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Phospholipid composition of the lung and liver of the hibernating ground squirrel, Citellus lateralis
Comp. Biochem. Physiol. Vol. 68a, pp. 203 to 208
0305-0491/81/0201-0203102.00/0
© Pergamon Press Lid 1981. Printed in Great Britain
PHOSPHOLIPID COMPOSITION OF THE LUNG AND LIVER OF THE HIBERNATING GROUND SQUIRREL, CITELLUS LATERALIS R. C. ALOIA Department of Anesthesiology, Loma Linda University, School of Medicine, and Pettis Memorial Veterans Hospital, Anesthesiology Service, Loma Linda, CA 92357, U.S.A. (Received 9 June 1980) Phospholipid analysis of the lung and liver tissue of the ground squirrel, Citellus lateralis, reveals significant differences in molar values of phospholipid classes between active and hibernating animals. 2. Specifically, the mol% value of phosphatidylcholine from lung tissue increases significantly in the hibernating physiological state. 3. Also, in liver tissue, the mol% value of phosphatidylethanolamine increases while that of phosphatidylserine, phosphatidylinositol and diphosphatidytglycerol decreases significantlY in the hibernating physiological state. 4. No differences were found in the absolute quantity of phospholipid in either tissue from animals in the active and hibernating physiological state.
Abstract--1.
INTROD UCTION
MATERIALS AND METHODS
Mammals which are capable of hibernation are unique organisms since they can voluntarily reduce their metabolism and lower their body temperature to below 5°C (Johanson, 1967). The golden manteled ground squirrel, for example, can lower its body temperature to 1 or 2°C and maintain it at this level for several weeks at a time during the hibernating season (Aloia & Pengelley, 1979). The various organs, cellular organelles and cell membranes of the organism are apparently capable of continued activity at these low temperatures since the animals can spontaneously arouse from the depressed metabolism state and raise their body temperature to normal levels against thermal gradients of 30--35°C. This cyclic phenomenon of hibernation, i.e. lowering the body temperature to below 5°C, maintaining at this level for periods of 10 days of more and then spontaneously raising it again to approximately 37°C, continues for the 4-5 months of the winter, hibernating season. One of the questions which has intrigued biologists for many years concerns the molecular mechanisms of adaptation of the cell membranes (Aloia, 1980). In other words, are there alterations in the composition of the cell membrane lipids which are commensurate with hibernation? It is known that poikilothermic organisms and bacteria alter their membrane lipid composition when adapted (or grown) at low temperatures (Hazel & Prosser, 1974; Fox, 1975). Furthermore, if there are lipid alterations, are they required to permit the animal to hibernate (see Aloia, 1979b)? Recent studies have shown that alterations in phospholipids and fatty acids of brain and the phospholipids of kidney were found when these organs from hibernating mammals were analyzed (Aloia, 1979a; Aloia, 1978; Goldman, 1975). This communication reports alterations in the phospholipids class composition which occur in lung and liver of the ground squirrel, Citellus lateralis, during hibernation.
Animals Ground squirrels (Citellus lateralis) were purchased from Mrs Bertha Rogers, Shingletown, CA 96088, U.S.A. housed in individual cages and fed Purina lab chow and water ad lib. They were maintained at 23 ___I°C on 12:12 light:dark cycle until 30th October at which time a population of squirrels matched for weight, sex and age was transferred to cold boxes at 2 + I°C, also on a 12:12 L:D regimen. Within 2wk most squirrels had exhibited preliminary bouts of hibernation. Accurate records of the days of hibernation and arousal were maintained and by January, the majority of the squirrels had exhibited several consecutive bouts of hibernation greater than 8 days in duration. Hibernators (Tb = 2-4°C) were sacrificed by decollation on days 4-7 of an established cycle of hibernation (>/8 days) during the months of January-March. Active animals (Tb = 37 ___l°C) were similarly sacrificed (after receiving Nembutal 50mg/kg-IP) during the months of JuneAugust. Immediately prior to sacrifice body weight and rectal temperature were recorded. Also animals were sacrificed between 1100 and 1145 hr. The liver and lung were removed from the animal and frozen in liquid nitrogen within 2-3 min after sacrifice. All organs were stored at -70°C until extracted. Lipid extraction and chromatography The lungs from 34 active and 37 hibernating squirrels, and the liver from 20 active and 21 hibernating squirrels were analyzed for their phospholipid composition. Most of the organs were pooled in groups of 2-4 for analysis. Lipids were extracted with chloroform:methanol (C/M), 2:1 (20vol/g organ wt) followed by chloroform:methanol:28% ammonia, 16:4:1 (Rouser et al., 1967). An aliquot was weighed on a Perkin-Elmer Ad-2 auto-balance to determine the percentage of lipid in the column eluent and aliquots of approx 400/~g were spotted on 4 TLC plates. A fifth, totals plate, which was not chromatograpbed, contained 6 aliquots and was used to determine recoveries of the chromatographic runs. TLC plates consisted of 33% silica gel H with 2.5% magnesium acetate. Spotting was accomplished in a spotting chamber maintained at 27°C at
