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Composition and biosynthesis of molecular species of retina phosphoglycerides
Neurochemistry
Vol. Pergamon Press Ltd.
I, pp. 381-392. 1980. Printed in Great Britain.
CO}~OSITION AND BIOSYNTHESIS OF MOLECULAR SPECIES OF RETINA PHOSPHOGLYCERIDES M. I. Avelda~o de Caldironi and N. G. Baz~n Instituto de Investigaciones Bioqufmicas, Universidad Nacional del Sur y Consejo Nacional de Investigaciones Cientfficas y Tgcnicas, Bahfa Blanca, Argentina.
ABSTRACT Recent studies on the fatty acid composition and labeling by glycerol of individual molecular species of retinal phosphoglycerides are presented. Docosahexaenoate-containing species are important constituents of retina phospholipids. 22:6 and saturated fatty acids appear in similar amounts in hexaenes (nearly 50% each). Very high percentages of 22:6 and other polyenoic fatty acids are found in molecular species containing negligible amounts of saturated fatty acids, indicating the occurrence of dipolyunsaturated phosphoglycerides, including didocosahexaenoate. On the other extreme, fully saturated species occur. 22:6-containing species are synthesized at high rates in most phosphoglycerides including phosphatidylinositol, which is mainly formed by tetraenoic classes. Oligoenes and saturates are synthesized de novo and once formed they may serve as precursors of others (e.g. tetraene~ by acyl-exchange reactions. Propranolol induces rapid modifications in the de novo biosynthesis of retina glycerolipids. Oligoenes and saturates are the most stimulated species in PI and PS, and the most inhibited ones in PC. In PE, the labeling of these species is stimulated, while the polyenes are inhibited. This remarkable example of metabolic heterogeneity in a single phosphoglyceride opens new possibilities in the study of the regulation of retinal membrane lipid biosynthesis. KEYWORDS Retina, glycerophospholipids, molecular species, glycerol uptake, docosahexaenoate, arachidonate, polyenes, lipid synthesis, propranolol.
INTRODUCTION Photoreceptor cells and especially their rod outer segments, have been the main sub ject of studies on retinal biochemistry to date, due to the development of methods-for obtaining high purity-ROS preparations and to the interest in the mechanisms of phototransduction. Several investigations have dealt with the composition of phosphoglycerides and various aspects of their metabolism, but these topics are far from being completely understood. During the past few years we have been studying minor lipids like diacylglycerols, free fatty acids and phosphatidic ecids from amphibian and mammalian retinas (Avelda~o and Baz~n, 1974, 1977). Differences in the total
~ . c . I . 1/1-4 x
381
382
N.
i.
Aveidano
de
Caidiroili
~n/d N.
C.
I~.lz;~l~
levels and fatty acid composition of these lipids in retina as compared with other tissues gave an insight about the structural and metabolic heterogeneity of retina phosphoglycerides. Parallel studies were carried out on the de novo biosynthetic pathways in the toad (Pascual de Baz£n and Baz~n, ]976 ) and the cattle retina in vitro (Giusto and Baz£n, 1979a). Every phospilolipid class, as well as neutral glycerides, were efficiently labeled with radioactive glycerol. The time course of the incorporation of this precursor showed a rapid labeling of phosphatidic acid, followed by that of phosphatidylinositol and diacylglycero]s. Subsequently, interesting differences between cattle and toad retinas were found: in the former the biosynthetic pathway quantitatively favoured the route P ~ DG ~ TG, whereas in the toad the biosynthesis of phosphatidylinositol predominated (Baz~n and {~o-workers, 1976a). Integrity of the retina seemed a sine qua non requisite for an efficient lipid labeling, since the synthesis of some of them was abolished and showed a different pattern in cell-free preparations (Baz£n and co-workers, unpublished). The entire retina in vitro has also proven a useful model to study the effects of drugs on the metabolism of phospholipids in the CNS (Baz~n and coworkers, 1976b). On the other hand, as shown in another chapter of this book, amphiphilic drugs may represent excellent tools to search for the regulation of lipid metabolism in the tissue. In retinas incubated with amphiphi]fc cationic drugs, an acute stimulation of acidic phosphoglyceride synthesis (PA, P] and PS) and a drastic inhibition of that of triacylglycerols and phospbatidylcholine takes place, phosphatidylethanolamine being inhibited to a lesser extent. Thus this was an interesting model to approach the problem of metabolic heterogeneity of retina phosphoglycerides. It is a well known fact that each phosphoglyceride class has its o~] defined fatty acid composition, which suggests some degree of selectivity of the biosynthetic enzymes towards certain molecular species of precursors. As an example, phospha~dy~nositol contains exceedingly high amounts of arachidonate and stearate [n the cattle retina, whereas phosphatidylethanolamine is mainly formed by docosahexaenoate and stearate This suggests that tetraenes are the major molecular species of P] and hexaenes those of PE. ~ i c h are the reactions leading to tbis specific composition? Do the biosynthetic enzymes select tetraenoic species of PA to be used in the route PA+ CDPDG+ PI on one hand and hexaenoic PAs for the route PA~ ])G~ PC(PE)on the other? In other tissues like liver and brain the time-course of pbosphatidylinositols labeling by various precursors injected in vivo showed that tetraenes may be synthesized by exchanging arachidonate for the fatty acid Jn the 2-position of glycerol in less unsaturat~.d molecules of PI (Holub and Kuksis, 1971a, [971b; Mac Doug~ll and others, 1975). Are these reactions operative in retina? This chapter summarizes some pieces of our recent work on the composition and biosynthesis of retina glycerolipid molecular species. It is shown that amphiphilie drugs can affect the labeling of the species of each phosphogly,e~i~]e t~ Jifferent extents, suggesting that some of them may be primarily inw~lve~] [n the ~Je ~o\,o pathways.
