Effects of chronic ethanol administration on poly-phosphoinositide metabolism in the mouse brain: variance with age

Neurochem. Int. Vol. 22, No. 1, pp. 11-17, 1993 Printed in Great Britain. All rights reserved

0197-0186/93 $5.00+0.00 Copyright © 1993 Pergamon Press Ltd

EFFECTS OF CHRONIC ETHANOL ADMINISTRATION ON POLY-PHOSPHOINOSITIDE METABOLISM IN THE MOUSE BRAIN: VARIANCE WITH AGE GRACE Y. SUN, I* MEENA NAVIDI, l F U - G E N YOA, l W . GIBSON WOOD 2 a n d ALBERT Y. SUN 3 JDepartments of Biochemistry and 3Pharmacology, University of Missouri, School of Medicine, Columbia, MO 65212, U.S.A. 2GRECC, VA Medical Center and Department of Pharmacology, University of Minnesota, School of Medicine, Minneapolis, MN 55417, U.S.A. (Received 21 April 1992 ; accepted 29 May 1992)

Al~raet--Using a procedure in which poly-phosphoinositides (poly-PI) in C57BI mouse brain were labeled with [32P]Pi or [32P]ATP, the effects of chronic ethanol administration and age on metabolism of these anionic phospholipids were examined. Within 4 h after intracerebral injection, both labeled precursors were effectively incorporated into membrane phospholipids with high proportions of labeling among phosphatidylcholine, phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate. With few exceptions, the phospholipid labeling patterns in different brain regions, e.g. cortex, hippocampus and hypothalamus, were similar. However, when the brain homogenate was subjected to differential and sucroseFicoll gradient centrifugation, different phospholipid labeling patterns were observed in the subcellular membrane fractions. Young adult mice given an ethanol (5% w/v) liquid diet for 2 months showed an increase in the levels of labeled phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate and phosphatidylserine in the cortex and hippocampus as compared to the pair-fed controls, but these changes were not observed in the hypothalamus. In another study, 12- and 26-month-old mice were administered either an ethanol (8 g/kg in two doses daily) or a control diet by gavage for 3 weeks. The 12month-old group given the ethanol diet showed an increase in labeled poly-Pl which was found largely in the synaptosomal fraction. Surprisingly, the 26-month-old mice given the same ethanol paradigm showed a decrease in labeled poly-PI. Consistent with our previous observations, the 26-month-old mice showed a higher proportion of labeled poly-PI in the synaptosomal fraction as compared to the younger age group. Taken together, these results suggest that chronic ethanol administration could result in an increase in biosynthesis of poly-PI in adult mice but aged mice showed a different response to the effect of ethanol.

Chronic ethanol ingestion can lead to the development of tolerance and physical dependence although the biochemical mechanisms underlying these phenomena have not been clearly elucidated. In earlier studies, attempts were made to examine the effects of ethanol on bulk membrane lipids (Sun and Sun, 1985), but most of the changes in fatty acid composition as well as in individual phospholipids were very small and difficult to detect. It is now recog-

nized that ethanol probably does not alter the bulk membrane lipids but instead, specific lipid domains within the lipid bilayer may exhibit special sensitivity to perturbation by ethanol (Wood and Schroeder, 1988 ; W o o d et al., 1989a, b, 1990). Our laboratory has obtained data consistent with an effect of chronic ethanol administration on brain acidic phospholipids including PS and phosphoinositides (PI, PIP and PIP2) (Sun and Sun, 1983 ; Sun et al., 1984, 1987b). In most instances, the ethanolinduced changes were found mainly in the synaptosomal fraction. Since poly-PI in brain are present only in trace quantities which are metabolically very active, it is difficult to accurately assess ethanol effects on their basal metabolism. Poly-PI are engaged in receptor-mediated signaling pathways and hydrolysis of PIP2 by phospholipase C results in the release of two second messengers, inositol 1,4,5-trisphosphate

* Author to whom all correspondence should be addressed, at: M121 Medical Science Building, Biochemistry Department, University of Missouri, Columbia, MO 65212, U.S.A. Abbreviations: PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol ; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; poly-PI, PIP2 and PIP ; PS, phosphatidylserine. 11

I2

(i~.x~: Y.