203
204
R.C. ALOIA tO •
°
AICIMIHacl HaO (3:4:1:1"0.5) Fig. I. Two-dimensional thin-layer chromatogram of lung phospholipids from the ground squirrel, Citellus lateralis in its active physiological condition. First dimension solvent: chloroform:methanol:28~ aqueous ammonia, 65:25:5. Second dimension solvent: acetone:chloroform:methanol:acetic acid: water, 3 :4:1 : 1:0.5. Symbols: PE--phosphatidylethanolamine; PC--phosphatidylcholine; SPH-sphingomyelin; PI--phosphatidylinositol; PS--phosphatidylserine; DPG--diphosphatidylglycerol; PG--phosphatidylglycerol; PA--phosphatidic acid; LBPA--lysobisphosphatidic acid; LDPG--lysodiphosphatidyl glycerol; LPC--lysophosphatidylcholine. 55~ relative humidity in a nitrogen atmosphere. After spotting, the 4 TLC plates were chromatographed using the solvent system of Rouser (Rouser et al., 1967), namely, chloroform:methanol:ammonia (28~; freshly prepared) 65:25:5, and, acetone:chloroform:methanol:acetic acid and water 3:4:1:1:0.5, in the first and second dimensions, respectively. Plates were then sprayed with sulfuric acid: formaldehyde, 97:3 and charred at 180°C for 45 min. Lipid spots were identified by comparison with known standard (Rouser et al., 1967), aspirated into thick-walled ignition tubes and digested with 70 perchloric acid for 75 min at 180°C. Color reagents were added (Ammonium molybdate and ascorbic acid) and the ignition tubes were heated at 105°C for 7 min. After centrifugation to pellet the silica, optical density of the supernatant was recorded with a Beckman Model 25 dualbeam spectrophotometer at 825 nm (see Aloia, 1978 and Rouser et al., 1970 for further detail). Using this system complete separation of all phospholipid classes was obtained (Fig. 1). RESULTS Figure 1 illustrates a typical 2D-TLC chromatogram of the lipids from the lung of a ground squirrel
in its active physiological state. It demonstrates that the chromatographic technique used in this study separates all of the phospholipid classes. A similar 2D-TLC chromatogram of liver lipids has been recently published (Fig. 1, Aloia & Pengelley, 1979). The levels of each phospholipid class analyzed are expressed as a molar percentage determined by a comparison of all chromatographed lipids with lipids from the "totals" plate which were not chromatographed. Table 1 shows the molto values of lung ljpids and Table 2 shows the m o l ~ values of liver lipids. Table 1 illustrates 16 representative groups of quadruplicate analyses (from a total 34) of lung tissue from active and hibernating squirrels. Comparison of the TPL values (total phospholipid) reveals that there is no significant difference in the quantity of phospholipid in lung tissue from active and hibernating animals. There are 3.71 + 0.38 and 3.23 + 0.52 m phosphorus/100 g wet wt found in lungs from active and hibernating animals, respectively. However, there is a significant difference in the molto values of the indi-
Table 1. Lung phospholipids 1 Organ pool
A
B
(n) 4
(2)
(2)
Sex
~
PE 5
17.28~o -I-0.61 54.48 -+0.16 0.77 -+0.07 12.86 +0.72 2.63 -+0.18
PC DPG SPI-t PI
(A) Active animalsz C D
E
F
G
H
(2)
(3)
(1)
(2)
(2)
(2)
~3
~'
~'
~&~
~S
~'
~
~
_+SD
19.70~ +0.16 50.32 -+0.12 0.85 +0.03 12.88 _+0.21 3.97 _+0.19
18.44~ _+0.21 52.76 -+0.68 0.66 -+0.06 12.92 _+0.16 2.70 _+0.05
18.99~o 18.08~o 18.06~o 20.06~o + 1.18 - + 0 . 3 7 +0.14 -+0.46 49.17 49.82 51.79 47.02 +0.31 +3.59 - + 0 . 9 4 +0.54 1.02 1.05 0.81 1.07 - + 0 . 0 8 +0.17 _+0.07 _+0.11 14.31 13.26 12.58 14.45 _+0.18 _+0.54 +0.37 _+0.54 3.01 1.75 2.84 3.36 _+0.10 _+0.18 _+0.04 _+0.11
19.72~o 18.53 -+0.44 + 1.00 46.66 50.806 +0.17 -+2.47 1.23 0.98 +0.11 _+0.21 14.71 13.00 +0.88 +0.95 2.88 3.00 +0.18 _+0.60
Phospholipids of lung and liver Table t
Organ pool
(n)" Sex
A (2)
(contd)
(A) Active animalsz C D (2) (3)
B (2)
205
E (1)
F (2)
G (2)
H (2) +SD
PS PG PA UNK s LPL 9
7.31 +-0.19 2.00 -+0.12 0.21 ___0.02 0.25 1.58
%Rec t°
8.10 _+0.17 1.40 -+0.03 0.26 -+0.05 0.50
8.07 _+0.08 2.43 _+0.21 0.25 -+0.04 0.39
8.04 _+0.58 2.36 _+0.42 0.32 _+0.16 0.92 _+0.46 1.37 2.01 2.51 1.52 2.11 2.33 2.06 _+0.47 101.03% 100.40% 99.02% 99.90% 10J.52% 102.89% 101.20_+ 1.21% 3.69 3.84 3.96 3.91 3.71 3.20 3.71 +0.38
2.01
100.30% 100.63~
TPL 11
3.42
3.87
Organ pool (n)4 Sex
A (2) ~
B (1) ~
PE s
UNK a
15.71% +0.62 57.70 +0.54 0.69 +0.07 10.81 +0.74 3.23 -/-0.07 6.66 __+0.06 2.12 -+0.29 0.41 _+0.05 0.71
LPL 9
1.77
PC DPG SPH PI PS PG PA
%Rec ~°
100.09%
8.61 +0.26 3.44 _+0.50 0.36 -+0.03 1.20
(B) Hibernating animals 2 C D E (1) (3) (4) ~ ~ & ~ ~
16.70% +0.68 56.26 + 1.08 0.75 +0.08 11.86 +0.10 2.36 +0.18 5.90 +_0.07 2.41 _+0.30 0.31 _+0.02 1.03 2.41
8.09 _+0.09 2.76 _+0.08 0.87 4-0.05 1.14
16.06% +0.43 57.13 +0.79 0.82 +0.13 10.00 +0.56 1.82 -I-0.40 6.86 _+0.12 2.94 _+0.16 0.60 _+0.09 0.94 2.84
14.96% +1.13 58.86 +0.84 0.74 +0.08 10.58 +0.17 2.79 _0.10 5.90 _+0.13 2.42 +0.88 1.14 + 0.08 0.76 2.59
20.319/o +0.10 49.48 +0.46 1.33 +0.03 11.90 +0.16 3.55 +0.17 8.20 +0.10 0.577 -+0.28 0.51 _+0.03 1.47 2.68
8.49 _+0.03 2.49 _+0.17 0.31 4-0.01 1.16
8.93 _+0.05 1.82 _+0.28 0.32 4- 0.04 0.79
8.83 +0.46 2.16 +0.17 0.31 + 0.03 1.17
F (2) ~
G (3) 5'
H (3) ~
20.53% +0.61 50.80 +0.18 0.60 +0.16 13.89 +0.09 2.52 +0.75 7.77 -+0.39 1.57 -+0.68 0.17 _+0.04 1.65 1.24