FATTY ACIDS AND MOLECULAR SPECIES OF RETINA GLYCEROLiPIDS The major fatty acids in cattle and toad retina £1 <'erolipid (:]asses aud in some me lecular species are presented in Table ]. High revels of docosahexaenoate characterize the composition of retina] phosphoglyeerides as it has been known for some years (for references see Daemen, 1973). This fatty acid is highly concentrated in toad retina glycerolipids, probably reflecting the contribution of the outer segments of rods. The high percentage of 22:6 in phosphatidylinositol (2%%) is surprising and contrasts with that found in cattle retina, where it only accounts for a 4% of the acyl groups. In both retinas, phosphatidic acid contains high levels of arachidonate and particularly of docosahexaenoate. This indicates that at least part of the polyenoic species of phosphoglycerides may be syi~thesized de novo by using the corresponding hexaeneic species of the precursor. The composition of diacylglycero]s shows a further relative enrichment in hexaenoic molecular
percentages
24+1.9 38,+2.1 8_+0.7
TG* PC PE
PC PE
PC PE
PC PE
PC PE
Monoenoic
Tetraenoic
Hexaenoic
Supraenoie
-
3"+0.4 3-+ 1.3
14-+0.4 9-+1.2
20,+0.6 6+0.7
35 -+0.8 26_+7.0
-
2+0.3 5,+ 3.0
25 + 0 . 5 29"+0.3
21 +_0.6 32-+0.3
11 _+ 0.5 28-+4.8
15 +_0.5 27 _+ 3.0
17"+2.0 15+0.7 24_+1.6
34+0.2 35+1.4 3-+0.5
26-+2.1 26_+ 1.6
18:0
-
2"+0.5 2 + 0.7
3,+0.i 5"+0.6
7 +0.3 8-+1.3
48 _+ 1.0 43+_5.7
-
18,+2.0 17_+0.2 5-+0.5
5"+0.2 7"+0.7 17_+ 2.3
11+0.6 9_+1.0
18:1
-
3-+0.1 4_+ 0.3
0.3-+ 0 . 0 4 0.3"+0.1
37 + 1.0 45+1.4
-
-
5+_0.7 5+0.1 8_+1.0
44+0.6 2_+0.2 7,+ 1.0
10,+0.6 27_+1.8
20:4
95
77+1.9"* 65 + 3.2
54+0.9 55-+1.9
-
-
-
17"+4.2 17!0.9 42-+1.3
4+0.4 39"+1.3 8 + 1.6
17+3.8 7+1.3
22:6
4 0.4
13 6
19 9
42 28
70 48
17_+2 23 ; 23 2;3
13 ; 13 0.6;0.8
5;7 19_+2.0
16:0
4 0.3
29 23
23 29
4 7
8 16
7+_0.7 14 ; 16 10;10
21 ; 23 13 ; 13 .
17 ; 18 20+1.1
18:0
.
N.D.
4.5 0.6
4 15
Iii 12
33 36
-
22+4.0 16 ; 15 8;8
9 ;9 1; I .
Ii ; I0 5_+1.0
18:1
Toad
.
9 3
0.4 0.4
39 43
-
-
60** 65
49 50
-
-
-
18-+1.9 28 ; 28 53;50
25 ; 26 61;58
37 ;36 42-+4.0
22:6
of Phospha-
4-+0.2 4 ;4 6;6
25 ; 25 2 ; 2
16 ; 14 7+1.0
20:4
Species
Lipids were isolated by TLC (Rouser and others, 1971; Rodrfguez de Turco and Baz~n, 1977; Baz~n and Joel, 1970). PC~ and PEs were resolved by argentation-TLC after converting the phospholipids into acetyl-diglycerides (Parkes and Thompson, 1973,1975). Fatty acids were analysed by GLC. All the fatty acids in the samples were considered for the 100% v a ~ ues. *, Avelda~o and Baz~n, 1974. **, other highly unsaturated fatty acids (22:4,22:5,24:6, etc) were also present. ***, the subfraction of the supraenes remaining at the origin of the AgNO3-TLC plates.
Supraene I, microsomal PC***
PC PE
Saturated
80_+0.5 45 _+ 2.0
8-+0.6 I+0.i 6 + 0.6
PI PS CL
B)
18-+2.8 18-+1.2
16:0
Cattle
of representative Fattx_Acids in Retina Glycerolipids ~ d in major Molecular tidylcholine (PC) and Phosphatidylethanolamine (PE).
