- Email: [email protected]
Age-related changes in rat liver phospholipid transfer activity
Experimental Gmmology, Vol. 25, pp. 55-60, Printed in the USA. All rights reserved.
AGE-RELATED
0531-5565190 $3.00 + .OO Copyright 0 1990 Pergamon Pressplc
1990
CHANGES IN RAT LIVER PHOSPHOLIPID TRANSFER ACTIVITY
TANIA T. MARKOVSKA,DIANA H. PETKOVA,A.B . MOMCHILOVA-PANKOVA and K. S. KOUMANOV Central Laboratoryof Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Abstract - The age-induced changes in the liver cytosol phospholipid transfer activity of male and female rats have been investigated. These changes were found to be closely related to the age-induced alterations in the two major microsomal phospholipids - phosphatidylcholine and phosphatidylethanolamine. Regression analysis indicated a linear correlation between the phospholipid transfer activity and the level of phosphatidyicholine (positive) and phosphatidylethanolamine (negative) in liver microsomes of both male and female rats. Key Words: aging, phospholipid
transfer activity, phosphatidylcholine,
phosphatidylethanolamine
INTRODUCTION LIPS TRANSFER proteins (LTP) have been reported in the cytosol of almost all investigated cells (Wirtz and Zilversmit, 1968; Possmayer, 1974; Butler and Thompson, 1975; Zborowski and Wojtczak, 1975; Dyatlovitskaya et al., 1978; Engle et aZ., 1978; Koumanov and Infante, 1986). These proteins perform transfer of lipids from their biosynthesis site, the endoplasmic reticulum, up to other subcellular membranes. Two phospholipid transfer proteins have been observed in rat liver cytosol. The first transfers specifically phosphatidylcholine (PC-TP) (Lumb et al., 1976; Poorthuis et al., 1980), and the second performs a nonspecific transport of almost all membrane phospholipids (nsPLTP) (Bloj and Zilversmit, 1977; Dyatlovitskaya et al., 1978; Crain and Zilversmit, 1980). The properties and the composition of these two LTP have been thoroughly investigated (Bloj and Zilversmit, 1977; Poorthuis et al., 1980). Although the physiological role of LTP is not entirely elucidated, their participation in maintenance of the membrane’s composition and structure is widely accepted (Dawson, 1973; Wirtz, 1974; Yaffe and Kennedy, 1983). It is well known that aging induces alterations in the membranes phospholipid composition (Grinna and Barber, 1972, 1976; Grinna, 1977b; Hegner et aE., 1979; Petkova et al., 1986), as well as in the activity of many membrane-bound enzymes (Grinna, 1977a; Nokubo, 1985; Pratz and Corman, 1985; Petkova et al., 1986, 1988). Aging was observed to lead to changes of the
Correspondence to: K.S. Koumanov. (Received 15 June 1988; Accepted 13 March 1989)
55
56
T.T. MARKOVSKA
ef a/
cholesterol/phospholipid ratio (CH/PL) in rat, mouse and monkey liver microsomes (Grinna, 1977b; Grinna and Barber, 1972, 1976; Howcroft and Martin, 1974; Maloney et al., 1986). Schmucker et al. (1984) demonstrated a decline in the fluidity of liver microsomes from male Fisher rats during aging. Armbrecht et al. (1982) however, reported some opposite findings. We recently demonstrated an increase of the CH/PL ratio and the structural order of the lipid bilayer in rat liver plasma membranes during aging (Petkova et al., 1986). The purpose of this article was to investigate the age-related alterations of liver cytosol lipid transfer activity and its eventual correlation with the level of the microsomal phospholipid fractions. MATERIALS
AND METHODS
Animals Male and female Wistar strain rats (purchased from the Department of Laboratory Animals, Biological Center, Bulgarian Academy of Sciences) were kept for 16 months in laboratory conditions (in a ventilated room at ambient temperature 22’ 2 2°C) and fed a standard laboratory diet. The animals were killed at the age of 3, 8, 11 and 16 months (15 rats per age group) and their livers were collected. Only rats with macroscopically normal livers were used. Isolation of rat liver mitochondria
and microsomes
Rat liver subcellular fractions were prepared from tissue homogenates by differential centrifugation in 0.2 M sucrose/ 1 mM EDTA/ 1OmM Tris-HCl, pH 7.4 (SET buffer). The tissue was disrupted by a teflon homogenizer. The crude homogenate was centrifuged at 1500 X g. The mitochondrial fraction was obtained by centrifugation of the supematant at 14 000 X g for 15 min at 4°C. The microsomal fraction was then recovered from the postmitochondrial supematant by centrifugation at 105 000 X g for 60 min at 4°C. Partial puri$cation
of rat liver phospholipid
transfer protein (Wirtz et al., 1972)
The postmicrosomal 105 000 X g supematant was adjusted to pH 5.1 using 3M HCI. After 1 h, the precipitate was sedimented by centrifugation for 15 min at 14 000 X g in a Janetzky K-24 (GDR) refrigerated centrifuge. The pH of the supematant was readjusted to pH 7.4 with 1M Tris-HCl and the supematant was used for phospholipid transfer protein activity determination. Preparation
of liposomes
Liposomes were prepared from I-palmitoyl-Z( 14C) oleoylphosphatidylcholine (Amersham), L-ol-phosphatidylcholine from bovine liver (Sigma) and a trace (less than 1%) of (3H)trioleoylglycero1 (Amersham) as unexchangeable marker. The lipids were dissolved in diethyl ether, and the solvent was evaporated under nitrogen. The lipids were suspended in SET buffer and sonicated in a UZDN (USSR) sonifier at 25 “C for 10 min. Assay of phospholipid Liposomes
transfer activio
(280 nmol phospholipids)
(Koumanov
and Infante,
were incubated
1986)
with rat liver mitochondria
(1400 nmol
AGING AND PHOSPHOLIPID TRANSFER ACTIVITY
57
3
11 8 16 Age in months FIG. 1. Age-related changes in liver cytosol phospholipid transfer activity (0) and level of PC (A) and PE (A) in microsomes of male rats. Each point is mean of 15 determinations k SD.