18.11% +0.18 53.98 +0.51 0.92 +0.12 11.51 +0.52 2.81 +0.06 8.17 -+0.09 1.77 _+0.34 0.30 _+0.06 0.35 2.03
18.12% +0.39 54.12 +0.66 1.08 +0.08 11.97 +0.21 2.78 -t-0.02 7.94 +_0.04 2.33 _+0.16 0.47 + 0.25 0.34 1.34
~3 -+ SD 17.61 +2.11 54.366 + 3.64 0.88 +0.23 11.45 + 1.24 2.80 +0.55 7.24 +0.85 1.99
+0.92 0.55 +0.52 1.10 +0.54 2.30 +0.49
99.60% 100.47% 100.27% 98.58~o 99.20% 99.00% 102.50% 100.60
_+1.5o% TPL 11
3.40
2.96
3.47
4.28
2.68
2.55
2.69
3.00
3.23 + 1.50
t All values are expressed as mol% of total lipid phosphorus: mean and standard deviation of quadruplicate determinations. 2 A total of 16 quadruplicate analyses of the lungs from 34 summer animals in their active physiological state (Tb = body temperature = 37°C) were analyzed individually or in groups of 2-4. A total of 18 quadruplicate analyses of the lungs from 37 winter animals in their hibernating physiological state (Tb = < 3°C) were analyzed individually or in groups of 2-4. The results of eight representative quadruplicate analyses are presented here. 3 Mean values __+standard deviation (',~ ___SD) of all quadruplicate analyses. 4 (n) = number of animal organs per pool. 5 See Fig. 1 for symbols. 6 Statistically significant difference between values from the active and hibernating groups of animals (P < 0.01). Determined by Student's t-test. TPG and PE are incompletely resolved; PG value is low. a UNK = unknown lipid spots which contained phosphorus, including the material which remained at the origin after chromatography. 9 LPL = lysophospholipids. This category includes the following lysoglycerophosphatides: lysobisphosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine and lysodiphosphatidyl glycerol. 10 % Rec = % recovery based on a comparison between the sum of phosphorus values in all chromatographic spots, and, the phosphorus value of the total's spots which were not chromatographed. 1~ TPL = total phospholipid: m mol of phosphorus/100 g wet wt. C.B.P. 68/2a--C
R. C. ALOIA
206
Table 2. Liver phospholipids 1 Organ pool (n) 4
Sex pE 5 PC DPG SPH PI PS PG PA UNK s LPL 9 ~oRec l°
A (3) & ~' 26.52~o _+0.40 48.26 ±0.69 4.28 _+0.18 3.51 ±0.30 9.02 _+0.12 3.12 +0.21 ND 0.28 _+0.09 2.02 2.68 99.61~o
(A) Active animals 2 C D (3) (4) 4 ,~
B (3) ~
F (3) "~
23.63~o ±0.00 51.19 _+1.61 3.99 -0.14 3.51 ±0.03 9.81 ±0.26 3.33 _+0.05 0.55 _+0.17 0.90 _+0.24 1.03
25.84~o _+ 1.46 50.78 _+1.50 4.28 _+0.16 3.73 _+0.17 9.08 +0.12 3.47 _+0.28 0.29 ±0.04 0.25 _+0.04 0.75
26.01~ _+0.18 50.23 _+0.86 4.33 _+0.09 2.63 _+0.45 9.23 _+0.33 3.76 _+0.04 0.50 _+0.12 0.44 ± 0.06 0.68
24.87~o _+0.14 49.42 _+1.11 4.06 _+0.25 3.47 _+0.06 9.46 _+0.12 3.44 _+0.27 0.37 _+0.08 0.44 _+0.20 2.51
26.81~o _+0.12 48.80 _+0.19 4.44 ±0.18 3.58 _+0.14 9.30 _+0.24 3.68 _+0.11 0.36 _+0.04 0.44 _+0.05 0.77
2.05
1.54
1.97
1.95
1.83
98.80~o 97.81~
100.67~
TPL l i
4.98
4.38
4.64
Organ pool (n)' Sex
A (3) 3` & 3`
B (3) 3'
(B) Hibernating C (2) ~
PE s
E (4) 3
4.66
4.65
animals 2 D E (3) (3) 3 ~
5.04
F (3) 3`
UNK s
29.62~o ±0.16 48.67 +0.34 3.61 +0.04 3.18 ±0.13 8.56 _+0.48 2.41 ±0.14 0.47 _+0.22 0.78 -+0.09 1.34
30.71~ ±0.46 48.22 +0.17 3.92 _+0.19 3.11 ±0.28 8.20 _+0.44 2.76 ±0.11 0.34 _+0.03 0.49 _+0.08 0.79
29.50~ + 1.45 49.09 +0.59 3.44 _+0.14 3.26 _+0.17 8.54 _+0.37 2.87 ±0.13 ND ND 0.65 _+0.03 1.11
29.32~ ± 1.26 48.46 +0.63 4.22 _+0.21 3.33 _+0.08 8.53 _+0.54 2.71 ±0.12 0.46 _+0.02 0.57 _+0.04 0.82
29.17~o +0.53 48.61 ±0.07 3.86 _+0.07 3.14 _+0.16 8.75 _+0.50 2.71 ±0.06 ND ND 0.66 _+0.09 1.45
LPL 9
1.90
1.36
1.48
1.54
1.57
1.65
DPG SPH PI PS PG PA
~oRec 1° TPL 11
98.32~o 102.14~o 102.44~o 97.85~ 4.86
4.14
4.69
4.76
25.617 ± 1.18 49.78 _+1.15 4.236 _+0.17 3.407 _+0.39 9.327 _+0.29 3.47 _+0.23 0.41 _+0.10 0.46 ± 0.23 1.29 _+0.78 2.00 _+0.38 99.71~o _+1.24~o 4.72 -+0.24
101.13~o 100.25~o
30.51~ + 1.24 47.89 + 1.06 4.04 +0.32 3.70 +0.00 8.42 _+0.18 2.48 +0.18 ND ND 0.29 -+0.08 0.40
PC
~3 (20)
101.80~o 99.02~ 4.76
4.86
G (4) ~
~3 (21)
29.09~o +0.35 46.76 +0.35 3.79 _+0.04 3.54 _+0.21 8.72 _+0.32 2.70 ±0.12 0.42 +0.18 0.62 ± 0.08 2.17
29.707 +0.65 48.24 ±0.75 3.846 _+0.26 3.32 "~ _+0.22 8.537 _+0.18 2.66 ±0.16 0.42 -+0.06 0.58 _+0.16 1.15 ±0.57 2.11 1.66 _+0.26 100.63~o 100.31~o _+ 1.90~o 4.71 4.68 +0.25
1 All values are expressed as molto of total lipid phosphorus: mean and standard deviation of quadruplicate determinations. 2 Active animal: Tb = 37°C; sacrificed in summer.. Hibernating animal: Tb _< 3°C; sacrificed in winter. 3 Mean values ± standard deviation (,X ± SD). 4n = number of animal organs/pool. 5 See Fig. 1 for symbols. 6 Statistically significant difference between values from active and hibernating groups of animals (P < 0.01). Determined by. Student's t-test. 7 Statistically significant difference between values from active and hibernating groups of animals (P < 0.001). Determined by Student's t-test.
Phospholipids of lung and liver
207
8 UNK = unknown lipid spots which contained phosphorus, including material which remained at the origin after chromatography. 9 LPL = lysophospholipids: includes, lysobisphosphatidic acid; lysophosphatidylcholine; lysodiphosphatidyl glycerol. 10 ~ Rec = ~ recovery based on a comparison between the sum of phosphorus values in all chromatographic spots, and, the phosphorus value of the total's spots which were not chromatographed. 11 TPL = total phospholipid: mmol of phosphorus/100 g wet wt.
