± 0.5
`0.2 ± 0.1
`20:4/20:4
`2.1 ± 0.7
`2.0 ± 0.9
`0.8 ± 0.3
`6.0 ± 1.0
`0.2 ± 0.1
`18:1/20:5
`0.5 ± 0.2
`2.0 ± 0.1
`0.5 ± 0.2
`0.2 ± 0.1
`16:1/16:1
`0.8 ± 0.4
`0.3 ± 0.2
`Trace
`18:1/22:6
`5.1 + 2.4
`2.5 ± 1.5
`14.1 ± 1.8
`14.4 ± 1.6
`16:0/22:6
`13.8 ± 2.1
`12.9 ± 3.9
`26.1 ± 2.5
`27.3 ± 2.1
`2.1 ± 1.0
`18:1/20:4
`3.4 ± 1.2
`7.2 ± 2.5
`2.3 ± 1.5
`2.5 ± 0.5
`16:0/20:4
`2.3 ± 1.5
`1.2 ± 0.9
`1.6 ± 1.4
`0.9 ± 0.3
`18:0/20:5
`1.6 ± 0.5
`4.0 ± 1.8
`0.7 ± 0.2
`18:0/22:6
`24.8 ± 4.4
`32.4 ± 2.5
`39.1 + 8.6
`49.1 ± 5.5
`8.5 ± 2.0
`18:0/20:4
`9.8 ± 1.0
`4.8 ± 2.5
`4.3 ± 2.1
`3.7 ± 1.2
`18:1/18:1
`6.6 ± 1.4
`2.1 ± 1.4
`0.4 ± 0.1
`16:0/18:1
`2.2 ± 0.8
`4.0 ± 1.7
`5.5 + 0.4
`3.6 ± 0.3
`16:0/16:0
`0.6 ± 0.4
`0.3 ± 0.2
`0.2 ± 0.1
`0.4 ± 0.3
`0.4 ± 0.2
`18:0/22:4
`0.4 ± 0.2
`0.1 ± 0.08
`Trace
`0.3 ± 0.1
`2.7 ± 1.5
`18:0/18:1
`2.0 ± 1.0
`Trace
`1.0 ± 0.9
`4.0 ± 3.3
`16:0/18:0
`1.6 ± 1.3
`3.8 ± 2.5
`0.3 ± 0.2
`0.7 ± 0.4
`18:0/18:0
`0.4 ± 0.2
`0.3 ± 0.2
`Others
`8.9
`1.5
`4.8
`0.8
`Cyrinus carpio was collected in summer (25°C) and winter (5°C) in
`Hungary, Acerina cernua in Finland (Vaasa, 5°C), and Catla catla in
`India (25°C).
`
`Molecular species composition of brain
`Table 3.
`phosphatidylcholines in fish seasonally or evolutionarily
`adapted to contrasting temperatures
`% total
`
`At 250C
`C. carpio
`C. catla
`(n = 7)
`(n = 6)
`1.7 _ 0.7
`0.6 ± 0.4
`0.2 ± 0.1
`0.2 ± 0.1
`1.6 ± 0.4
`0.5 ± 0.1
`0.5 ± 0.2
`0.4 ± 0.1
`Trace
`0.1 ± 0.08
`1.3 ± 0.3
`3.0 ± 1.2
`8.4 ± 5.1
`27.3 ± 10.2
`2.6 + 2.0
`2.3 ± 2.1
`2.6 ± 0.7
`1.2 ± 0.3
`0.4 ± 0.3
`0.7 ± 0.2
`19.5 ± 3.1
`23.5 ± 15.0
`2.3 ± 2.0
`Trace
`13.5 + 2.4
`2.5 ± 1.5
`2.0 ± 1.2
`1.8 ± 1.4
`20.4 ± 14.5
`30.5 ± 18.7
`2.8 ± 1.7
`4.1 ± 2.1
`3.7 ± 2.7
`0.8 ± 0.5
`8.7 + 4.9
`9.4 ± 3.3
`1.6 ± 1.4
`Trace
`2.2
`3.0
`
`At 50C
`Acyl
`A. cernua
`C. carpio
`(n = 5)
`(n = 8)
`groups
`1.9 ± 0.9
`22:6/22:6
`1.9 ± 0.8
`0.2 ± 0.1
`20:5/20:5
`0.4 ± 0.1
`1.6 ± 0.4
`20:4/20:4
`1.8 ± 0.8
`0.3 ± 0.2
`18:1/20:5
`1.2 ± 0.2
`0.2 ± 0.1
`16:1/16:1
`0.8 ± 0.3
`18:1/22:6
`3.5 ± 1.3
`5.6 ± 0.6
`11.2 ± 5.1
`16:0/22:6
`24.4 ± 6.7
`1.7 ± 0.6
`18:1/20:4
`1.0 ± 0.5
`16:0/20:4
`3.2 + 0.9
`2.9 ± 0.7
`1.0 ± 0.2
`18:0/20:5
`1.8 ± 0.5
`18:0/22:6
`10.0 ± 3.1
`12.6 + 2.7
`18:0/18:2
`2.7 ± 2.0
`Trace
`18:0/20:4
`12.7 ± 8.5
`2.5 ± 1.1
`18:1/18:1
`3.4 ± 0.1
`1.6 ± 0.8
`16:0/18:1
`21.5 + 12.8