phospholipids) from an uniform source (3-month-old rats) and post pH 5.1 supernatant (150 kg protein) from liver cytosol obtained from rats of different ages. A final volume of 1.5 ml was reached using SET buffer. Incubations were performed for 20 min at 37 “C in a water bath with constant agitation. The reaction was stopped by chilling the mixture in an ice bath and sedimenting the mitochondria at 14 000 x g for 15 min. Aliquots of the liposome-containing supernatant were put into plastic vials containing a scintillation mixture for aqueous samples. The vials were stirred and the radioactivity counted in an automatic LKB-1215 Rackbeta II scintillation counter. The 14W3H ratio was used for calculation of the phospholipid transfer activity which was expressed as nmol phospholipids transferred/mm per mg protein.
Analysis Phospholipids were extracted (Folch ef al., 1957) from rat liver mitochondria and microsomes and were subsequently chromatographed (only microsomal lipids) on silica gel 60 thin-layer plates (Merck). Chloroform/methanol/isopropanol/O.25% KCYtriethylamine (30: 9:25:6:18, v/v) were used as developing solvents (Touchstone et al., 1980). The amounts of phospholipid phosphorus (Kahovkova and Odavic, 1969) and protein (Lowry et al., 1951) were determined. Statistical analysis was carried out using Student’s t test. Linear regression analysis has also been performed.
RESULTS The age-induced alterations in the activity of liver cytosol LTP and in the level of the two most abundant liver microsomal phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), are presented in Fig. 1. It is evident that the phospholipid transfer in male rats was enhanced up to 11 months and was decreased in 16-month-old animals. It is noteworthy that this activity was changed in parallel with the PC level in liver microsomal membranes. The
58
T.T. MARKOVSKA
3
0 Age
er al.
11 in months
lb
changes in liver cytosol phospholipid transfer activity (0) and level of PC (A) and PE (A) in microsomes of female rats. Each point is mean of 15 determinations t SD.
FIG.2. Age-related
elevating LTP activity corresponded to an increasing PC content up to 11 months of age. In 16-month-old rats, both the transfer activity and the PC level were decreased. Thus, a positive linear correlation was observed between these two parameters, with a correlation coefficient of Y = 0.725. The alterations of PE were just opposite. Up to 11 months of age, the PE level was decreased and was enhanced at about 16 months, reaching the value observed in 3-month-old rats. The negative correlation between the PE level and the LTP activity showed a coefficient r= - 0.780. Figure 2 shows the results obtained in analogical investigations using female rats. In this case, the alterations were almost similar to the ones reported for male rats; both the PC level and the LTP activity were gradually enhanced up to 16 months with an unsignificant diminution at the 1 lth month. The PE level was decreased up to 11 months, and after this age remained almost unchanged. In female rats, we found linear correlations between the LTP activity and the level of PC (r = 0.991) and PE (Y = -0.755). It should be noted that for all ages under investigation, both the content of PC and PE as well as the activity of LTP were higher in male compared to female rats.