li)r mobilization of intracellular ( a "+ store and diacylglycerol for activation of protein kinase C (Berridge, 1987; Fowler and Tiger, 1991). Previous studies using brain slice p r e p a r a t i o n s have shown that n e u r o t r a n s m i t t e r - i n d u c e d poly-PI hydrolysis was rather insensitive to the effects o f ethanol in t~itro and in z'ico ( H o f f m a n et al., 1986; Gonzales a n d Crews, 1988: G o n z a l e s et al., 1986- Crews et al., 1986). It is possible that agonist-induced poly-PI response elicited by brain slices are not c o m p a r a b l e to that in the intact brain. Consequently, it is desirable to be able to observe changes occurring directly in the brain ill fifo. In a previous study (Sun et al., 1987a), we a t t e m p t e d to label the phospholipids in brain by i.p. injection of [32p]Pi into young rats. Results suggested an increase in i n c o r p o r a t i o n of label into poly-PI due to chronic ethanol administration. Nevertheless, this procedure was limited to experiments with young rats (less t h a n 6 weeks of age) because i n c o r p o r a t i o n of label into the brain phospholipids decreased dramatically with age. We have subsequently a t t e m p t e d to improve the labeling procedure a n d f o u n d that these labeled precursors could be effectively incorporated into brain phospholipids after intracerebral injection (Sun and Lin, 1989). Using this procedure, age-related differences in labeling of PIP2 could be observed in the mouse brain (Sun a n d Lin, 1990). The purpose of the present experiments was to further examine the effect of ethanol a n d age on PIP a n d PIP~ metabolism with respect to specificity a m o n g brain regions and subcellular m e m b r a n e fractions.

EXPERIMENTAL PROCEDURES

Animals and ethanol administration Experimental protocol 1. Four-week-old male C57B1/6J mice (weighing 13 g) were purchased from Taconic Farm (Germantown, NY). Animals were housed individually in Sinclair Farm Small Animal Facilities after arrival and were given lab chow (Purina, Ralston, MO) and tap water ad lib for 2 weeks prior to initiation of an experiment. Animals were then adapted to a nutritionally complete liquid diet (Sustacal, chocolate flavor, Evansville, IN) for a week and then divided into an ethanol group (n = 8) and a pair-fed control group (n = 8). The ethanol group was given the Sustacal diet containing 5% (w/v) ethanol ad lib. The pair-fed group was given the same diet but containing an isocaloric amount of glucose instead of ethanol. Mice were weighed periodically and the diets were administered for a period of 2 months. At the end of the feeding paradigm, non-withdrawn animals were injected intracerebrally with [32p]Pi (20 ,uCi, NEN, Boston, MA) under brief ether anesthesia. Injections were made using a 27 gauge x sL" needle adapted with a polyethylene tubing to give a 3 mm penetration depth (Sun and

SiN ,,/ a/. kin, 1989). Allcr injection, the labcl was allowed to equilibrate in the brain for 4 11prior to sacrificing the animals. ILwer#nenml protocol 2. C57BI/6NNIA male mice 12 and 26 months of age were obtained from Jackson Laboratory (Bar Harbor, ME). Upon arrival, animals were housed individually in Sinclair Farm Small Animal Facilities and were given lab chow and tap water ad lib for 2 weeks. Mice were administered ethanol (totaling 8 I0 g-kg) by gavage m the form of a nutritionally complete liquid diet (Similac, Ross Laboratories, Columbus. OH). Ethanol (increasing from 8 to 10 g/kg) was adminislered via intragastric intubation in two fractional doses (9 a.m. and 4 p.m.) for 3 weeks as in procedures described previously (Chandrasekhar e,' a/.. 1988a). Animals were also supplemented with lab chow ad lih. At the end of the dietary treatment, each animal was injected intracerebrally with [7A+P]ATP (10 tK;i, NEN, Boston, MA) 4 h prior to killing and brain dissection. Dissection O/ brain re.qion.~ and i.~olation O/ slthcelluho" fi'aclions Animals were killed by decapitatkm and the cerebral cortex was quickly removed and submersed in an ice-cold solution of 0.32 M sucrose, 1 mM EDTA and 50 mM Tris HCI (pH 7.4). In experiments involving dissection of brain regions (hypothalamus, hippocampus and cerebral cortex), the brain was placed on ice and dissection was carried out as quickly as possible (within 1 min). Brain tissue was homogenized in ice-cold sucrose Tris solution with a glass homogenizer tilted with a Teflon pestle. Aliquots of the homogenate were routinely taken for counting of radioactivity and for protein determination. In the experiment with different age groups, the entire cerebrum was dissected and tissue homogenate was subjected to sucrose Ficoll gradient centrifugation according to the procedure described by Sun el al. (1988). Subcellular fractions such as synaptosomes, myelin, somal plasma membranes (PM) and microsomal fractions were isolated for analysis of membrane phospholipids and aliquots of each subcellular fraction were taken for deterruination of radioactivity. Portions of the major fractions (Pc and synaptosome) were also taken for protein determilmlion by the method of Lowry el al. (1951) using bovine serum albumin (Sigma, St. Louis. MO) as standard Lipid extraction and ~eparalion The procedures for lipid extraction and septtration by HPTLC were similar to that described by Sun and Lin (1989). The neutral and acidic phospholipids in the tissue homogenates were extracted by a 2-step solvent extraction procedure to ensure complete extraction of the poly-P1. Phospholipids were separated by HPTLC (Silica gel 60, E. Merck, Darmstadt, Germany) using a 2-D separation procedure with three solvent systems and a short exposure to HC1 fumes for separation of the plasmaiogens from the diacyl type phospholipids (Sun and Lin, 1989). After solvenl development, the HPTLC plates were exposed to iodine vapor for visualization of the lipid spots. Individual lipids were marked and scraped into scintillation vials for determination of radioactivity by a Beckman LS 5800 liquid scintillation spectrometer (Beckman Inst., Fnllerton, CA). RESULTS