`24.6 ± 14.6
`16:0/16:0
`3.4 ± 1.1
`1.7 ± 1.2
`18:0/18:1
`3.5 ± 2.8
`0.8 ± 0.6
`16:0/18:0
`9.4 ± 3.2
`8.5 _ 4.7
`18:0/18:0
`1.4 ± 0.5
`0.2 ± 0.1
`Others
`11.3
`5.2
`See legend to Table 2.
`warm-acclimated carp (Fig. 1), indicating a slightly less
`ordered environment of the spin probes in the former. The
`homeoviscous efficacy, calculated according to Wodtke and
`Cossins (4), was estimated to be around 10%6. When cold- or
`warm-acclimated fish were shifted to the opposite extreme of
`the temperature in steps of 0.5°C/hr, neither the fatty acid
`composition (data not shown) nor the effective rotation
`correlation time (Fig. 1) of the isolated phospholipids was
`adapted to the new temperature. In contrast, vesicles from
`cold-acclimated temperature-upshifted animals become less
`ordered in the lower temperature regions and vice versa. In
`a separate set of experiments, the isolated brain cells were
`labeled with the spin probe to study the responses given by
`the intact membranes. To avoid thermal shock to the cells,
`both the preparation and the labeling procedure were done at
`the acclimation temperature; moreover, the ESR tempera-
`
`4.00
`
`3.40
`
`2.80 F
`
`2.20 F
`
`1.60 F
`
`1.00
`
`3
`
`13
`
`18
`
`23
`
`28
`
`33
`
`Temp., °C
`
`Temperature dependence of the rotational correlation
`FIG. 1.
`time (TR) of 14-PGSL embedded in phospholipid vesicles prepared
`from brains of cold (5°C) and warm (25°C) temperature-acclimated
`carps or shifted to the other temperature extreme (means ± SD of
`five experiments). *, Warm-acclimated; A, shift down; v, cold-
`acclimated; O, shift up.
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`Proc. Natl. Acad. Sci. USA 91 (1994)
`
`8237
`
`Inset). We interpret these results as a manifestation of an
`active adaptation of the physical properties of the intact brain
`cell membrane to the new temperature. Without this adap-
`tation, opposite results would have been obtained-i.e., a
`decrease in the anisotropy parameter in temperature-
`upshifted cells, and vice versa, as demonstrated in model
`experiments using phospholipid vesicles (data not shown).
`
`(A
`U
`
`I an
`4.UU
`
`3.40 -
`
`2.80
`
`2.20 -
`
`1.60
`
`I nn
`
`0
`
`.*'t %
`
`1%. -
`
`5
`
`10
`
`15
`20
`Temp., 0C
`
`25
`
`tational correlation
`Temperature dependence of the ro
`FIG. 2.