DISCUSSION Our investigations on phospholipid transfer activity in liver cytosol from male and female rats showed that it undergoes significant alterations in the course of development and aging. During the first months of male rat development, the phospholipid transfer activity was significantly enhanced and was decreased after 11 months of age, almost reaching the level observed in 3-month-old animals. It should be emphasized that the alterations in this activity showed a positive correlation with the age-related changes of the PC content in liver microsomes. Our results showed that the microsomal level of PC was elevated up to 11 months and was decreased after this age. The de novo synthesis of PC is known to take place mainly in the endoplasmic reticulum (Bell and Coleman, 1980) and the newly synthesized PC molecules are being transferred to the other subcellular membranes which do not synthesize their own phospholipids. This transfer is
AGING AND PHOSPHOLIPID TRANSFER
ACTIVITY
59
performed essentially by LTP. As already mentioned, a highly specific PC-TP has been reported in rat liver cytosol (Lumb et al., 1976; Poorthuis et al., 1980). It is quite likely that the increase or decrease of the PC level in the endoplasmic reticulum might influence the activity of PC-TP. The elevated PC level in the endoplasmic reticulum might induce activation and/or increased synthesis of the PC-TP, and vice-versa; the decreased content of PC might reduce the activity or the quantity of LTP. For determination of the LTP activity in vitro, we used both mitochondria and liposomes from a common origin, that is, the donor and acceptor membranes for all investigations were identical. Thus, the observed age-related changes in the rate of intermembrane phospholipid transfer were due to alterations in the activity and/or the amount of LTP in liver cytosol, and not to changes in the phospholipid composition of the donor and acceptor membranes. Of course, the changes in vivo in the composition and physicochemical properties of the donor and acceptor membranes in the cell could also influence the phospholipid transfer. There are numerous data in literature devoted to this problem (Hellings et al., 1974; Wirtz et al., 1976; Kamp et al., 1977; Machida and Ohnishi, 1978). As for the negative correlation between the LTP activity and the PE level in microsomes, it might be due to the already reported fact that PE content higher than 20 mol% inhibits the LTP activity (Wirtz et al., 1976; Kamp et al., 1977). In this respect its diminution could induce just the opposite effect. Furthermore, together with the age-induced changes in the phospholipid transfer activity, we also observed some sex-related alterations. In cytosol from male rats, this activity was higher compared to female ones. In male rats, however, the PC level corresponding to the different ages was higher compared to females which might be one of the reasons for the sex-related differences in the lipid transfer activity. Of course, other mechanisms such as, for example, the influence of sex hormones, might be responsible for the reported age-related changes in the LTP activity and the level of PC and PE. Therefore, the elucidation of the age-induced alterations of LTP as well as the factors influencing these alterations would contribute to the better understanding of the complex processes of membrane aging. REFERENCES ARMBRECHT, H.J., BIRNBAUM, L.S., ZENSER, T.V., and DAVIS, B.B. Changes in hepatic microsomal membrane fluidity with age. Exp. Gerontol. 17, 41-48, 1982. BELL, R.M. and COLEMAN, R.A. Enzymes of glycerolipid synthesis in eukariotes. Ann. Rev. Biochem. 1980, 49-459, 1980. BLOJ, B. and ZILVERSMIT, D.B. Rat liver proteins capable of transfetring phosphatidylethanolamine. Purification and transfer activity for other phospholipids. J. Biol. Chem. 252, 1613-1619, 1977. BUTLER, M.M. and THOMPSON, W. Transfer of phosphatidylserine from liposomes or microsomes to mitochondria. Stimulation by a cell supematant factor. Biochim. Biophys. Acta 388, 52-57, 1975. GRAIN, R.C. and ZILVERSMIT, D.B. Two nonspecific phospholipid exchange proteins from beef liver. Biochemistry 19, 1433-1439, 1980. DAWSON, R.M.C. The exchange of phospholipids between membranes. Subcell. Biochem. 2, 69-89, 1973. DYATLOVITSKAYA, E.V., TIMOFEEVA, N.G., and BERGELSON, L.D. A universal lipid exchange protein from rat hepatoma. Eur. J. Biochem. 82, 463471, 1978. ENGLE, M.J., VAN GOLDE, L.M.G., and WIRTZ, K.W.A. Transfer of phospholipids between subcellular fractions of the lung. FEBS Z.&t.86, 277-281, 1978. FOLCH, J., LEE, M., and SLOANE-STANLEY, G.H. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497-507, 1957. GRINNA, L.S. Age-related alterations in membrane lipid and protein interactions: Arrhenius studies of microsomal