PA DG*
A)
Glycerolipid
TABLE 1
mo o0 ~-
o
~" m
384
N. I .
A v e l d ~ n o de C a l d i r o n i
and N. G. Baz<~7
s p e c i e s , a s c o m p a r e d t o PA, i n t h e t o a d r e t i n a . This is consistent with the idea of selectivity suggested above, since it is evident that phosphatidate phosphohyd r o l a s e i s m o r e a c t i v e t o w a r d s h e x a e n o i c t h a n t o w a r d s o t h e r s p e c i e s o f PA on a p e r -
centual basis. In the cattle retina, on the other hand, arachidonate-containing species predominate in diacylglycerols. This is quite an amazing feature since tetraenes are not the major species in the lipids known to be formed from diacylglycerols, namely PC, PE and TG, and may reflect the contribution to thediglyceride pool of degradative reactions involving tetraenoic phosphoglycerides (Aveldafio and Baz~n, 1974). A relationship between phosphatidylinositol and diglycerides has been previously proposed in the rat brain (Keough and others, 1972). Diacylglycerols enriched in arachidonate and stereate are rapidly produced in brain shortly after decapitation (AveldaNo and Baz~n, 1975). Other reactions, such as the "back reaction" of choline (ethanolamine) phosphotransferase ~Kanoh and Ohno, 1976) could contribute to this complex pool. The molecular species of phosphatidylcholine and ethanolamine were separated by argentation chromatography after converting the phosphoglycerides into acetyldiglycerides (Parkes and Thompson, 1973, 1975). These non polar derivatives offer the advantages of giving far better and more reproducible resolution into molecular species than the intact phosphoglycerides, besides requiring lower activation temperatures. About eight groups of molecular species were identified in retina: saturates, monoenes, di-, tri-, tetra-, penta-, hexa-, and "supra"-enes. The saturates, containing exclusively saturated fatty acids, accounted for 18% of the phosphatidylcholines and i% of tile phosphatidylethanolamines (Table 2). It is evident from Table i that a great proportion of PC saturated species is dipalmitoyl-PC, i.e. 16:0 bound to positions 1 and 2 of the glycerol backbone, since palmitate accounts for an 80% of the fatty acids of this fraction. Other subspecies in the saturates may be 18:0-16:0 as well as other minor possibilities. In phosphatidylethanolamine, saturated molecular species are very low (Table 2), and their composition, (Table ]b), indicates that dipalmitoyl species are lower in PE than in PC on a percentual basis. Monoepes are formed by a monoenoic fatty acid (oleic, palmitoleic, etc) and a saturated one, giving rise to phospholipids containing one double bound per molecule. These species are important constituents of phosphatidylcholine, and are very low in PE (Table 2). The saturated fatty acids are palmitic and stearic, the former predominating in monoenoic-PCs and the latter in monoenoic-PEs (Table ib). Major subspecies in the dienoic fraction are monoenoic-monoenoic and saturated-dienoic fatty acid combinations. The tetraenoic species are predominantly formed by arachidonate-stesrate and arachidonate--palmitate pairs (Table ib), the former predominating in PE and the second being more important among tetraenoic PCs. Other combinations like 18:1-20:3 are also probable constituents of this fraction. Major subspecies of the hexaenoic fraction may be docosahexaenoate-stearate and docosahexaenoate-palmitate on the basis of the fatty acid composition shown in Table lb. Combinations like 18:1-22:5 may also occur in this fraction. Hexaenes are the predominant species of cattle retina phosphatidylethanolamines and the second in importance among chemical classes of PC (Table 2). Hexaenoic phosphatidylcholines predominate in the toad retina. Molecular species even more unsaturated than the hexaenes occurred in the phosphoglycerides of both toad (Avelda~o and Baz£n, 1977), and cattle retina. These species, which make up a 6% of the phosphatidylcholines, contribute to a 13% of the phosphatidylethanolamines in the mammalian retina. In the amphibian they form nearly half of the phosphatidylethanolamines (Table 2). Their fatty acid composition shows very high levels of docosahexaenoate (Table lb) as well as of other polyenoic acyl groups, whereas the content of saturated fatty acids is very low. This indicates that some of the species that form this fraction are dipolyunsaturated, some of which may contain docosahexaenoate in both positions of the glycerol backbone.
385
Retina Phosphoglycerides TABLE 2
Molecu!ar Species Composition of Retina Phosphoglycerides
Species Saturates Monoenes Dienes Tri- + Tetraenes Pentaenes Hexaenes Supraenes
Phosphatidylcholines
Phosphatidylethanolamines
Cattle (4)
Cattle (4)
18.3 28.5 4.7 11.9 3.4 26.9 6.3
+ ~ ~ + + ~ +
0.8 0.7 0.2 0.2 0.4 i.O 0.2
Toad (i) 7.7 30.1 4.9 12.3 2.8 38.2 4.3
1.0 3.9 2.7 12.O 3.2 63.9 13.0
+ + ~ + + + ~
O.i 0.4 0.3 0.5 0.5 2.0 1.9
Toad (i) 2.0 1.8 1.5 14.3 3.1 28.1 49.2
Results are expressed as weight %, and were obtained after resolving the acetyldiglycerides on silver ion-TIC and subjecting the me thyl esters derived from each species to GLC. Methyl-nonadecanoate was used as internal standard. In one case in which these species were separated, it was possible to find heptaenoic molecular species (mainly containing 18:1 and 22:6) all the way down to a species remaining at the origin of the AgNO3-TLC plates which contained 95% docosahexaenoate. The pattern of molecular species found in retina phosphoglycerides is therefore more complex than the one reported for most tissues, going from fully saturated to exceedingly unsaturated molecules. Since the latter are not present in other neural tissues in signigicant amounts, it is possible that these species of phosphoglycerides are highly concentrated in the outer segments of photoreceptors. Triacylglycerols containing docosahexaenoate in the three positions of the glycerol backbone were reported to occur in the tapetum lucidum of the sand trout (Nicol and others, 1972). However this is the first case, to our knowledge, that didocosahexaenoyl-phosphoglycerides are found in microsomes from a mammalian tissue. LABELING OF MOLECULAR SPECIES BY RADIOACTIVE GLYCEROL The polyenoic molecular species take up relatively more radioactivity from 14Cglycerol in the toad than in the cattle retina glycerolipids. This is in agreement with the higher proportion and higher unsaturation degree of phosphatidic acid and diglycerides in the toad retina. The distribution of radioactivity in phosphatidyleholines and -ethanolamines (Fig. i) is similar to the distribution of mass (Table 2) suggesting no large differences in specific activities of the species in both animals. This was not the case for phosphatidylinositol, at least in the cattle retina, since a predominant proportion of the radioactivity appeared in the fraction including penta-, hexa-, and supraenes. Taken together, these species com prise less than 15% of the total mass of the lipid, which indicates their very high biosynthetic rate. The "supraenoic" fraction is synthesized in retina at considerably high rates (Fig. i). These species are labeled in diacylglycerols at early incubation times, suggesting that supraenoic phosphoglycerides may be synthesized from the corresponding species of 1,2-diacylglycerols. These species also occur and are labeled from glycerol in phosphatidylinositol and phosphatidylserine (Figs. 4 and 5).