vidual phospholipid classes. This is seen for phosphatidylcholine*, which increases from 50.80 + 2.47 m o l ~ in active animals to 54.36 4- 3.64mO1~o in hibernating animals. Table 2 illustrates all of the groups of quadruplicate analyses of liver tissue from active and hibernating animals. Examination of this table indicates that, similar to lung tissue, the quantity of phospholipid (mmol of phosphorus/100 g wet wt) in livers from animals in both active and hibernating states is essentially the same, being 4.72 4- 0.24 and 4.68 + 0.25, respectively (see TPL in Table 2). However, there are significant differences in the molar percent values of several individual phospholipid classes. In the hibernating state the membrane phospholipid composition of liver cells changes such that PE increases 169/o (25.61-29.709/o), while DPG, PI, and PS decrease by 9~o (4.23-3.84~o), 99/0 (9.32-8.53~o) and 239/0 (3.47-2.669/o), respectively. All of these changes in phospholipid class composition are significant. DISCUSSION The primary conclusion to be drawn from these results is that both the lung and liver tissue of the ground squirrel, Citellus lateralis undergo alterations in steady state levels of several phospholipid classes during hibernation. In the lung tissue there is a 7.1~ increase in the molar percent value of PC. In the liver tissue there is a 169/ooincrease in the level of PE and a 9, 23, and 9 ~ reduction in PI, PS, and DPG, respectively. There does not appear to be an alteration in the total quantity of membrane mass during the hibernating state, since in both tissues, the absolute quantity of phosphorus/100 g wet wt is similar in both active and hibernating physiological state. Comparison of the phospholipid class composition of the lung and liver of active and hibernating squirrels with the homologous organs in mammals which are incapable of hibernation reveals some interesting relationships. For example, the molar percent level of PE in rat, mouse, bovine, and human lung was found to be 21.2 4- 0.99/0 (Baxter et al., 1969), which is clearly higher than 17.6 4- 2.1 and 18.5 + 1~, found in hibernating and active ground squirrel lung (Table 1). However, the molar value for lung PE taken from squirrels which were warm-adapted at 35°C for 3 yr was 21.2 4- 0.9, exactly the same as the mean value for the four non-hibernating mammalian species (Aloia & Pengelley, 1979). Similarly for PC, even though the molar ~ values for the hibernating and active squirrels were significantly different from each other, being 54.4 4- 3.6~o and 50.8 4- 2.59/0, respectively, they were * PC = phosphatidylcholine; PE = phosphatidylethanol amine; PI = phosphatidylinositol;PS = phosphatidylserine; DPG = diphosphatidylglycerol.
clearly higher than the corresponding value of 43.9 + 3.3~, which represents the mean molar ~o value for rat, mouse, bovine, and human lung (Baxter et al., 1969). The molar 3/o value for lung PC from the warm-adapted squirrels (35°C/3 yr) is 47.9 + 0.5~, which is intermediate between the active and hibernating squirrel value, and, that of the non-hibernating mammalian species (Aloia & Pengelly, 1979). Thus, it appears that the molar ~ values of PE and PC from the lung of both active and hibernating animals are either above or below the range of corresponding values from non-hibernating mammals. On the other hand, the molar ~ values of phospholipid classes of non-hibernating mammals are similar to the corresponding values from hibernators in one physiological state but not the other. For example, the molar ~ value of PE from the liver of active squirrels is 25.61 ___ 1.18, which falls within the range of 22.1-27.99/o (24.14 + 1.5~o); the values derived from the mouse, rat, bovine, and human liver (Rouser et al., 1969). However, the molar ~ value of 29.7 + 0.65~ (range = 29.09-30.719/o) for PE from the liver of hibernating squirrels is clearly higher than the mean and range values of the liver of the non-hibernating species. The reverse situation seems to exist for liver PI values. The mean molar 9/o value of PI from the liver of the active and hibernating squirrels is 9.32 4- 0.29~o (range = 9.02-9.81~) and 8.53 + 0.18 (range = 8.2-8.75), respectively. It is clear that the corresponding value from the liver of non-hibernating species being 8.28 4- 0.429/0 (range = 7.3-8.9~) is similar only to the value of liver PI from hibernating animals. Interpretation of these alterations in steady state levels of phospholipid classes is difficult. They may or may not be required for the cell membranes of the squirrel to adapt to low temperatures, or, to maintain appropriate viscosity or fluidity (Sinensky, 1974). The increased quantity of PC found in the lung tissue of hibernating animals may be interpreted as an adaptive alteration, since, for a given fatty acid composition this species of phospholipid melts at approximately 25°C lower than the other major phospholipid in lung, namely PE (Chapman & Wallach, 1968). However, interpretations such as this may not be valid if the membranes of active squirrels are already adapted to function at reduced temperatures. This may indeed be the case since the cell membranes of other mammals (rat, rabbit) have been shown by differential scanning calorimetry to remain in a fluid condition to approximately 0°C (Hackenbrock et al., 1976; Martonosi, 1974; Blayzk & Steim, 1972). It is noteworthy that the alterations in phospholipid class composition which are observed in liver tissue during hibernation are confined to those phospholipids which are negatively charged at neatral pH (Schroeder, 1978), and are located predominately on
208
R.C. ALOIA
the inner monolayer of t h e m e m b r a n e (Marinetti, 1977; Bretscher, 1972). Since m e m b r a n e - b o u n d enzymes, such as N a + - K +-ATPase have been shown to be maximally activated by negatively charged phospholipids, e.g. PS (Kimelberg & Papahadjopoulos, 1974), it may be that the alterations in these phospholipid classes are required to sustain maximal activity under the altered environmental conditions of hibernation.
Acknowledgements--This investigation was supported in part by the National Science Foundation (BMS 75-07667), American Heart Association (75-711), Riverside County Chapter of the American Heart Association and the Department of Anesthesiology, Loma Linda University School of Medicine. The author specifically thanks Drs George Rouser, Gene Kritchevsky and Bernard J. Brandstater for their advice and assistance. Special thanks are also due to Mr Dan Morrison, and Mr Frank Awender for their dedicated technical assistance. REFERENCES
ALOIA R. C. (1979a) Membrane structure and function during hibernation. In Chemical Zoology. Vol. 11, pp. 49 75. Academic Press, New York. ALOIA R. C. (1979b) Brain lipid composition of the hibernating and active ground squirrel, Citellus lateralis, d. therm. Biol. 4, 223-231. ALOiA R. C. (1980) The role of membrane fatty acids in mammalian hibernation. Fedn Proc. Fedn Am. Socs exp. Biol. in press. ALOIA R. C. & PENGELLEV E. T. (1979) Lipids, Membranes and Hibernation. In Chemical Zoology. Vol. 11, pp. 148. Academic Press, New York. ALOIA R. C. (1978) Phospholipid composition of hibernating ground squirrel (Citellus lateralis) kidney and low temperature membrane function. Comp. Biochem. Physiol. 60B, 14-27. BAXTER C. F., ROUSER G. & SIMON G. (1969) Variations among vertebrates of lung phospholipid class composition. Lipids 4, 243-244. BLAZVK J. F. & STEtM J. (1972) Phase transitions in mammalian membranes. Biochim. biophys. Acta 266, 737-741. BRETSCHER M. (1972) Asymmetrical lipid bilayer structure for biological membranes. Nature, New Biol. 236, 11-13. CHAPMAN D. & WALLACH D. F. H. (1968) Recent physical
studies of phospholipids and natural membranes. In Biological Membranes, pp. 125-202. Academic Press, New York. Fox C. F. (1975) Phase transitions in model systems and membranes. In Biochemistry of Cell Walls and Membranes. Series I, Vol. 2, pp. 279-307. Butterworths, Baltimore, MD. GOLDMAN S. S. (1975) Cold resistance of the brain during hibernation III. Evidence of a lipid adaptation. Am. J. Physiol. 228 (3), 834-838. HACKENBROCK C. R., HOCHLI M. & CHAU R. M. (1976) Calorimetric and freeze fracture analysis of lipid phase transitions and lateral translational motion of intramembrane particles in mitochondrial membranes. Biochim. biophys. Acta 455, 466-484. HAZEL J. R. & PROSSERC. L. (1974) Molecular mechanisms of temperature compensation in poikilotherms. Physiol. Rev. 54, 620~77. KIMELSERG H. D. & PAPAHADJOPOULOSD. (1974) Effect of phospholipid acyl chain fluidity, phase transition and cholesterol on (Na + + K+)- stimulated adenosine triphosphatase. J. biol. Chem. 249, 1071-1080. JOHANSSON B. W. (1967) Heart and Circulation in hibernators. In Mammalian Hibernation III, pp. 200-219. Oliver & Boyd, London. MARINETTI G. (1977) Arrangement of phosphatidylserine and phosphatidylethanolamine in the erythrocyte membrane. Biochim. biophys. Acta 465, 198 209. MARTONOSl M. R. (1974) Thermal analysis of sarcoplasmic reticulum membranes. FEBS Lett. 47(2), 327 329. ROUSER G., KRITCHEVSKY G., SIAKOTOS A. N. & YAMAMOTO A. (1970) Lipid composition of the brain and its subcellular structures. In Neuropathology: Methods and Diagnosis, pp. 691-753. Little & Brown, Boston, MA. ROUSER G., SIMON G. & KRITCHEVSKY G. (1969) Species variation in phospholipid class distribution of organs: 1. kidney, liver and spleen. Lipids 4(6), 599-606. ROUSER G., KRITCHEVSKY G. & YAMAMOTO A. (1967) Column chromatography and associated procedures for separation and determination of phospholipids and glycolipids. In Chromatographic Analysis of Lipids. Vol. 1, pp. 99-162. Marcel Decker, New York. SINENSKY M. (1974) Homeoviscous adaptation a homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli. Proc. hath. Acad. Sci., U.S.A. 71(2), 522-525. SCHROEDER F. (1978) Isothermal regulation of membrane fluidity in murine fibroblasts with altered phospholipid polar head groups. Biochim. biophys. Aeta 511,356-376.