60
T T. MARKOVSKA
er al
glucose-6-phosphatase. Gerontology 23, 342-349, 1977a. GRINNA, L.S. Changes in cell membrane during aging. Gerontology 23, 452-464, 1977b. GRINNA, L.S. and BARBER, A.A. Age-related changes in membrane lipid content and enzyme activities. Biochim. Biophys. Acta 288, 347-353, 1972. GRINNA, L.S. and BARBER, A.A. Lipid changes in microsomal and mitochondrial membranes of rat liver during aging. Fed. Proc. 35, 1425, 1976. HAWCROFI, D.W. and MARTIN, P.A. Studies on age-related changes in the lipids of mouse liver microsomes. Mech. Ageing Dev. 3, 121-130, 1974. HEGNER, D., PLAIT, D., HECKERS, H., and SCHLOEDER, U. Age-dependent physicochemical and biochemical studies of human red cell membranes. Mech. Ageing Dev. 10, 117-130, 1979. HELLINGS, J.A., KAMP, H.H., WIRTZ, K.W.A., and VAN DEENEN, L.L.M. Transfer of phosphatidylcholine between liposomes. Eur. J. Biochem. 47, 601-605, 1974. KAHOVKOVA, J. and ODAVIC, R. A simple method for analysis of phospholipids separated on thin-layer chromatography. J. Chromatogr. 40, 90-95, 1969. KAMP, H.H., WIRTZ, K.W.A., BAER, P.R., et al. Specificity of the phosphatidylcholine exchange protein from bovine liver. Biochemistry 16, 1310-1316, 1977. KOUMANOV, K. and INFANTE, R. Phospholipid-transfer proteins in human liver and primary liver carcinoma. Biochim. Biophys. Acta 876, 526-532, 1986. LOWRY, R.H., ROSENBROUGH, N.J., FARR, A.L., and RANDALL, R.J. Protein measurement with the Folin phenol reagent. /. Biol. Chem. 193, 265-275, 1951. LIMB, R.H., KLOOSTERMAN, A.D., WIRTZ, K.W.A., and VAN DEENEN, L.L.M. Some properties of phospholipid exchange proteins from rat liver. Eur. J. Biochem. 69, 15-22, 1976. MACHIDA, K. and OHNISHI, S.I. A spin labeled study of phosphatidylcholine exchange protein. Regulation of the activity by phoshpatidylserine and calcium ion. Biochim. Biophys. Acta 507, 156-164, 1978. MALONEY, A.G., SCHMUCKER, D.L., VESSEY, D.S., and WANG, R.K. The effects of aging on the hepatic microsomal mixed-function oxidase system of male and female monkeys. Hepatology 6, 282-287, 1986. NOKUBO, M. Physical-chemical and biochemical differences in rat liver plasma membranes in aging F-344 rats. J. Gerontol. 40, 409-414, 1985. PETKOVA, D.H., MOMCHILOVA, A.B., and KOUMANOV, K.S. Age-related changes in rat liver plasma membrane phospholipase A2 activity. Exp. Gerontol. 21, 187-193, 1986. PETKOVA, D.H., MOMCHILOVA-PANKOVA, A.B., MARKOVSKA, T.T., and KOUMANOV, K.S. Age-related changes in rat liver plasma membrane sphingomyelinase activity. Exp. Gerontol. 23, 19-24, 1988. POORTHUIS, B.J.H.M., VAN DER KRIFT, T.P., TEERLINK, T., AKEROYD, R., HOSTETLER, K.Y., and WIRTZ, K.W.A. Phospholipid transfer activities in Morris hepatomas and the specific contribution of the phosphatidylcholine exchange protein. Biochim. Biophys. Actu 600, 376-386, 1980. POSSMAYER, F. Evidence for a specific phosphatidylinositol transferring protein in rat brain. Brain Res. 74, 167-174, 1974. PRATZ, J. and CORMAN, B. Age-related changes in enzyme activities, protein content and lipid composition of kidney brush-border membranes. Biochim. Biophys. Acta 814, 265-273, 1985. SCHMUCKER, D.L., VESSEY, D.A., WANG, R.K., JAMES, J.L., and MALONEY, A.G. Age-dependent alterations in the physicochemical properties of rat liver microsomes. Mech. Ageing Dev. 27, 207-217, 1984. TOUCHSTONE, J.C., CHEN, J.C., and BEAVER, K.M. Improved separation of phospholipids in thin-layer chromatography. Lipids 15, 61-62, 1980. WIRTZ, K.W.A. Transfer of phospholipids between membranes. Biochim. Biophys. Acta 344, 95-l 17, 1974. WIRTZ, K.W.A. and ZILVERSMIT, D.B. Exchange of phospholipids between liver mitochondria and microsomes in vitro. J. Biol. Chem. 243, 3596-3602, 1968. WIRTZ, K.W.A., KAMP, H.H., and VAN DEENEN, L.L.M. Isolation of a protein from beef liver which specifically stimulates the exchange of phosphatidylcholine. Biochim. Biophys. Acta 274, 696-617, 1972. WIRTZ, K.W.A., GEURTS VAN KESSEL, W.S.M., KAMP, H.H., and DEMEL, R.A. The protein mediated transfer of phosphatidylcholine between membranes. The effect of membrane lipid composition and ionic composition of the medium. Eur. J. Biochem. 61, 515-523, 1976. YAFFE, M.P. and KENNEDY, E.P. Intracellular phospholipid movement and the role of phospholipid transfer protein in animal cells. Biochemistry 22, 1497-1507, 1983. ZBOROWSKI, J. and WOJTCZAK, L. Net transfer of phosphatidylinositol from microsomes and mitochondria to liposomes catalyzed by exchange protein from rat liver. FEES I&t. 51, 317-320, 1975.