`brain cells of carps
`time (ri) of 14-PGSL embedded in membranes ol
`hifted to the other
`adapted to summer or winter temperature or s
`riments).*, Warm-
`temperature extreme (means ± SD of five expel
`), shift up.
`acclimated; A, shift down; *, cold-acclimated; c
`for theIdcold-
`ture scannings were run in the heating c)
`cl
`~cleforhe cld-
`:he warm-adapted
`adapted cells, and in the cooling cycle for t
`cells. This experimental approach, in shar
`p contrast to the
`ed an =80%,o com-
`isolated phospholipids (Fig. 1), demonstrate
`r for the temper-
`pensation of the membrane structural orde
`ature (Fig. 2). There were no significant s
`differences in the
`m-acclimated and
`effective rotation correlation times of warr
`Is and vice versa
`cold-acclimated temperature-upshifted cell
`(Fig. 2).
`ient of the mem-
`This experiment suggested that adjustm
`new temperature
`brane physical state of the brain cells to the
`)nfirm this, brain
`was a rapid and reversible process. To cc
`e cooled under in
`cells prepared from warm-adapted fish wer
`se of the fluores-
`vitro conditions to 10'C and the time cour
`the plasma mem-
`cence anisotropy of DPH-PA embedded in
`brane was followed, after the removal of ali
`iquots of the cells
`Atropy parameter
`from the incubation medium. The anise
`-re exposed to the
`started to decrease shortly after the cells we
`)nstant low value
`reduced temperature, and it assumed a cc
`11 population was
`after 15-20 min (Fig. 3). When the same ce
`ached the original
`rewarmed, the anisotropy increased and reE
`Its were obtained
`value within a few minutes. Similar resul
`fish brains were
`when cells from warm- or cold-adapted
`spectively, in the
`exposed to cold or warm temperature, ret
`rpical experiment
`cell of the spectrophotofluorimeter. In a ty
`dropped into the
`in which cells from cold-adapted fish were
`spectrophotofluorimeter cell set to 250C,
`the fluorescence
`anisotropy started to increase within a fen
`ov minutes (Fig. 3
`0.32r
`0.30 F
`
`T
`
`T
`
`DISCUSSION
`i U 1
`Fatty acid composition in mammalian brain cell is fairly
`1f--=-|stable: only long-term dietary effects can cause some changes
`in it (26-28), especially in the phosphatidylethanolamine
`fraction (27, 29). Long-chain polyunsaturated fatty acids
`31
`30
`present in high concentration in mammalian nerve cells
`originate in the liver and are transported to the brain by the
`circulatory system (30). The same is also undoubtedly true
`for fish, but the effect of the environmental temperature must
`also be taken into consideration. Ethanolamine phosphoglyc-
`erides gave the most sensitive response to changes of the
`environmental temperature. Despite the expected slowing
`down of metabolic processes with decreasing temperature, a
`turnover of molecular species composition of the phosphati-
`dylethanolamines took place in the brains of thermally ac-
`climated carp. The changes were characterized by accumu-
`lation of 1-monounsaturated/2-polyunsaturated and dioleoyl
`species with a fall in the environmental temperature (Table
`2). It seems conceivable that these changes are a direct effect
`of temperature on the lipid metabolism of carp brain and do
`not stem from the diet, since these fish stop feeding at or
`below 10TC. Previous studies demonstrated increases in the
`formation (31) and level (32) of long-chain polyunsaturated
`fatty acids in carp liver acclimated or exposed to cold, and it
`is tempting to speculate that a proportion of these fatty acids
`might have been transported to the brain during the accli-
`mation to reduced temperatures. Fish brain cells have been
`shown to take up and selectively incorporate unsaturated
`fatty acids from the incubation medium in vitro (33, 34). The
`level of 18:1/22:6 phosphatidylethanolamine in the brain cells
`of carp acclimated to reduced temperatures resembles that
`which has been found in the brains of boreal and subtropical
`fish species (Table 2) as well as in brains of cod and rainbow
`trout adapted to low (5-70C) temperatures (20, 23). It requires
`further investigations to decide whether the accumulation of
`this species in response to cooling is a result of a selective
`deacylation/reacylation reaction, an intensive desaturation
`of the existing 18:0/22:6 species, or its transfer from the liver.