O u r previous study (Sun a n d Lin, 1989) described the uptake of [7-32p]ATP by phospholipids in rat

13

Chronic ethanol and age on poly-PI metabolism in mouse brain brain. Initially, [32p]ATP was used because this labeled precursor was thought to give better labeling of poly-PI through the PI- and PIP-kinase route. Nevertheless, a comparison of the results from injections of [32P]ATP and [32p]Pi indicated no major differences in the phospholipid labeling patterns between the two precursors. In both cases, radioactivity was readily taken up by phosphoinositides (PI, PIP and PIP2) as well as other phospholipids in a time-dependent manner (Sun and Lin, 1989). In the first experimental protocol, we examined the uptake of [32p]Pi by adult C57BI/6J mice that were given either the ethanol or the pair-fed liquid diet for 2 months. At 4 h after injection, the phospholipid labeling pattern was examined in three different brain regions, namely the cortex, hippocampus and hypothalamus. As shown in Fig. 1, although the phospholipid labeling patterns among the three brain regions in the controls are similar with major proportions of the labels in PI, PC and PIP2, small regional differences can also be observed. For example, the hypothalamus showed a higher proportion of labeled PC and a lower proportion of labeled PIP2 and PA as compared to the cortex and hippocampus. Since there were no major differences in the percent distribution of radioactivity in PE between the ethanol group and controls among the three brain regions, labeled PE was used as an internal standard for assessing the relative changes in phospholipid labeling due to chronic ethanol administration. Data in Fig. 2 show the phospholipid labeling patterns between the ethanol-treated and control groups in the

[ ] Control (n=8) 4 -

[ ] Alcohol (n=8)

Cortex

13..J EL

i I f " / / PIP2

/

1

/ PI

I PC

PEpL

PC

PEpL

t PIP

PA

PS

f

Hippocampus

J_ 2 /

4

/

uJ ,_1 EL

/ 2

t

PIP2

PIP

r--d=1 PS PA

Hypothalamus

2.0 1.5 1.0 0.5 0

r=tm PIP2

PIP

PA

PI

T

3.5 3.0 2.5

PIP2 PIP PA PS PI PC aPE PE Fig. 1. Percent distribution of radioactivity among the phospholipids in mouse cortex, hypothalamus, and hippocampus regions. C57BI/6J mice were injected intracerebrally with [32p]Pi and brain regions were dissected in ice 4 h after injection. Phospholipids were separated by 2-D HPTLC as described in text. The data are percent distribution of radioactivity (mean ___SD, n = 5) in each group of the phospholipids recovered from the HPTLC plate.

/ / / / / /

5

4.0

lO

/ / / / / /

UJ

PS

"1"

/ / / / I / / / / Pl

I

/

•

i/ I/ / / / / PC

PEpL

Fig. 2. Ratio of radioactivity of individual phospholipids in different brain regions with respect to chronic ethanol administration. Mice were administered ethanol in the form of a liquid diet for 2 months and injected intracerebrally with [32p]Pi. Controls were pair-fed the same diet supplemented with an isocaloric amount of sucrose. Results are ratios of radioactivity of individual phospholipids against that of PE (mean+SD, n = 5). *Values from the ethanol group are significantly different from controls based on analysis of variance, P < 0.05.