AGE-RELATED
0531-5565190 $3.00 + .OO Copyright 0 1990 Pergamon Pressplc
1990
CHANGES IN RAT LIVER PHOSPHOLIPID TRANSFER ACTIVITY
TANIA T. MARKOVSKA,DIANA H. PETKOVA,A.B . MOMCHILOVA-PANKOVA and K. S. KOUMANOV Central Laboratoryof Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Abstract - The age-induced changes in the liver cytosol phospholipid transfer activity of male and female rats have been investigated. These changes were found to be closely related to the age-induced alterations in the two major microsomal phospholipids - phosphatidylcholine and phosphatidylethanolamine. Regression analysis indicated a linear correlation between the phospholipid transfer activity and the level of phosphatidyicholine (positive) and phosphatidylethanolamine (negative) in liver microsomes of both male and female rats. Key Words: aging, phospholipid
transfer activity, phosphatidylcholine,
phosphatidylethanolamine
INTRODUCTION LIPS TRANSFER proteins (LTP) have been reported in the cytosol of almost all investigated cells (Wirtz and Zilversmit, 1968; Possmayer, 1974; Butler and Thompson, 1975; Zborowski and Wojtczak, 1975; Dyatlovitskaya et al., 1978; Engle et aZ., 1978; Koumanov and Infante, 1986). These proteins perform transfer of lipids from their biosynthesis site, the endoplasmic reticulum, up to other subcellular membranes. Two phospholipid transfer proteins have been observed in rat liver cytosol. The first transfers specifically phosphatidylcholine (PC-TP) (Lumb et al., 1976; Poorthuis et al., 1980), and the second performs a nonspecific transport of almost all membrane phospholipids (nsPLTP) (Bloj and Zilversmit, 1977; Dyatlovitskaya et al., 1978; Crain and Zilversmit, 1980). The properties and the composition of these two LTP have been thoroughly investigated (Bloj and Zilversmit, 1977; Poorthuis et al., 1980). Although the physiological role of LTP is not entirely elucidated, their participation in maintenance of the membrane’s composition and structure is widely accepted (Dawson, 1973; Wirtz, 1974; Yaffe and Kennedy, 1983). It is well known that aging induces alterations in the membranes phospholipid composition (Grinna and Barber, 1972, 1976; Grinna, 1977b; Hegner et aE., 1979; Petkova et al., 1986), as well as in the activity of many membrane-bound enzymes (Grinna, 1977a; Nokubo, 1985; Pratz and Corman, 1985; Petkova et al., 1986, 1988). Aging was observed to lead to changes of the
Correspondence to: K.S. Koumanov. (Received 15 June 1988; Accepted 13 March 1989)
55
56
T.T. MARKOVSKA
ef a/
cholesterol/phospholipid ratio (CH/PL) in rat, mouse and monkey liver microsomes (Grinna, 1977b; Grinna and Barber, 1972, 1976; Howcroft and Martin, 1974; Maloney et al., 1986). Schmucker et al. (1984) demonstrated a decline in the fluidity of liver microsomes from male Fisher rats during aging. Armbrecht et al. (1982) however, reported some opposite findings. We recently demonstrated an increase of the CH/PL ratio and the structural order of the lipid bilayer in rat liver plasma membranes during aging (Petkova et al., 1986). The purpose of this article was to investigate the age-related alterations of liver cytosol lipid transfer activity and its eventual correlation with the level of the microsomal phospholipid fractions. MATERIALS
AND METHODS
Animals Male and female Wistar strain rats (purchased from the Department of Laboratory Animals, Biological Center, Bulgarian Academy of Sciences) were kept for 16 months in laboratory conditions (in a ventilated room at ambient temperature 22’ 2 2°C) and fed a standard laboratory diet. The animals were killed at the age of 3, 8, 11 and 16 months (15 rats per age group) and their livers were collected. Only rats with macroscopically normal livers were used. Isolation of rat liver mitochondria
and microsomes
Rat liver subcellular fractions were prepared from tissue homogenates by differential centrifugation in 0.2 M sucrose/ 1 mM EDTA/ 1OmM Tris-HCl, pH 7.4 (SET buffer). The tissue was disrupted by a teflon homogenizer. The crude homogenate was centrifuged at 1500 X g. The mitochondrial fraction was obtained by centrifugation of the supematant at 14 000 X g for 15 min at 4°C. The microsomal fraction was then recovered from the postmitochondrial supematant by centrifugation at 105 000 X g for 60 min at 4°C. Partial puri$cation
of rat liver phospholipid
transfer protein (Wirtz et al., 1972)
The postmicrosomal 105 000 X g supematant was adjusted to pH 5.1 using 3M HCI. After 1 h, the precipitate was sedimented by centrifugation for 15 min at 14 000 X g in a Janetzky K-24 (GDR) refrigerated centrifuge. The pH of the supematant was readjusted to pH 7.4 with 1M Tris-HCl and the supematant was used for phospholipid transfer protein activity determination. Preparation
of liposomes
Liposomes were prepared from I-palmitoyl-Z( 14C) oleoylphosphatidylcholine (Amersham), L-ol-phosphatidylcholine from bovine liver (Sigma) and a trace (less than 1%) of (3H)trioleoylglycero1 (Amersham) as unexchangeable marker. The lipids were dissolved in diethyl ether, and the solvent was evaporated under nitrogen. The lipids were suspended in SET buffer and sonicated in a UZDN (USSR) sonifier at 25 “C for 10 min. Assay of phospholipid Liposomes
transfer activio
(280 nmol phospholipids)
(Koumanov
and Infante,
were incubated
1986)
with rat liver mitochondria
(1400 nmol
AGING AND PHOSPHOLIPID TRANSFER ACTIVITY
57
3
11 8 16 Age in months FIG. 1. Age-related changes in liver cytosol phospholipid transfer activity (0) and level of PC (A) and PE (A) in microsomes of male rats. Each point is mean of 15 determinations k SD.