386
H. i. Aveld~no
de Caldironi
40
aud N. C. ii~z:in
Phosphatidylcholines
•~ l.O
I
Phospha tidylot hanolamines
g 2O
>6
r~ L. 0
6
5
4
3
2
1
0
1
0
Diacylgtycerots
40
2O t-
>6
o
Q_
40 20
6
~ 5
>6, 6,5 Fig.
4
3
2
itols
4
2,3
1,0
i.- Comparison of the distribution of radioactivity from labeled glycerol among molecular species of cattle ( ~ ) and toad ( • ) retina glycerolipids. The retinas were incubated for 30 min in the medium of Ames and Hastings (1956) in the presence of 5 ~Ci each of 14C_glycerol. The molecular species of PC, P E a n d ])G were resolved as acetyldiglycerides (Parkes and Thompson, 1973, 1975) and the Pls by spotting the intact phosphoglyceride (Holub and Kuksis, 1971 c).
The time-course of labeling of PCs and PEs shows that the polyenes are synthesized at relatively higher rates than the oligoenes (Fig. 2). This is also the case in diacylglycerols, where monoenes and saturates are produced at nearly linear rates while the uptake of radioactivity in the polyenes began to be decreased. In spite of the fact that tetraenes are the major fraction of diglycericies, they are not the most labeled, as observed in rat brain (MacDougall and others, 1975~. When retinas were incubated in the absence of labeled glycerol, after a preincubat ion with the precursor (Fig. 3) total diglyceride radioactivity rapidly dropped, indicating prelabelled diacylglycerols were being used for the biosynthesis of PE and PC. Again, the biosynthesis of polyenoic species of phosphoglycerides is fast er than that of oligoenes, which are still increasing at 40 min incubation. It has been shown in rat liver thac 2-1inoleyl-PC and 2-hexaeuos'l-PE ar~ the major products of the de novo pathway from 1,2-diacylglycerols (Kanoh and Ohno, 1976).
Retina Phosphogiycerides
I
r
~ E
20 ~
I
D6 I
I
i
I
"~
o>6
Z
o ~'40 c
o cD
PE
PC
¢-
387
i
I
i
I
I
i
1
i
uO "1 62
x~
20
x3
10
30 rain
10 30 of i n c u b a t i o n
10
30
14 Fig. 2.- Incorporati6n of radioactivity from C-glycerol in cattle retina phosphatidylcholines, -ethanolamines, and diacylglycerols. The retinas were incubated for the specified intervals in the presence of the precursor (5 ~Ci/retina).
PC ]
6o
c~
I
S
DG
PE I
i
, ,-~----o
O~, 6 •
m "~
6
z~ 5
40 20
o
t
i
I
I
iX
E~
•~ 40 20
10
30
10 min
of
30
10
30
incubation
Fig. 3.- Labeling of phosphatidylcholines,-ethanolamines, and diacylglycerols after removing radioactive glycerol. Cattle retinas were incubated for 15 min with 5 ~Ci/retina of 14C-glycerol. After extensive washings, incubation continued in glycerolfree media.
388
M. I. Aveld~no de Caldironi and N. G. Baz~n
The data in figs. 3 and 4 suggest that hexaenoic phosphoglycerides may be synthesized de novo in the retina, as suggested by their high rates of labeling and by the fact that hexaenoic diglycerides are exhausted faster than other species upon withdrawal of glycerol. The same possibility is suggested from the high levels of docosahexaenoate in phosphatidic acid (Table 1 and Giusto and Baz~n, 1979b) The changes in diacylglycerols do not account for the changes in phosphatidylcholi nes and-ethanolamines, but it should be kept in mind that an important proportion of the diglycerides are used for the synthesis of triglycerides in the cattle ret ina. Preliminary data suggest that saturated species of TG concentrate most of the glycerol in our in vitro model. In separate experiments carried out with 3H-glycerol, the distribution of the label among phosphatidylinositols changed as a function of time after removal of the precursor (Fig. 4). After a maximum taking place at 5 min, the percentage of radioactivity in oligoenes and saturates shows a continuous drop, while that in the tetraenes is increased. This suggests that tetraenoic phosphatidylinositols may be synthesized in retina from oligoenoic species trough acyl-exchange reactions introducing arachidonate as shown in liver and brain (Holub and Kuksis, 1971a, 1971b; MacDougall et al., 1975).