14

GRA('t ~". SI:N el ~11.

three brain regions. In the cortex, there were significant (P < 0.05) increases in labeled PIPe, PIP a n d PS in the ethanol group as c o m p a r e d to controls. The h i p p o c a m p u s also showed a similar increase in labeled PIPe and PS but little or no changes were found in the h y p o t h a l a m u s between the ethanol and control groups. The second experimental protocol was designed to c o m p a r e the phospholipid labeling patterns a m o n g different subcellular m e m b r a n e fractions isolated from 12- or 26-month-old mice that were given either the ethanol or control diet by gavage (for 3 weeks). Data in Table I show the recovery of radioactivity and protein (as percent o f total h o m o g e n a t e ) a m o n g different subcellular fractions with respect to ethanol and age. In general, there are no obvious differences in the recovery of radioactivity c o m p a r i n g the ethanol group with controls. However, some differences were observed with respect to age. F o r example, synaptosomes from the 26-month-old groups tend to show a decrease in the recovery of protcin (P < 0.05) and radioactivity (not significant) as c o m p a r e d to the 12m o n t h - o l d groups. In contrast, recovery of radioactivity in the P M fraction was significantly higher in the older age groups as c o m p a r e d to the 12-monthold group (Table 1). As reported previously (Sun and Lin, 1989), intracerebral injection of [32P]ATP resulted in unique phospholipid labeling patterns a m o n g different subcellular fractions. O f special interest are the high p r o p o r t i o n s of label in PIPe a n d PA in the myelin fraction comprising over 50% of the total radio-

activity of the phospholipids in that fraction (Table 2). F u r t h e r m o r e , labeled P I P , in the s y n a p t o s o m e and PM fractions were 3-fold higher than in the microsomal fraction, consistent with the role o f this phospholipid in receptor-mediated signal t r a n s d u c t i o n mechanisms. Changes in p h o s p h o l i p i d labeling due to ethanol were found only in the s y n a p t o s o m a l fraction. W h e n the phospholipid labeling patterns from brain h o m o g e n a t e and synaptosomes of the 26m o n t h - o l d group were c o m p a r e d to the 12-month-old group, the aged group showed a higher p r o p o r t i o n of' labeled PIP2 a n d lower p r o p o r t i o n s of labeled PI, PC and PE t h a n that in the 12-month-old group (Fig. 3). Again, these changes are found mainly in the synaptosomal fraction and no other subeellular fractions exhibit similar changes (Tables 2 and 3). W h e n the synaptosomal phospholipid labeling patterns in the 12-month-old mouse groups were compared with respect to chronic ethanol administration, there was an increase in the p r o p o r t i o n s of labeled PIP and P I P , a n d a decrease in labeled PI and PE in the ethanol group as c o m p a r e d to the pair-led control group (Table 2). W h e n the same comparison was made with the 26-month-old groups, there was instead a decrease in labeled PIPe and P A and an increase in labeled PC in the ethanol group as c o m p a r e d to the controls (Table 3). W h e n the lipids in the myelin fraction were analyzed, we consistently observed an u n k n o w n lipid spot appearing left of the PA, However, the labeling of this " u n k n o w n " was not altered with respect to ethanol a d m i n i s t r a t i o n and age.

Table 1. Percent recovery of radioactivity and proteins in individual subcellular fi'actions from the 12- and 26-month-old control and ethanol mice 26-month-old

12-month-old

Fractions

('

P~ Syn PM Mye Mic

17.82+ 1.47 7.92_+0.80 1.t 6 + 0. I I 1.39_+0.22 2.14-+0.30

P2 Syn

31.52 + 1.53 17.37±0.65

A Radioactivity, % of total 18.23_+0.81 7.65_+ 1.02 1.03+ 0.10 1.16+0.16 2.49+0.14 Proteins, % of total 35.59 _+2.73 16.53_+1.66