phospholipids) from an uniform source (3-month-old rats) and post pH 5.1 supernatant (150 kg protein) from liver cytosol obtained from rats of different ages. A final volume of 1.5 ml was reached using SET buffer. Incubations were performed for 20 min at 37 “C in a water bath with constant agitation. The reaction was stopped by chilling the mixture in an ice bath and sedimenting the mitochondria at 14 000 x g for 15 min. Aliquots of the liposome-containing supernatant were put into plastic vials containing a scintillation mixture for aqueous samples. The vials were stirred and the radioactivity counted in an automatic LKB-1215 Rackbeta II scintillation counter. The 14W3H ratio was used for calculation of the phospholipid transfer activity which was expressed as nmol phospholipids transferred/mm per mg protein.
Analysis Phospholipids were extracted (Folch ef al., 1957) from rat liver mitochondria and microsomes and were subsequently chromatographed (only microsomal lipids) on silica gel 60 thin-layer plates (Merck). Chloroform/methanol/isopropanol/O.25% KCYtriethylamine (30: 9:25:6:18, v/v) were used as developing solvents (Touchstone et al., 1980). The amounts of phospholipid phosphorus (Kahovkova and Odavic, 1969) and protein (Lowry et al., 1951) were determined. Statistical analysis was carried out using Student’s t test. Linear regression analysis has also been performed.
RESULTS The age-induced alterations in the activity of liver cytosol LTP and in the level of the two most abundant liver microsomal phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), are presented in Fig. 1. It is evident that the phospholipid transfer in male rats was enhanced up to 11 months and was decreased in 16-month-old animals. It is noteworthy that this activity was changed in parallel with the PC level in liver microsomal membranes. The
58
T.T. MARKOVSKA
3
0 Age
er al.
11 in months
lb
changes in liver cytosol phospholipid transfer activity (0) and level of PC (A) and PE (A) in microsomes of female rats. Each point is mean of 15 determinations t SD.
FIG.2. Age-related
elevating LTP activity corresponded to an increasing PC content up to 11 months of age. In 16-month-old rats, both the transfer activity and the PC level were decreased. Thus, a positive linear correlation was observed between these two parameters, with a correlation coefficient of Y = 0.725. The alterations of PE were just opposite. Up to 11 months of age, the PE level was decreased and was enhanced at about 16 months, reaching the value observed in 3-month-old rats. The negative correlation between the PE level and the LTP activity showed a coefficient r= - 0.780. Figure 2 shows the results obtained in analogical investigations using female rats. In this case, the alterations were almost similar to the ones reported for male rats; both the PC level and the LTP activity were gradually enhanced up to 16 months with an unsignificant diminution at the 1 lth month. The PE level was decreased up to 11 months, and after this age remained almost unchanged. In female rats, we found linear correlations between the LTP activity and the level of PC (r = 0.991) and PE (Y = -0.755). It should be noted that for all ages under investigation, both the content of PC and PE as well as the activity of LTP were higher in male compared to female rats.