40.. > O
30 20
0
10 oO~2..o_o>6
o
I
I
L_ 0
U
E
I
101-
I
I
I
I
I
Q I
3 I
I
I
,
0
20 40 60 0 20 40 60 min of inc ubation
Fig. 4.- Distribution of radioactivity among molec ular species of phosphatidylinositol, after removing radioactive3glycerol. The retinas were incubated with H-glycerol during IO min. Then it was washed out and incubation proceeded for an additional period of 40 min.
389
Retina Phosphoglycerides EFFECT OF PROPRANOLOL ON THE LABELING OF MOLECULAR SPECIES OF RETINA PHOSPHOLIPIDS
Short-term incubations of retinas with propranolol result in a stimulated synthesis of phosphatidylinositol and phosphatidylserine (Ilincheta and others, this bookS. Table 3 shows that the incubation for only iO min with the drug causes a drastic unbalance in the synthesis of molecular species of both lipids, saturated and oligoenoic spe cies being more stimulated than the polyenes. TABLE 3 Effect of Propranolol on the Percentage of Radioactivity appearing in the Saturated + Oligoenoic Phosphatidylinositols and Phosphatidylserines.
Controls
(4)
Propranolol
(4)
Phosphatidylinositols
25.4 + 3.1
46.2+3.7
Phosphatidylserines
39.9 + 7.5
69.9+4.4
The retinas were incubated for iO min in the presence or absence of propranolol (0.5 mM). 3H-glycerol was added and in cubation proceeded for an additional period of IO minutes. Lipids were extracted and molecular species resolved on AgNO3-TLC, in the form of acetyldiglycerides. The radioactivities appearing in the polyenes on one hand and in the saturates + oligoenes on the other were added, and percentages calculated. The changes elicited by propranolol in the labeling of phosphatidylinositols, -se rines, -cholines and -ethanolamines in a microsomal preparation obtained after in cubating bovine retinas for 30 min in the presence of glycerol are shown in Fig. 5. The labeling of monoenoic and saturated PCs is inhibited 15 fold while the oth er species are inhibited about iO fold as compared to controls. The stimulation of these species in PI is about 7 fold, while the rest of the species undergo a 3 to 5 fold stimulation during this period. The highest changes in saturated and oligo enoic species appear in phosphatidylserine: they are increased about i0 times in the presence of other species stimulation of 2 fold. In contrast with phosphatidylcholine and triacylglycerol, the inhibition of the labeling of phosphatidylethanolamine by propranolol is weaker. Moreover, depend ing on the time of preincubation with the drug, no inhibition at all has occurred in some experiments. When the effect of propranolol was surveyed in phosphatidyl ethanolamines (Fig. 5), the polyenes were inhibited while the saturates and oligo enes were stimulated. This is a remarkable example of metabolic heterogeneity in a single phosphoglyceride and with a single precursor, and suggests a different metabolic origin of molecular classes of phosphatidylethanolamine. The inhibited species could mainly arise from diacylglycerols through ethanolamine phosphotransferase activity as it is probably the case for phosphatidylcholines through choline phosphotransferase. Due to the definite stimulation of the oligoenoic+saturated species in phosphatidylserine, which attain very high specific ac tivities if the mass involved is considered, a precursor-product relationship among
390
M. i. Aveld~no de Caidirotli and N. C. BazSll
both phosphoglycerides in retina is suggested, specifically involving these specie~ 3H-labeled serine is actively taken up by phosphatidylserine and phosphatidylethanolamine, perhaps through phosphatidylserine decarboxylation, the overall process being stimulated by propranolol (Ilincheta and others, this volume).
PHOSPHATIDYLCHOLINES
PHOSPHATIDYL -
INOSITOLS
1200
!
6 C _
800 o L
0
_
>6
"5
oo
/ /
×
E
800
5
1
400
Io
;~6
¢ ¢
PHOSPHATIDYL E THANOLAMINE S
I
JJlit
PHOSPHATIDYLSERINES
'
I
400
Fig. 5.- Effect of propranolol on the labeling of molecular species of microsomal phosphoglycerides obtained from cattle retinas incubated 30 min with 3H-glycerol. The retinas were preincubated for 10 min before the addition of glycerol; the incubating media contained no CaCI2 and 0.5 mM ( • ), or no ( [] ) propranolol. Microsomes were prepared, lipids were extracted and molecular species resolved.
When an entire tissue like the retina is incuabated 30 min with an u b i q u i t o u s precursor like glycerol, besides assesing de novo synthesis the contribution from one or more of the following reactions to the f~nal distribution of radioactivity among species of phosphoglycerides may be expected: a) acyl-exchange reactions (any phospholipid); b) N-methylation (species of PE+PCs); c) reversion of the bio synthetic routes (radioactive PCs or PEs*labeled DG, which in turn could be recycled); d) decarboxylation (PCs+PEs); e) base-exchange reactions (PCs~-~PEs+~PSs); f) other reactions. The specificities of some of these reactions have been investigated in other tissues but the retina has still many answers to give, by using suitable precursors and pharmacological tools, on the regulation of the intermediary metabolism of its lipids.