C

A

18.08± 1.20 7.30_+2.11 1.67* -+0.28 0.93-+0.19 2.63+0.34

19.39+ 0.79 6.33_+0.29 1.80" ± 0.22 1.31 -+0.30 2.81 _+0.59

33.05 ± 0.99 13.13" ± 1 . 9 l

37.62 -+3.58 13.07"_+1.12

C57B1/NNIA mice 12 and 26 months of age were administered ethanol (A) and control (C) diet as described in text. They were injected intracerebrally with [32P]ATPand at 4 h after injection, the cortex was dissected and homogenized in sucrose Tris buffer (total homogenate). A portion of the total homogenate was taken for protein determination and for counting of radioactivity. The brain homogenate was then subjected to subcellular fractionation to obtain individual fractions. Results are recovery of proteins or radioactivity of the individual fractions as percent of the total (mean_+SD, n = 4 ) . Statistical evaluation based on analysis of variance indicated significant differences comparing the 26-month-old (ethanol and control) with the respective 12-month-old groups, *P < 0.05.

Chronic ethanol and age on poly-PI metabolism in mouse brain 30 I " Total homogenata /

.,= L --

*

[]

12-month-old

,.T.,

"°I i

Ni

1

r'lll

0 ~

PIPl 30 F

15

PIP

PA

PS

PI

PC

aPE dPE

Synapt°s°mas

*

*

10 5 0

PIP2 PIP

PA

PS

PI

PC

aPE dPE

Fig. 3. Comparison of the phospholipid labeling patterns in mouse brain with respect to age. Mice 12 and 26 months of age were injected intracerebrally with [32P]ATP (10 #Ci) 4 h prior to killing and dissection of brain cortex. Results are expressed as percent distribution of radioactivity of phospholipids recovered from the HPTLC plates. *Values from the 26-month-old group are significantly different from those in the 12-month-old group based on analysis of variance, P < 0.05. DISCUSSION Although poly-PI are important membrane phospholipids associated with the signal transduction pathway (Berridge, 1987), little is known regarding effects of drugs, ethanol and aging on their metabolism in the brain in vivo. This is because experiments to study poly-PI metabolism in brain are frequently confronted with difficulties due to their active metabolism and presence in trace amounts. Furthermore, different pools of these phospholipids exhibit different metabolic activity among different subcellular fractions (Sun et al., 1990). Through intracerebral injection with [32p]Pi or [32p]ATP, it is now possible to label the poly-PI as well as other phospholipids in brain for studies of their metabolism (Sun and Lin, 1989). We found that incorporation of label into the phosphoinositides (PI, PIP and PIP2) was rapid and reached a plateau after 2 h, whereas labeling of other phospholipids was less rapid and continued to increase with time for 16 h. A 4 h labeling period was

15

used in these experiments since this time period was sufficient to allow maximal labeling of the poly-PI and a comparison of the poly-PI labeling with that of other phospholipids. Results of the present study that used two different experimental paradigms indicated similar increases in labeled poly-PI in the mouse cortex with respect to chronic ethanol administration. Although the changes are not striking, they are consistently found in the synaptosomal fraction in the cortex and hippocampus. These results seem to indicate that the ethanolinduced change in poly-PI labeling is associated with events occurring in the neurons. The increase in labeling of poly-PI is in agreement with results from our earlier study in which rat brain phospholipids were labeled through i.p. injection of [32p]Pi (Sun et al., 1987a). Although i.p. injection is less invasive to the animals as compared to intracerebral injection which was carried out under light ether anesthesia, the i.p. procedure is limited to studies with young rats that are less than 1 month of age. Mice subjected to the intracerebral injection procedure recovered from the ether anesthesia within 1 min after injection, and there were no obvious signs of behavioral differences between the ethanol and control groups in their response to ether anesthesia. Therefore, it is reasonable to conclude that the injection procedure is not a factor for the differences in poly-PI metabolism between the ethanol and control groups. The increase in labeled poly-PI in brain synaptosomes due to chronic ethanol administration can be explained either by the increase in their biosynthesis through PI and PIP-kinase or a decrease in their hydrolysis by phospholipase C, an event known to be regulated by receptor agonists (neurotransmitters). Since animals did not undergo ethanol withdrawal nor were they stimulated by any agonists during this experiment, it is reasonable to conclude that the increase in poly-PI labeling is the result of an adaptive increase in their biosynthesis. The decrease in labeling of PI in the ethanol group seems to further support the notion that more labeled PI is channeled to form PIP and PIP2. It is conceivable that the increase in poly-PI labeling due to chronic ethanol is different from that observed in rats administered intoxicating doses of ethanol (Chandrasekhar et al., 1988b). Unlike the acute study where the increase in poly-PI was marked by a decrease in levels of diacylglycerols, there were no differences in the levels of Ins(1,4,5)P3 or diacylglycerols in the chronic ethanol mouse group (data not shown). Results from this study also show changes in the phospholipid labeling pattern with respect to age.