DISCUSSION Our investigations on phospholipid transfer activity in liver cytosol from male and female rats showed that it undergoes significant alterations in the course of development and aging. During the first months of male rat development, the phospholipid transfer activity was significantly enhanced and was decreased after 11 months of age, almost reaching the level observed in 3-month-old animals. It should be emphasized that the alterations in this activity showed a positive correlation with the age-related changes of the PC content in liver microsomes. Our results showed that the microsomal level of PC was elevated up to 11 months and was decreased after this age. The de novo synthesis of PC is known to take place mainly in the endoplasmic reticulum (Bell and Coleman, 1980) and the newly synthesized PC molecules are being transferred to the other subcellular membranes which do not synthesize their own phospholipids. This transfer is
AGING AND PHOSPHOLIPID TRANSFER
ACTIVITY
59
performed essentially by LTP. As already mentioned, a highly specific PC-TP has been reported in rat liver cytosol (Lumb et al., 1976; Poorthuis et al., 1980). It is quite likely that the increase or decrease of the PC level in the endoplasmic reticulum might influence the activity of PC-TP. The elevated PC level in the endoplasmic reticulum might induce activation and/or increased synthesis of the PC-TP, and vice-versa; the decreased content of PC might reduce the activity or the quantity of LTP. For determination of the LTP activity in vitro, we used both mitochondria and liposomes from a common origin, that is, the donor and acceptor membranes for all investigations were identical. Thus, the observed age-related changes in the rate of intermembrane phospholipid transfer were due to alterations in the activity and/or the amount of LTP in liver cytosol, and not to changes in the phospholipid composition of the donor and acceptor membranes. Of course, the changes in vivo in the composition and physicochemical properties of the donor and acceptor membranes in the cell could also influence the phospholipid transfer. There are numerous data in literature devoted to this problem (Hellings et al., 1974; Wirtz et al., 1976; Kamp et al., 1977; Machida and Ohnishi, 1978). As for the negative correlation between the LTP activity and the PE level in microsomes, it might be due to the already reported fact that PE content higher than 20 mol% inhibits the LTP activity (Wirtz et al., 1976; Kamp et al., 1977). In this respect its diminution could induce just the opposite effect. Furthermore, together with the age-induced changes in the phospholipid transfer activity, we also observed some sex-related alterations. In cytosol from male rats, this activity was higher compared to female ones. In male rats, however, the PC level corresponding to the different ages was higher compared to females which might be one of the reasons for the sex-related differences in the lipid transfer activity. Of course, other mechanisms such as, for example, the influence of sex hormones, might be responsible for the reported age-related changes in the LTP activity and the level of PC and PE. Therefore, the elucidation of the age-induced alterations of LTP as well as the factors influencing these alterations would contribute to the better understanding of the complex processes of membrane aging. REFERENCES ARMBRECHT, H.J., BIRNBAUM, L.S., ZENSER, T.V., and DAVIS, B.B. Changes in hepatic microsomal membrane fluidity with age. Exp. Gerontol. 17, 41-48, 1982. BELL, R.M. and COLEMAN, R.A. Enzymes of glycerolipid synthesis in eukariotes. Ann. Rev. Biochem. 1980, 49-459, 1980. BLOJ, B. and ZILVERSMIT, D.B. Rat liver proteins capable of transfetring phosphatidylethanolamine. Purification and transfer activity for other phospholipids. J. Biol. Chem. 252, 1613-1619, 1977. BUTLER, M.M. and THOMPSON, W. Transfer of phosphatidylserine from liposomes or microsomes to mitochondria. Stimulation by a cell supematant factor. Biochim. Biophys. Acta 388, 52-57, 1975. GRAIN, R.C. and ZILVERSMIT, D.B. Two nonspecific phospholipid exchange proteins from beef liver. Biochemistry 19, 1433-1439, 1980. DAWSON, R.M.C. The exchange of phospholipids between membranes. Subcell. Biochem. 2, 69-89, 1973. DYATLOVITSKAYA, E.V., TIMOFEEVA, N.G., and BERGELSON, L.D. A universal lipid exchange protein from rat hepatoma. Eur. J. Biochem. 82, 463471, 1978. ENGLE, M.J., VAN GOLDE, L.M.G., and WIRTZ, K.W.A. Transfer of phospholipids between subcellular fractions of the lung. FEBS Z.&t.86, 277-281, 1978. FOLCH, J., LEE, M., and SLOANE-STANLEY, G.H. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497-507, 1957. GRINNA, L.S. Age-related alterations in membrane lipid and protein interactions: Arrhenius studies of microsomal