Retina Phosphoglycerides
391
REFERENCES Ames, A.lll, and B. Hastings (1956). Studies on water and electrolytes in nervous tissue, l.Rabbit retina: Methods and interpretation of data, J. Neurophysiol., 19, 201-212. Aveldafio, M.l.,and N.G. Baz~n (1974). Free fatty acids, diacyl- and triacylglycerols and total phospholipids in vertebrate retina: comparison with brain, choroid and plasma. J. Neurochem., 23, 1127-1135. Avelda~o, M.I., and N.G. Baz~n (1975). Rapid production of diacylglycerols enriched in arachidonate and stearate during early brain ischemia. J. Neurochem., 25, 919920. Avelda~o de Caldironi, M.I.,and N.G. Baz~n (1977). Acyl groups, molecular species and labeling by 14C-glycerol and 3H-arachidonic acid of vertebrate retina glycerolipids. In N.G. Baz~n, R.R. Brenner, and N.M. Giusto (Eds) Function and Biosynthesis of Lipids. Plenum Press, N.Y. pp. 397-404. Baz~n, N.G., M.I. Avelda~o~ H.E. Pascual de Baz~n, and N.M. Giusto (1976a). Metabolism of retina acylglycerides and arachidonic acid. In R. Paoletti, G. Porcelatti,and G. Jacini (Eds). Lipids., Raven Press,N.Y. pp. 89-97. Baz~n, N.G., M.G. Ilincheta de Boschero, N.M. Giusto, and H.E. Pascual de Baz~n (1976b). De novo glycerolipid biosynthesis in the toad and cattle retina. Redirecting of the pathway by propranolol and ph~ntolamine. In G. Porcellati, L. Amaducci, and C. Galli (Eds.). Function and metabolism of phospholipids in the central and peripheral nervous systems. Plenum Press, N.Y., pp. 139-148. Baz~n, N.G.,and C.D. Joel (1970). Gradient-thickness thin layer chromatography for the isolation and analysis of trace amounts of free fatty acids in large lipid samples. J. Lipi~ Res., ii, 42-47. Daemen, F.J.M. (1973). Vertebrate rod outer segment membranes. Biochem. Biophys. Acta, 300: 255-288. Giusto, N.M., and N.G. Baz~n (1979a). Phospholipids and acylglycerols biosynthesis 14 14 and CO 2 production from ( C)-glycerol in the bovine retina: the effects of incubation time, oxygen and glucose. Exp. Eye Res., 29, 155-168. Giusto, N.M., and N.G. Baz~n (1979b). Phosphatidic acid of retinal microsomes contains a high proportion of docosahexaenoate. Biochem. Biophys. Res. Commun., (in press). Holub, B.J.,and A. Kuksis (1971a). Structural and metabolic interrelationships among gl~cerop|~ha~des ofratliver in vivo. Can.J.Biochem., 49; 1347-1356. Holub, B.J.,and A. Kuksis (1971b). Differential distribution of orthophosphate 32p and glycerol- 14C among molecular species of phosphatidylinositols of rat liver in vivo. J. Lipid Res., 12, 699-705. Holub, B.J., and A. Kuksis (1971c) Resolution of intact phosphatidilinositols by argentation thin-layer-chromatography. J. Lipid Res., 12, 510-512. Kanoh, H., and K. Ohno (1976). The role of 1,2-diacyiglycerol: CDP-choline (ethanolamine) phosphotransferase in forming phospholipid species in rat liver. In P. Paoletti, G. Porcellati, and G. Jacini (Eds). Raven Press, N.Y., pp. 39-48. Keough, K.M.W., G. MacDonald, and W. Thompson (1972). A possible relation between phosphoinositides and the diglyceride pool in rat brain. Biochim. Biophys.Acta, 270, 337-347. MacDonald, G., R.R. Baker, and W. Thompson (1975). Selective synthesis of molecular classes of phosphatidic acid, diacylglycerol and phosphatidylinositol in rat brain J, Neurochem., 24, 655-661. Nicol, J.A.C.~ H.J. Arnott, G.R. Mizzuno, E.C, Ellison, and J.R. Chipault (1972). Oeurrence of glyceryl tridocosahexaenoate in the eye of the sand trout Cynoscion arenarius. Lipids, 7, 171-177. Parkes, J.G.,and W. Thompson (1973). Structural and metabolic relation between molecular classes of phosphatidylcholine in mitochondria and endoplasmic reticulum of guinea pig liver. J.biol. Chem. 245, 6655-6662. Parkes, J.G.,and W. Thompson (1975). Phosphatidilethanolamine in liver mitochondria and endoplasmic reticulum. Molecular species distribution and turnover.
392
M. I. Aveld~no de Caldironi and N. G. Baz~n
Can. J. Biochem., 53, 698-705. Pascual de Baz~n, H.E.-~and N.G. Baz~n (1976). Phospholipid composition and (14C)~ glycerol incorporation into glycerolipids of toad retina and brain, J. Neurochem., 27, 1051-1057, Rodrlguez de Turco, E.,and N.G. Baz~n (1977). Simple preparative and analytical thin-layer chromatographic method for the rapid isolation of phosphatidic acid from tissue lipid extracts. J_. Chromatosr., 137, 194-197, Rouser, G., S. Fleischer, and A° Yamamoto (1970)-. Two dimensional thin layer chroma. tographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids, 5, 494~496.
Vol. Pergamon Press Ltd.
I, pp. 381-392. 1980. Printed in Great Britain.