0305-0491/81/0201-0203102.00/0
© Pergamon Press Lid 1981. Printed in Great Britain
PHOSPHOLIPID COMPOSITION OF THE LUNG AND LIVER OF THE HIBERNATING GROUND SQUIRREL, CITELLUS LATERALIS R. C. ALOIA Department of Anesthesiology, Loma Linda University, School of Medicine, and Pettis Memorial Veterans Hospital, Anesthesiology Service, Loma Linda, CA 92357, U.S.A. (Received 9 June 1980) Phospholipid analysis of the lung and liver tissue of the ground squirrel, Citellus lateralis, reveals significant differences in molar values of phospholipid classes between active and hibernating animals. 2. Specifically, the mol% value of phosphatidylcholine from lung tissue increases significantly in the hibernating physiological state. 3. Also, in liver tissue, the mol% value of phosphatidylethanolamine increases while that of phosphatidylserine, phosphatidylinositol and diphosphatidytglycerol decreases significantlY in the hibernating physiological state. 4. No differences were found in the absolute quantity of phospholipid in either tissue from animals in the active and hibernating physiological state.
Abstract--1.
INTROD UCTION
MATERIALS AND METHODS
Mammals which are capable of hibernation are unique organisms since they can voluntarily reduce their metabolism and lower their body temperature to below 5°C (Johanson, 1967). The golden manteled ground squirrel, for example, can lower its body temperature to 1 or 2°C and maintain it at this level for several weeks at a time during the hibernating season (Aloia & Pengelley, 1979). The various organs, cellular organelles and cell membranes of the organism are apparently capable of continued activity at these low temperatures since the animals can spontaneously arouse from the depressed metabolism state and raise their body temperature to normal levels against thermal gradients of 30--35°C. This cyclic phenomenon of hibernation, i.e. lowering the body temperature to below 5°C, maintaining at this level for periods of 10 days of more and then spontaneously raising it again to approximately 37°C, continues for the 4-5 months of the winter, hibernating season. One of the questions which has intrigued biologists for many years concerns the molecular mechanisms of adaptation of the cell membranes (Aloia, 1980). In other words, are there alterations in the composition of the cell membrane lipids which are commensurate with hibernation? It is known that poikilothermic organisms and bacteria alter their membrane lipid composition when adapted (or grown) at low temperatures (Hazel & Prosser, 1974; Fox, 1975). Furthermore, if there are lipid alterations, are they required to permit the animal to hibernate (see Aloia, 1979b)? Recent studies have shown that alterations in phospholipids and fatty acids of brain and the phospholipids of kidney were found when these organs from hibernating mammals were analyzed (Aloia, 1979a; Aloia, 1978; Goldman, 1975). This communication reports alterations in the phospholipids class composition which occur in lung and liver of the ground squirrel, Citellus lateralis, during hibernation.
Animals Ground squirrels (Citellus lateralis) were purchased from Mrs Bertha Rogers, Shingletown, CA 96088, U.S.A. housed in individual cages and fed Purina lab chow and water ad lib. They were maintained at 23 ___I°C on 12:12 light:dark cycle until 30th October at which time a population of squirrels matched for weight, sex and age was transferred to cold boxes at 2 + I°C, also on a 12:12 L:D regimen. Within 2wk most squirrels had exhibited preliminary bouts of hibernation. Accurate records of the days of hibernation and arousal were maintained and by January, the majority of the squirrels had exhibited several consecutive bouts of hibernation greater than 8 days in duration. Hibernators (Tb = 2-4°C) were sacrificed by decollation on days 4-7 of an established cycle of hibernation (>/8 days) during the months of January-March. Active animals (Tb = 37 ___l°C) were similarly sacrificed (after receiving Nembutal 50mg/kg-IP) during the months of JuneAugust. Immediately prior to sacrifice body weight and rectal temperature were recorded. Also animals were sacrificed between 1100 and 1145 hr. The liver and lung were removed from the animal and frozen in liquid nitrogen within 2-3 min after sacrifice. All organs were stored at -70°C until extracted. Lipid extraction and chromatography The lungs from 34 active and 37 hibernating squirrels, and the liver from 20 active and 21 hibernating squirrels were analyzed for their phospholipid composition. Most of the organs were pooled in groups of 2-4 for analysis. Lipids were extracted with chloroform:methanol (C/M), 2:1 (20vol/g organ wt) followed by chloroform:methanol:28% ammonia, 16:4:1 (Rouser et al., 1967). An aliquot was weighed on a Perkin-Elmer Ad-2 auto-balance to determine the percentage of lipid in the column eluent and aliquots of approx 400/~g were spotted on 4 TLC plates. A fifth, totals plate, which was not chromatograpbed, contained 6 aliquots and was used to determine recoveries of the chromatographic runs. TLC plates consisted of 33% silica gel H with 2.5% magnesium acetate. Spotting was accomplished in a spotting chamber maintained at 27°C at