16

GRA(~ h. %tN

~'t

./.

Table 2. Percenl distribution of radioaclivity among phospholipids in brain homogenatc and subcclluEar fractions of control and alcoholic mice (12-momh-old) Fractions

PIP,

PIP

PA

PS

Pt

PC

aPE

dPE

9.4*+0.7 11.0+0.6

2.3+0.3 2.0-~ 0.4

17.0+2.4 16.3 ? 1.2

28.1"i 1.2 25.4+0.9

5.4 ! 1.2 6.8 + 0.7

8.9_+0.8 10.2+0.5

I Ink

Tom/brain homo.qenale.

A C ~|'tlaplosooH'.~

20.1 +/I.5 18.6+ 1.4

10.1 ~ 1.4 10.6+_0.6

.

A ('

12.0"_+1.7 9.0+1.0

10.1"+0.7 7.3~ 1.0

5.9~ 0.4 6.3t.0.3

2.6_+0.2 20.2*+0.6 2 . 8 + - 0 . 0 25.1+.0.4

31.5i 1.6 29.5+2.1

5.0-+0.6 12.5",,0.5 5.6+0.8 15.2 J_-0.3

7.7~ 0.6 8.4+1.4

21.7+ 1.6 21.4+1.8

1.5+0.2 1.6÷0.2

6.6_+0.2 6.7+0.5

14.2+0.6 13.7! 1.1

6.0±0.6 5.1+.0.4

6.8+0.4 6.8:~0.5

3.7 f 0.2 4.0+0.3

2.7+0.1 2.6!0.1

23.4+[).5 23.0+ 1.2

30.7_+1.9 29.3 ,~ 1.2

5.8 +0.7 5,1 +0.3

14.3_+[).7 14.8+0.4

3.5±0.5 3.7+0.7

2.0+0.2 2.3+0.1

1.6-+0.2 1.4+0.1

31.5+0.9 32.3,,1.0

39.8_+1.0 37.5+1.5

4.8+0.7 5.2 f 0.4

12.6,,0.6 13.9+0.4

Myelin :

A (

33.6+2.7 33.4+2.6

6.9--0.5 7.2+0.6

2.53 0.3 2.6 ~-0.3

P/a~'ma memhrane~ "

A C

12.6_+.0.9 13.7+_0.4

Microsomes.

A C

4.2+0.4 3.8+_0.8

Twelve-month-old mice were either administered ethanol (A) or an isocatoric diet with sucrose (C) by gavage for 3 weeks as described in text. Animals were injected intracerebrally with [3'P]ATP (10 pC±) 4 h prior to killing and dissection of brain cortex. Data represent percent distribution (mean -- SD, n = 4) of radioactivity among phospholipids in brain homogenate and subcellular fractions. Statistical evaluation based on analysis of variance indicated significant differences comparing the ethanol group with controls. *P < 0.05. Unk, unknown lipid.

U n l i k e the increase in p o l y - P l in the c h r o n i c e t h a n o l g r o u p , aged mice seem to s h o w a specific increase in labeled PIP2 b u t n o t PIP, a n d this increase is m a r k e d by a general decrease in labeled PI, PC a n d PE. T h e increase in labeled PIP2 in the aged mice is in g o o d a g r e e m e n t with results o b t a i n e d f r o m o u r p r e v i o u s s t u d y in w h i c h [32p]Pi w a s injected into 27- a n d 10m o n t h - o l d mice ( S u n a n d Lin, 1990). In b o t h studies, the age-related increase in labeled PIP2 is f o u n d m a i n l y in the s y n a p t o s o m e s a n d n o t in o t h e r s u b cellular fractions. In a n o t h e r s t u d y c o m p a r i n g the

acidic p h o s p h o l i p i d s in m o u s e s y n a p t o s o m e s with age, we also o b s e r v e d an increase in the levels o f p o l y - P I with age in spite o f the fact t h a t o t h e r acidic p h o s p h o l i p i d s (e.g. PI a n d PS) decreased with age (Sun e t a l . , 1987b). A l t h o u g h the p h y s i o l o g i c a l implic a t i o n o f the increase in PIP2 in the aged b r a i n r e m a i n s to be f u r t h e r elucidated, it is r e a s o n a b l e to believe t h a t this age effect is o f i m p o r t a n c e due to the role o f this highly c h a r g e d p h o s p h o l i p i d as s u b s t r a t e for the signal t r a n s d u c t i o n p a t h w a y . It is interesting t h a t C r e w s e t al. 11986) did n o t o b s e r v e o b v i o u s c h a n g e s