60
T T. MARKOVSKA
er al
glucose-6-phosphatase. Gerontology 23, 342-349, 1977a. GRINNA, L.S. Changes in cell membrane during aging. Gerontology 23, 452-464, 1977b. GRINNA, L.S. and BARBER, A.A. Age-related changes in membrane lipid content and enzyme activities. Biochim. Biophys. Acta 288, 347-353, 1972. GRINNA, L.S. and BARBER, A.A. Lipid changes in microsomal and mitochondrial membranes of rat liver during aging. Fed. Proc. 35, 1425, 1976. HAWCROFI, D.W. and MARTIN, P.A. Studies on age-related changes in the lipids of mouse liver microsomes. Mech. Ageing Dev. 3, 121-130, 1974. HEGNER, D., PLAIT, D., HECKERS, H., and SCHLOEDER, U. Age-dependent physicochemical and biochemical studies of human red cell membranes. Mech. Ageing Dev. 10, 117-130, 1979. HELLINGS, J.A., KAMP, H.H., WIRTZ, K.W.A., and VAN DEENEN, L.L.M. Transfer of phosphatidylcholine between liposomes. Eur. J. Biochem. 47, 601-605, 1974. KAHOVKOVA, J. and ODAVIC, R. A simple method for analysis of phospholipids separated on thin-layer chromatography. J. Chromatogr. 40, 90-95, 1969. KAMP, H.H., WIRTZ, K.W.A., BAER, P.R., et al. Specificity of the phosphatidylcholine exchange protein from bovine liver. Biochemistry 16, 1310-1316, 1977. KOUMANOV, K. and INFANTE, R. Phospholipid-transfer proteins in human liver and primary liver carcinoma. Biochim. Biophys. Acta 876, 526-532, 1986. LOWRY, R.H., ROSENBROUGH, N.J., FARR, A.L., and RANDALL, R.J. Protein measurement with the Folin phenol reagent. /. Biol. Chem. 193, 265-275, 1951. LIMB, R.H., KLOOSTERMAN, A.D., WIRTZ, K.W.A., and VAN DEENEN, L.L.M. Some properties of phospholipid exchange proteins from rat liver. Eur. J. Biochem. 69, 15-22, 1976. MACHIDA, K. and OHNISHI, S.I. A spin labeled study of phosphatidylcholine exchange protein. Regulation of the activity by phoshpatidylserine and calcium ion. Biochim. Biophys. Acta 507, 156-164, 1978. MALONEY, A.G., SCHMUCKER, D.L., VESSEY, D.S., and WANG, R.K. The effects of aging on the hepatic microsomal mixed-function oxidase system of male and female monkeys. Hepatology 6, 282-287, 1986. NOKUBO, M. Physical-chemical and biochemical differences in rat liver plasma membranes in aging F-344 rats. J. Gerontol. 40, 409-414, 1985. PETKOVA, D.H., MOMCHILOVA, A.B., and KOUMANOV, K.S. Age-related changes in rat liver plasma membrane phospholipase A2 activity. Exp. Gerontol. 21, 187-193, 1986. PETKOVA, D.H., MOMCHILOVA-PANKOVA, A.B., MARKOVSKA, T.T., and KOUMANOV, K.S. Age-related changes in rat liver plasma membrane sphingomyelinase activity. Exp. Gerontol. 23, 19-24, 1988. POORTHUIS, B.J.H.M., VAN DER KRIFT, T.P., TEERLINK, T., AKEROYD, R., HOSTETLER, K.Y., and WIRTZ, K.W.A. Phospholipid transfer activities in Morris hepatomas and the specific contribution of the phosphatidylcholine exchange protein. Biochim. Biophys. Actu 600, 376-386, 1980. POSSMAYER, F. Evidence for a specific phosphatidylinositol transferring protein in rat brain. Brain Res. 74, 167-174, 1974. PRATZ, J. and CORMAN, B. Age-related changes in enzyme activities, protein content and lipid composition of kidney brush-border membranes. Biochim. Biophys. Acta 814, 265-273, 1985. SCHMUCKER, D.L., VESSEY, D.A., WANG, R.K., JAMES, J.L., and MALONEY, A.G. Age-dependent alterations in the physicochemical properties of rat liver microsomes. Mech. Ageing Dev. 27, 207-217, 1984. TOUCHSTONE, J.C., CHEN, J.C., and BEAVER, K.M. Improved separation of phospholipids in thin-layer chromatography. Lipids 15, 61-62, 1980. WIRTZ, K.W.A. Transfer of phospholipids between membranes. Biochim. Biophys. Acta 344, 95-l 17, 1974. WIRTZ, K.W.A. and ZILVERSMIT, D.B. Exchange of phospholipids between liver mitochondria and microsomes in vitro. J. Biol. Chem. 243, 3596-3602, 1968. WIRTZ, K.W.A., KAMP, H.H., and VAN DEENEN, L.L.M. Isolation of a protein from beef liver which specifically stimulates the exchange of phosphatidylcholine. Biochim. Biophys. Acta 274, 696-617, 1972. WIRTZ, K.W.A., GEURTS VAN KESSEL, W.S.M., KAMP, H.H., and DEMEL, R.A. The protein mediated transfer of phosphatidylcholine between membranes. The effect of membrane lipid composition and ionic composition of the medium. Eur. J. Biochem. 61, 515-523, 1976. YAFFE, M.P. and KENNEDY, E.P. Intracellular phospholipid movement and the role of phospholipid transfer protein in animal cells. Biochemistry 22, 1497-1507, 1983. ZBOROWSKI, J. and WOJTCZAK, L. Net transfer of phosphatidylinositol from microsomes and mitochondria to liposomes catalyzed by exchange protein from rat liver. FEES I&t. 51, 317-320, 1975.