203
204
R.C. ALOIA tO •
°
AICIMIHacl HaO (3:4:1:1"0.5) Fig. I. Two-dimensional thin-layer chromatogram of lung phospholipids from the ground squirrel, Citellus lateralis in its active physiological condition. First dimension solvent: chloroform:methanol:28~ aqueous ammonia, 65:25:5. Second dimension solvent: acetone:chloroform:methanol:acetic acid: water, 3 :4:1 : 1:0.5. Symbols: PE--phosphatidylethanolamine; PC--phosphatidylcholine; SPH-sphingomyelin; PI--phosphatidylinositol; PS--phosphatidylserine; DPG--diphosphatidylglycerol; PG--phosphatidylglycerol; PA--phosphatidic acid; LBPA--lysobisphosphatidic acid; LDPG--lysodiphosphatidyl glycerol; LPC--lysophosphatidylcholine. 55~ relative humidity in a nitrogen atmosphere. After spotting, the 4 TLC plates were chromatographed using the solvent system of Rouser (Rouser et al., 1967), namely, chloroform:methanol:ammonia (28~; freshly prepared) 65:25:5, and, acetone:chloroform:methanol:acetic acid and water 3:4:1:1:0.5, in the first and second dimensions, respectively. Plates were then sprayed with sulfuric acid: formaldehyde, 97:3 and charred at 180°C for 45 min. Lipid spots were identified by comparison with known standard (Rouser et al., 1967), aspirated into thick-walled ignition tubes and digested with 70 perchloric acid for 75 min at 180°C. Color reagents were added (Ammonium molybdate and ascorbic acid) and the ignition tubes were heated at 105°C for 7 min. After centrifugation to pellet the silica, optical density of the supernatant was recorded with a Beckman Model 25 dualbeam spectrophotometer at 825 nm (see Aloia, 1978 and Rouser et al., 1970 for further detail). Using this system complete separation of all phospholipid classes was obtained (Fig. 1). RESULTS Figure 1 illustrates a typical 2D-TLC chromatogram of the lipids from the lung of a ground squirrel
in its active physiological state. It demonstrates that the chromatographic technique used in this study separates all of the phospholipid classes. A similar 2D-TLC chromatogram of liver lipids has been recently published (Fig. 1, Aloia & Pengelley, 1979). The levels of each phospholipid class analyzed are expressed as a molar percentage determined by a comparison of all chromatographed lipids with lipids from the "totals" plate which were not chromatographed. Table 1 shows the molto values of lung ljpids and Table 2 shows the m o l ~ values of liver lipids. Table 1 illustrates 16 representative groups of quadruplicate analyses (from a total 34) of lung tissue from active and hibernating squirrels. Comparison of the TPL values (total phospholipid) reveals that there is no significant difference in the quantity of phospholipid in lung tissue from active and hibernating animals. There are 3.71 + 0.38 and 3.23 + 0.52 m phosphorus/100 g wet wt found in lungs from active and hibernating animals, respectively. However, there is a significant difference in the molto values of the indi-
Table 1. Lung phospholipids 1 Organ pool
A
B
(n) 4
(2)
(2)
Sex
~
PE 5
17.28~o -I-0.61 54.48 -+0.16 0.77 -+0.07 12.86 +0.72 2.63 -+0.18
PC DPG SPI-t PI
(A) Active animalsz C D
E
F
G
H
(2)
(3)
(1)
(2)
(2)
(2)
~3
~'
~'
~&~
~S
~'
~
~
_+SD
19.70~ +0.16 50.32 -+0.12 0.85 +0.03 12.88 _+0.21 3.97 _+0.19
18.44~ _+0.21 52.76 -+0.68 0.66 -+0.06 12.92 _+0.16 2.70 _+0.05
18.99~o 18.08~o 18.06~o 20.06~o + 1.18 - + 0 . 3 7 +0.14 -+0.46 49.17 49.82 51.79 47.02 +0.31 +3.59 - + 0 . 9 4 +0.54 1.02 1.05 0.81 1.07 - + 0 . 0 8 +0.17 _+0.07 _+0.11 14.31 13.26 12.58 14.45 _+0.18 _+0.54 +0.37 _+0.54 3.01 1.75 2.84 3.36 _+0.10 _+0.18 _+0.04 _+0.11
19.72~o 18.53 -+0.44 + 1.00 46.66 50.806 +0.17 -+2.47 1.23 0.98 +0.11 _+0.21 14.71 13.00 +0.88 +0.95 2.88 3.00 +0.18 _+0.60
Phospholipids of lung and liver Table t
Organ pool
(n)" Sex
A (2)
(contd)
(A) Active animalsz C D (2) (3)
B (2)
205
E (1)
F (2)
G (2)
H (2) +SD
PS PG PA UNK s LPL 9
7.31 +-0.19 2.00 -+0.12 0.21 ___0.02 0.25 1.58
%Rec t°
8.10 _+0.17 1.40 -+0.03 0.26 -+0.05 0.50
8.07 _+0.08 2.43 _+0.21 0.25 -+0.04 0.39
8.04 _+0.58 2.36 _+0.42 0.32 _+0.16 0.92 _+0.46 1.37 2.01 2.51 1.52 2.11 2.33 2.06 _+0.47 101.03% 100.40% 99.02% 99.90% 10J.52% 102.89% 101.20_+ 1.21% 3.69 3.84 3.96 3.91 3.71 3.20 3.71 +0.38
2.01
100.30% 100.63~
TPL 11
3.42
3.87
Organ pool (n)4 Sex
A (2) ~
B (1) ~
PE s
UNK a
15.71% +0.62 57.70 +0.54 0.69 +0.07 10.81 +0.74 3.23 -/-0.07 6.66 __+0.06 2.12 -+0.29 0.41 _+0.05 0.71
LPL 9
1.77
PC DPG SPH PI PS PG PA
%Rec ~°
100.09%
8.61 +0.26 3.44 _+0.50 0.36 -+0.03 1.20
(B) Hibernating animals 2 C D E (1) (3) (4) ~ ~ & ~ ~
16.70% +0.68 56.26 + 1.08 0.75 +0.08 11.86 +0.10 2.36 +0.18 5.90 +_0.07 2.41 _+0.30 0.31 _+0.02 1.03 2.41
8.09 _+0.09 2.76 _+0.08 0.87 4-0.05 1.14
16.06% +0.43 57.13 +0.79 0.82 +0.13 10.00 +0.56 1.82 -I-0.40 6.86 _+0.12 2.94 _+0.16 0.60 _+0.09 0.94 2.84
14.96% +1.13 58.86 +0.84 0.74 +0.08 10.58 +0.17 2.79 _0.10 5.90 _+0.13 2.42 +0.88 1.14 + 0.08 0.76 2.59
20.319/o +0.10 49.48 +0.46 1.33 +0.03 11.90 +0.16 3.55 +0.17 8.20 +0.10 0.577 -+0.28 0.51 _+0.03 1.47 2.68