Table 3. Percent distribution of radioactivib anaong phospholipids m brain homogenate and subcelhdar fractions of alcoholic mice (26month-old) Fraclions

PIP,

PlP

PA

PS

PI

PC

aPE

dPE 9.1±0.7 8.0+0.6

I ink

"lbtal brain homogenate :

A (' S)'naplosol'glc.s

A ('

19.7"+-2.8 23.8_+1.2

11.1-+1.6 10.6" t0.1 12.7+-2.2 11.9_+1.0

2.1+0.4 1.9+0.2

13.1!1.7 26.1"+ 1.4 12.9+_1.5 22.5+1.7

7.0-+[.3 7.6+1.6

."

7.7*_+ 1.4 12.0-+ 1.9

6.5_+0.8 7.6±0.3

6.7" +0.4 7.9 t : 0 . 8

2.8_+0.3 2.6+0.3

24.0 ± 0.9 33.5*_+0.5 22.5.+_0.8 28.7+0.8

5.0
29.7_+4.4 31.3+2.7

7.6+0.3 8.7 J: 0.6

21.3±1.0 24.0+2.0

1.8+0.2 1.5+0.1

8.2"-+1.1 16.2"+_2.0 5.8_+0.5 12.1+0.3

5.1+0.4 6.7±1.3

13.9_+0.9 13.0_+0.05

Myelin :

A C

7.6-+1.2 6.9_+0.3

2.6+0.5 2.9+0.3

Plasma membranes.

A C

13.3_+0.7 12.7+2.2

8.0±0.5 9.0--0.9

3.9+_0.2 3.7~_0.1

2.9_+0.1 2.8+_0.1

19.8+_0.9 22.7+0.9

32.3_+0.5 29.9,,I.4

5.6_+1.1 5.0+_0.8

14.1_+0.3 14.3-+1.0

4.0--0.9 4.4+ 1.9

3.5--0.7 2.7 J 0.1

2.2+_0.2 2.1 +0.1

1.4!0.1 1.5+0.0

30.3_+2.2 32.8+_1.4

40.0_+1.2 38.0+2.4

4.8_+0.7 4.6_+0.2

13.6+_0.4 14.1 ,,0.1

Microsomes :

A C

Twenty-six-month-old mice were administered the ethanol (A) and control (C) diet as described in text. Animals were injected intracerebrally wi{h [32P]ATP (10 #Ci) 4 h prior to killing and dissection of brain cortex. Data represent percent distribution (mean--SD, n = 4) of radioactivity among phospholipids in brain homogenate and subcellular fractions. Statistical evaluation based on analysis of variance indicated significant differences comparing the ethanol group with controls, *P < 0.05.

Chronic ethanol and age on poly-PI metabolism in mouse brain in receptor-stimulation o f p h o s p h o i n o s i t i d e hydrolysis in b r a i n slices p r e p a r e d from aged rats. O n the o t h e r h a n d , previous studies by W o o d et al. (1982) have indicated t h a t aged mice were more sensitive to the intoxicating effects o f acute ethanol a d m i n istration. The differences in sensitivity m a y be due to the increased level of PIP2, subsequently creating a local d o m a i n with e n h a n c e d level of m e m b r a n e b o u n d Ca 2+. O u r study here also indicated age differences in the poly-PI response to chronic e t h a n o l treatment. In fact, the 2 6 - m o n t h - o l d mice given the same ethanol regime showed changes opposite to those in the 12m o n t h - o l d group. A n interesting observaion is the increase in labeling o f P C after a d m i n i s t r a t i o n o f e t h a n o l to the aged g r o u p a n d n o t the 12-month-old group. Obviously, m o r e studies are needed to better u n d e r s t a n d the complex p h e n o m e n a underlying the effects o f alcohol a n d age o n m e t a b o l i s m of brain poly-PI a n d o t h e r phospholipids. Acknowledgements--This research project was supported in part by research grants AA 06661 (GYS), AA 02025 (AYS) and AA 07292 (WGW) from NIAAA.

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