`In another connection, it has been shown that carp liver cells
`preferentially esterify 18:1 at the sn-i position of phosphati-
`dylethanolamine (32) under in vitro conditions in the cold
`and, moreover, that high levels of this species characterize
`the phosphatidylethanolamines in the livers of marine and
`freshwater fish evolutionarily adapted to low temperatures
`(24).
`Parallel to changes in the acyl composition of the brain
`phosphatidylethanolamines, there was also a high level (70-
`80%) of compensation of membrane fluidity for temperature
`changes, as measured by spin label and fluorescence polar-
`ization techniques. Although the individual membranes were
`not separated in this study, based on the data obtained for the
`brains of rat (28) and Channa punctatus (8) and for the nerve
`of carp (9), we propose that the fatty acid composition of carp
`brain total phospholipids is close to that of the plasma
`membranes. Thus, it can be speculated that both labels
`(14-PGSL and DPH-PA) indicate the ordering state of these
`structures. However, it still remains to be elucidated whether
`the observed changes in acyl-group composition of phos-
`phatidylethanolamines are directly related to the observed
`high degree of compensation of membrane physical state for
`temperature change. Some physical properties of the pre-
`
`I
`
`0.23
`
`6.:
`°0.22
`
`0.27
`
`I-4
`
`0
`
`.if- 0.25
`
`0.22
`
`-
`
`Shift down
`>l
`10
`
`1
`
`0.201
`0
`
`0.21
`
`0
`
`T
`
`30
`
`10
`
`20
`
`Time, min
`T
`
`I
`
`20
`
`30
`
`40
`Time, min
`
`50
`
`80
`
`Iftup
`Shil
`lo> 20
`20
`10
`
`80
`80
`
`90
`90
`
`ro on fluorescence
`Effect of temperature shift in vitr
`FIG. 3.
`Lnes of brain cells
`anisotropy of DPH-PA embedded in membra
`ffive experiments).
`prepared from warm-adapted fish (means
`SD o
`PH-PA in brain cell
`(Inset) Change in fluorescence anisotropy of Di
`to exposure to the
`membranes of a cold-adapted carp in response
`other temperature extreme (250C).
`
`000004
`
`
`
`8238
`
`Biochemistry: Buda et al.
`
`Proc. Natl. Acad. Sci. USA 91 (1994)
`
`dominant phosphatidylcholine species (16:0/18:1, 16:0/22:6,
`and 18:0/22:6) are rather close (35, 36) and the same might be
`true also for the phosphatidylethanolamines. Indeed, the
`compensation was only about 10% when total phospholipids
`were assayed (Fig. 1).
`A principal difference between the extracted total phos-
`pholipids and the intact membranes is the presence of the
`proteins in the latter. One of the functional roles of 1-mono-
`unsaturated/2-polyunsaturated phosphatidylethanolamines
`might be the prevention of the contraction of the bilayer due
`to reduced thermal motion and thereby the maintenance of a
`higher degree of disorder in cold. We speculate that certain
`nonlipid membrane components, such as membrane pro-
`teins, are responsible for the observed high degree of com-
`pensation of membrane ordering state for the temperature.
`Due to their specific molecular architecture and their effects
`on packing properties of the bilayer these phosphatidyleth-
`anolamines might contribute to these proteins assuming the
`appropriate configuration in cold. Phosphatidylcholines such
`as 16:0/18:1 or 16:0/22:6 may not share this property of
`phosphatidylethanolamines and serve merely as a matrix of
`the membranes.
`
`We thank Dr. A. B. Das for collecting brains of Catla catla, Dr.
`T. Wiik for collecting brains ofAcerina cernua, and Dr. D. R. Tocher
`for help in the separation of brain cells. Thanks go also to the Tisza
`Fish Farm, Szeged, for supplying fish for the experiments and to D.
`Marsh, Gottingen, Germany, for 14-PGSL. This work has been
`supported by the Hungarian National Scientific Research Founda-
`tion under Contracts T5163 and 903/90.
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`000005