8.49 _+0.03 2.49 _+0.17 0.31 4-0.01 1.16
8.93 _+0.05 1.82 _+0.28 0.32 4- 0.04 0.79
8.83 +0.46 2.16 +0.17 0.31 + 0.03 1.17
F (2) ~
G (3) 5'
H (3) ~
20.53% +0.61 50.80 +0.18 0.60 +0.16 13.89 +0.09 2.52 +0.75 7.77 -+0.39 1.57 -+0.68 0.17 _+0.04 1.65 1.24
18.11% +0.18 53.98 +0.51 0.92 +0.12 11.51 +0.52 2.81 +0.06 8.17 -+0.09 1.77 _+0.34 0.30 _+0.06 0.35 2.03
18.12% +0.39 54.12 +0.66 1.08 +0.08 11.97 +0.21 2.78 -t-0.02 7.94 +_0.04 2.33 _+0.16 0.47 + 0.25 0.34 1.34
~3 -+ SD 17.61 +2.11 54.366 + 3.64 0.88 +0.23 11.45 + 1.24 2.80 +0.55 7.24 +0.85 1.99
+0.92 0.55 +0.52 1.10 +0.54 2.30 +0.49
99.60% 100.47% 100.27% 98.58~o 99.20% 99.00% 102.50% 100.60
_+1.5o% TPL 11
3.40
2.96
3.47
4.28
2.68
2.55
2.69
3.00
3.23 + 1.50
t All values are expressed as mol% of total lipid phosphorus: mean and standard deviation of quadruplicate determinations. 2 A total of 16 quadruplicate analyses of the lungs from 34 summer animals in their active physiological state (Tb = body temperature = 37°C) were analyzed individually or in groups of 2-4. A total of 18 quadruplicate analyses of the lungs from 37 winter animals in their hibernating physiological state (Tb = < 3°C) were analyzed individually or in groups of 2-4. The results of eight representative quadruplicate analyses are presented here. 3 Mean values __+standard deviation (',~ ___SD) of all quadruplicate analyses. 4 (n) = number of animal organs per pool. 5 See Fig. 1 for symbols. 6 Statistically significant difference between values from the active and hibernating groups of animals (P < 0.01). Determined by Student's t-test. TPG and PE are incompletely resolved; PG value is low. a UNK = unknown lipid spots which contained phosphorus, including the material which remained at the origin after chromatography. 9 LPL = lysophospholipids. This category includes the following lysoglycerophosphatides: lysobisphosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine and lysodiphosphatidyl glycerol. 10 % Rec = % recovery based on a comparison between the sum of phosphorus values in all chromatographic spots, and, the phosphorus value of the total's spots which were not chromatographed. 1~ TPL = total phospholipid: m mol of phosphorus/100 g wet wt. C.B.P. 68/2a--C
R. C. ALOIA
206
Table 2. Liver phospholipids 1 Organ pool (n) 4
Sex pE 5 PC DPG SPH PI PS PG PA UNK s LPL 9 ~oRec l°
A (3) & ~' 26.52~o _+0.40 48.26 ±0.69 4.28 _+0.18 3.51 ±0.30 9.02 _+0.12 3.12 +0.21 ND 0.28 _+0.09 2.02 2.68 99.61~o
(A) Active animals 2 C D (3) (4) 4 ,~
B (3) ~
F (3) "~
23.63~o ±0.00 51.19 _+1.61 3.99 -0.14 3.51 ±0.03 9.81 ±0.26 3.33 _+0.05 0.55 _+0.17 0.90 _+0.24 1.03
25.84~o _+ 1.46 50.78 _+1.50 4.28 _+0.16 3.73 _+0.17 9.08 +0.12 3.47 _+0.28 0.29 ±0.04 0.25 _+0.04 0.75
26.01~ _+0.18 50.23 _+0.86 4.33 _+0.09 2.63 _+0.45 9.23 _+0.33 3.76 _+0.04 0.50 _+0.12 0.44 ± 0.06 0.68
24.87~o _+0.14 49.42 _+1.11 4.06 _+0.25 3.47 _+0.06 9.46 _+0.12 3.44 _+0.27 0.37 _+0.08 0.44 _+0.20 2.51
26.81~o _+0.12 48.80 _+0.19 4.44 ±0.18 3.58 _+0.14 9.30 _+0.24 3.68 _+0.11 0.36 _+0.04 0.44 _+0.05 0.77
2.05
1.54
1.97
1.95
1.83
98.80~o 97.81~
100.67~
TPL l i
4.98
4.38
4.64
Organ pool (n)' Sex
A (3) 3` & 3`
B (3) 3'
(B) Hibernating C (2) ~
PE s
E (4) 3
4.66
4.65
animals 2 D E (3) (3) 3 ~
5.04
F (3) 3`
UNK s
29.62~o ±0.16 48.67 +0.34 3.61 +0.04 3.18 ±0.13 8.56 _+0.48 2.41 ±0.14 0.47 _+0.22 0.78 -+0.09 1.34
30.71~ ±0.46 48.22 +0.17 3.92 _+0.19 3.11 ±0.28 8.20 _+0.44 2.76 ±0.11 0.34 _+0.03 0.49 _+0.08 0.79
29.50~ + 1.45 49.09 +0.59 3.44 _+0.14 3.26 _+0.17 8.54 _+0.37 2.87 ±0.13 ND ND 0.65 _+0.03 1.11
29.32~ ± 1.26 48.46 +0.63 4.22 _+0.21 3.33 _+0.08 8.53 _+0.54 2.71 ±0.12 0.46 _+0.02 0.57 _+0.04 0.82
29.17~o +0.53 48.61 ±0.07 3.86 _+0.07 3.14 _+0.16 8.75 _+0.50 2.71 ±0.06 ND ND 0.66 _+0.09 1.45
LPL 9
1.90
1.36
1.48
1.54
1.57
1.65
DPG SPH PI PS PG PA
~oRec 1° TPL 11
98.32~o 102.14~o 102.44~o 97.85~ 4.86
4.14
4.69
4.76
25.617 ± 1.18 49.78 _+1.15 4.236 _+0.17 3.407 _+0.39 9.327 _+0.29 3.47 _+0.23 0.41 _+0.10 0.46 ± 0.23 1.29 _+0.78 2.00 _+0.38 99.71~o _+1.24~o 4.72 -+0.24
101.13~o 100.25~o
30.51~ + 1.24 47.89 + 1.06 4.04 +0.32 3.70 +0.00 8.42 _+0.18 2.48 +0.18 ND ND 0.29 -+0.08 0.40
PC
~3 (20)
101.80~o 99.02~ 4.76
4.86
G (4) ~
~3 (21)
29.09~o +0.35 46.76 +0.35 3.79 _+0.04 3.54 _+0.21 8.72 _+0.32 2.70 ±0.12 0.42 +0.18 0.62 ± 0.08 2.17
29.707 +0.65 48.24 ±0.75 3.846 _+0.26 3.32 "~ _+0.22 8.537 _+0.18 2.66 ±0.16 0.42 -+0.06 0.58 _+0.16 1.15 ±0.57 2.11 1.66 _+0.26 100.63~o 100.31~o _+ 1.90~o 4.71 4.68 +0.25
1 All values are expressed as molto of total lipid phosphorus: mean and standard deviation of quadruplicate determinations. 2 Active animal: Tb = 37°C; sacrificed in summer.. Hibernating animal: Tb _< 3°C; sacrificed in winter. 3 Mean values ± standard deviation (,X ± SD). 4n = number of animal organs/pool. 5 See Fig. 1 for symbols. 6 Statistically significant difference between values from active and hibernating groups of animals (P < 0.01). Determined by. Student's t-test. 7 Statistically significant difference between values from active and hibernating groups of animals (P < 0.001). Determined by Student's t-test.