CO}~OSITION AND BIOSYNTHESIS OF MOLECULAR SPECIES OF RETINA PHOSPHOGLYCERIDES M. I. Avelda~o de Caldironi and N. G. Baz~n Instituto de Investigaciones Bioqufmicas, Universidad Nacional del Sur y Consejo Nacional de Investigaciones Cientfficas y Tgcnicas, Bahfa Blanca, Argentina.
ABSTRACT Recent studies on the fatty acid composition and labeling by glycerol of individual molecular species of retinal phosphoglycerides are presented. Docosahexaenoate-containing species are important constituents of retina phospholipids. 22:6 and saturated fatty acids appear in similar amounts in hexaenes (nearly 50% each). Very high percentages of 22:6 and other polyenoic fatty acids are found in molecular species containing negligible amounts of saturated fatty acids, indicating the occurrence of dipolyunsaturated phosphoglycerides, including didocosahexaenoate. On the other extreme, fully saturated species occur. 22:6-containing species are synthesized at high rates in most phosphoglycerides including phosphatidylinositol, which is mainly formed by tetraenoic classes. Oligoenes and saturates are synthesized de novo and once formed they may serve as precursors of others (e.g. tetraene~ by acyl-exchange reactions. Propranolol induces rapid modifications in the de novo biosynthesis of retina glycerolipids. Oligoenes and saturates are the most stimulated species in PI and PS, and the most inhibited ones in PC. In PE, the labeling of these species is stimulated, while the polyenes are inhibited. This remarkable example of metabolic heterogeneity in a single phosphoglyceride opens new possibilities in the study of the regulation of retinal membrane lipid biosynthesis. KEYWORDS Retina, glycerophospholipids, molecular species, glycerol uptake, docosahexaenoate, arachidonate, polyenes, lipid synthesis, propranolol.
INTRODUCTION Photoreceptor cells and especially their rod outer segments, have been the main sub ject of studies on retinal biochemistry to date, due to the development of methods-for obtaining high purity-ROS preparations and to the interest in the mechanisms of phototransduction. Several investigations have dealt with the composition of phosphoglycerides and various aspects of their metabolism, but these topics are far from being completely understood. During the past few years we have been studying minor lipids like diacylglycerols, free fatty acids and phosphatidic ecids from amphibian and mammalian retinas (Avelda~o and Baz~n, 1974, 1977). Differences in the total
~ . c . I . 1/1-4 x
381
382
N.
i.
Aveidano
de
Caidiroili
~n/d N.
C.
I~.lz;~l~
levels and fatty acid composition of these lipids in retina as compared with other tissues gave an insight about the structural and metabolic heterogeneity of retina phosphoglycerides. Parallel studies were carried out on the de novo biosynthetic pathways in the toad (Pascual de Baz£n and Baz~n, ]976 ) and the cattle retina in vitro (Giusto and Baz£n, 1979a). Every phospilolipid class, as well as neutral glycerides, were efficiently labeled with radioactive glycerol. The time course of the incorporation of this precursor showed a rapid labeling of phosphatidic acid, followed by that of phosphatidylinositol and diacylglycero]s. Subsequently, interesting differences between cattle and toad retinas were found: in the former the biosynthetic pathway quantitatively favoured the route P ~ DG ~ TG, whereas in the toad the biosynthesis of phosphatidylinositol predominated (Baz~n and {~o-workers, 1976a). Integrity of the retina seemed a sine qua non requisite for an efficient lipid labeling, since the synthesis of some of them was abolished and showed a different pattern in cell-free preparations (Baz£n and co-workers, unpublished). The entire retina in vitro has also proven a useful model to study the effects of drugs on the metabolism of phospholipids in the CNS (Baz~n and coworkers, 1976b). On the other hand, as shown in another chapter of this book, amphiphilic drugs may represent excellent tools to search for the regulation of lipid metabolism in the tissue. In retinas incubated with amphiphi]fc cationic drugs, an acute stimulation of acidic phosphoglyceride synthesis (PA, P] and PS) and a drastic inhibition of that of triacylglycerols and phospbatidylcholine takes place, phosphatidylethanolamine being inhibited to a lesser extent. Thus this was an interesting model to approach the problem of metabolic heterogeneity of retina phosphoglycerides. It is a well known fact that each phosphoglyceride class has its o~] defined fatty acid composition, which suggests some degree of selectivity of the biosynthetic enzymes towards certain molecular species of precursors. As an example, phospha~dy~nositol contains exceedingly high amounts of arachidonate and stearate [n the cattle retina, whereas phosphatidylethanolamine is mainly formed by docosahexaenoate and stearate This suggests that tetraenes are the major molecular species of P] and hexaenes those of PE. ~ i c h are the reactions leading to tbis specific composition? Do the biosynthetic enzymes select tetraenoic species of PA to be used in the route PA+ CDPDG+ PI on one hand and hexaenoic PAs for the route PA~ ])G~ PC(PE)on the other? In other tissues like liver and brain the time-course of pbosphatidylinositols labeling by various precursors injected in vivo showed that tetraenes may be synthesized by exchanging arachidonate for the fatty acid Jn the 2-position of glycerol in less unsaturat~.d molecules of PI (Holub and Kuksis, 1971a, [971b; Mac Doug~ll and others, 1975). Are these reactions operative in retina? This chapter summarizes some pieces of our recent work on the composition and biosynthesis of retina glycerolipid molecular species. It is shown that amphiphilie drugs can affect the labeling of the species of each phosphogly,e~i~]e t~ Jifferent extents, suggesting that some of them may be primarily inw~lve~] [n the ~Je ~o\,o pathways.