Phospholipids of lung and liver
207
8 UNK = unknown lipid spots which contained phosphorus, including material which remained at the origin after chromatography. 9 LPL = lysophospholipids: includes, lysobisphosphatidic acid; lysophosphatidylcholine; lysodiphosphatidyl glycerol. 10 ~ Rec = ~ recovery based on a comparison between the sum of phosphorus values in all chromatographic spots, and, the phosphorus value of the total's spots which were not chromatographed. 11 TPL = total phospholipid: mmol of phosphorus/100 g wet wt.
vidual phospholipid classes. This is seen for phosphatidylcholine*, which increases from 50.80 + 2.47 m o l ~ in active animals to 54.36 4- 3.64mO1~o in hibernating animals. Table 2 illustrates all of the groups of quadruplicate analyses of liver tissue from active and hibernating animals. Examination of this table indicates that, similar to lung tissue, the quantity of phospholipid (mmol of phosphorus/100 g wet wt) in livers from animals in both active and hibernating states is essentially the same, being 4.72 4- 0.24 and 4.68 + 0.25, respectively (see TPL in Table 2). However, there are significant differences in the molar percent values of several individual phospholipid classes. In the hibernating state the membrane phospholipid composition of liver cells changes such that PE increases 169/o (25.61-29.709/o), while DPG, PI, and PS decrease by 9~o (4.23-3.84~o), 99/0 (9.32-8.53~o) and 239/0 (3.47-2.669/o), respectively. All of these changes in phospholipid class composition are significant. DISCUSSION The primary conclusion to be drawn from these results is that both the lung and liver tissue of the ground squirrel, Citellus lateralis undergo alterations in steady state levels of several phospholipid classes during hibernation. In the lung tissue there is a 7.1~ increase in the molar percent value of PC. In the liver tissue there is a 169/ooincrease in the level of PE and a 9, 23, and 9 ~ reduction in PI, PS, and DPG, respectively. There does not appear to be an alteration in the total quantity of membrane mass during the hibernating state, since in both tissues, the absolute quantity of phosphorus/100 g wet wt is similar in both active and hibernating physiological state. Comparison of the phospholipid class composition of the lung and liver of active and hibernating squirrels with the homologous organs in mammals which are incapable of hibernation reveals some interesting relationships. For example, the molar percent level of PE in rat, mouse, bovine, and human lung was found to be 21.2 4- 0.99/0 (Baxter et al., 1969), which is clearly higher than 17.6 4- 2.1 and 18.5 + 1~, found in hibernating and active ground squirrel lung (Table 1). However, the molar value for lung PE taken from squirrels which were warm-adapted at 35°C for 3 yr was 21.2 4- 0.9, exactly the same as the mean value for the four non-hibernating mammalian species (Aloia & Pengelley, 1979). Similarly for PC, even though the molar ~ values for the hibernating and active squirrels were significantly different from each other, being 54.4 4- 3.6~o and 50.8 4- 2.59/0, respectively, they were * PC = phosphatidylcholine; PE = phosphatidylethanol amine; PI = phosphatidylinositol;PS = phosphatidylserine; DPG = diphosphatidylglycerol.
clearly higher than the corresponding value of 43.9 + 3.3~, which represents the mean molar ~o value for rat, mouse, bovine, and human lung (Baxter et al., 1969). The molar 3/o value for lung PC from the warm-adapted squirrels (35°C/3 yr) is 47.9 + 0.5~, which is intermediate between the active and hibernating squirrel value, and, that of the non-hibernating mammalian species (Aloia & Pengelly, 1979). Thus, it appears that the molar ~ values of PE and PC from the lung of both active and hibernating animals are either above or below the range of corresponding values from non-hibernating mammals. On the other hand, the molar ~ values of phospholipid classes of non-hibernating mammals are similar to the corresponding values from hibernators in one physiological state but not the other. For example, the molar ~ value of PE from the liver of active squirrels is 25.61 ___ 1.18, which falls within the range of 22.1-27.99/o (24.14 + 1.5~o); the values derived from the mouse, rat, bovine, and human liver (Rouser et al., 1969). However, the molar ~ value of 29.7 + 0.65~ (range = 29.09-30.719/o) for PE from the liver of hibernating squirrels is clearly higher than the mean and range values of the liver of the non-hibernating species. The reverse situation seems to exist for liver PI values. The mean molar 9/o value of PI from the liver of the active and hibernating squirrels is 9.32 4- 0.29~o (range = 9.02-9.81~) and 8.53 + 0.18 (range = 8.2-8.75), respectively. It is clear that the corresponding value from the liver of non-hibernating species being 8.28 4- 0.429/0 (range = 7.3-8.9~) is similar only to the value of liver PI from hibernating animals. Interpretation of these alterations in steady state levels of phospholipid classes is difficult. They may or may not be required for the cell membranes of the squirrel to adapt to low temperatures, or, to maintain appropriate viscosity or fluidity (Sinensky, 1974). The increased quantity of PC found in the lung tissue of hibernating animals may be interpreted as an adaptive alteration, since, for a given fatty acid composition this species of phospholipid melts at approximately 25°C lower than the other major phospholipid in lung, namely PE (Chapman & Wallach, 1968). However, interpretations such as this may not be valid if the membranes of active squirrels are already adapted to function at reduced temperatures. This may indeed be the case since the cell membranes of other mammals (rat, rabbit) have been shown by differential scanning calorimetry to remain in a fluid condition to approximately 0°C (Hackenbrock et al., 1976; Martonosi, 1974; Blayzk & Steim, 1972). It is noteworthy that the alterations in phospholipid class composition which are observed in liver tissue during hibernation are confined to those phospholipids which are negatively charged at neatral pH (Schroeder, 1978), and are located predominately on
208
R.C. ALOIA
the inner monolayer of t h e m e m b r a n e (Marinetti, 1977; Bretscher, 1972). Since m e m b r a n e - b o u n d enzymes, such as N a + - K +-ATPase have been shown to be maximally activated by negatively charged phospholipids, e.g. PS (Kimelberg & Papahadjopoulos, 1974), it may be that the alterations in these phospholipid classes are required to sustain maximal activity under the altered environmental conditions of hibernation.
Acknowledgements--This investigation was supported in part by the National Science Foundation (BMS 75-07667), American Heart Association (75-711), Riverside County Chapter of the American Heart Association and the Department of Anesthesiology, Loma Linda University School of Medicine. The author specifically thanks Drs George Rouser, Gene Kritchevsky and Bernard J. Brandstater for their advice and assistance. Special thanks are also due to Mr Dan Morrison, and Mr Frank Awender for their dedicated technical assistance. REFERENCES
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