FATTY ACIDS AND MOLECULAR SPECIES OF RETINA GLYCEROLiPIDS The major fatty acids in cattle and toad retina £1 <'erolipid (:]asses aud in some me lecular species are presented in Table ]. High revels of docosahexaenoate characterize the composition of retina] phosphoglyeerides as it has been known for some years (for references see Daemen, 1973). This fatty acid is highly concentrated in toad retina glycerolipids, probably reflecting the contribution of the outer segments of rods. The high percentage of 22:6 in phosphatidylinositol (2%%) is surprising and contrasts with that found in cattle retina, where it only accounts for a 4% of the acyl groups. In both retinas, phosphatidic acid contains high levels of arachidonate and particularly of docosahexaenoate. This indicates that at least part of the polyenoic species of phosphoglycerides may be syi~thesized de novo by using the corresponding hexaeneic species of the precursor. The composition of diacylglycero]s shows a further relative enrichment in hexaenoic molecular
percentages
24+1.9 38,+2.1 8_+0.7
TG* PC PE
PC PE
PC PE
PC PE
PC PE
Monoenoic
Tetraenoic
Hexaenoic
Supraenoie
-
3"+0.4 3-+ 1.3
14-+0.4 9-+1.2
20,+0.6 6+0.7
35 -+0.8 26_+7.0
-
2+0.3 5,+ 3.0
25 + 0 . 5 29"+0.3
21 +_0.6 32-+0.3
11 _+ 0.5 28-+4.8
15 +_0.5 27 _+ 3.0
17"+2.0 15+0.7 24_+1.6
34+0.2 35+1.4 3-+0.5
26-+2.1 26_+ 1.6
18:0
-
2"+0.5 2 + 0.7
3,+0.i 5"+0.6
7 +0.3 8-+1.3
48 _+ 1.0 43+_5.7
-
18,+2.0 17_+0.2 5-+0.5
5"+0.2 7"+0.7 17_+ 2.3
11+0.6 9_+1.0
18:1
-
3-+0.1 4_+ 0.3
0.3-+ 0 . 0 4 0.3"+0.1
37 + 1.0 45+1.4
-
-
5+_0.7 5+0.1 8_+1.0
44+0.6 2_+0.2 7,+ 1.0
10,+0.6 27_+1.8
20:4
95
77+1.9"* 65 + 3.2
54+0.9 55-+1.9
-
-
-
17"+4.2 17!0.9 42-+1.3
4+0.4 39"+1.3 8 + 1.6
17+3.8 7+1.3
22:6
4 0.4
13 6
19 9
42 28
70 48
17_+2 23 ; 23 2;3
13 ; 13 0.6;0.8
5;7 19_+2.0
16:0
4 0.3
29 23
23 29
4 7
8 16
7+_0.7 14 ; 16 10;10
21 ; 23 13 ; 13 .
17 ; 18 20+1.1
18:0
.
N.D.
4.5 0.6
4 15
Iii 12
33 36
-
22+4.0 16 ; 15 8;8
9 ;9 1; I .
Ii ; I0 5_+1.0
18:1
Toad
.
9 3
0.4 0.4
39 43
-
-
60** 65
49 50
-
-
-
18-+1.9 28 ; 28 53;50
25 ; 26 61;58
37 ;36 42-+4.0
22:6
of Phospha-
4-+0.2 4 ;4 6;6
25 ; 25 2 ; 2
16 ; 14 7+1.0
20:4
Species
Lipids were isolated by TLC (Rouser and others, 1971; Rodrfguez de Turco and Baz~n, 1977; Baz~n and Joel, 1970). PC~ and PEs were resolved by argentation-TLC after converting the phospholipids into acetyl-diglycerides (Parkes and Thompson, 1973,1975). Fatty acids were analysed by GLC. All the fatty acids in the samples were considered for the 100% v a ~ ues. *, Avelda~o and Baz~n, 1974. **, other highly unsaturated fatty acids (22:4,22:5,24:6, etc) were also present. ***, the subfraction of the supraenes remaining at the origin of the AgNO3-TLC plates.
Supraene I, microsomal PC***
PC PE
Saturated
80_+0.5 45 _+ 2.0
8-+0.6 I+0.i 6 + 0.6
PI PS CL
B)
18-+2.8 18-+1.2
16:0
Cattle
of representative Fattx_Acids in Retina Glycerolipids ~ d in major Molecular tidylcholine (PC) and Phosphatidylethanolamine (PE).
PA DG*
A)
Glycerolipid
TABLE 1
mo o0 ~-
o
~" m
384
N. I .
A v e l d ~ n o de C a l d i r o n i
and N. G. Baz<~7
s p e c i e s , a s c o m p a r e d t o PA, i n t h e t o a d r e t i n a . This is consistent with the idea of selectivity suggested above, since it is evident that phosphatidate phosphohyd r o l a s e i s m o r e a c t i v e t o w a r d s h e x a e n o i c t h a n t o w a r d s o t h e r s p e c i e s o f PA on a p e r -
centual basis. In the cattle retina, on the other hand, arachidonate-containing species predominate in diacylglycerols. This is quite an amazing feature since tetraenes are not the major species in the lipids known to be formed from diacylglycerols, namely PC, PE and TG, and may reflect the contribution to thediglyceride pool of degradative reactions involving tetraenoic phosphoglycerides (Aveldafio and Baz~n, 1974). A relationship between phosphatidylinositol and diglycerides has been previously proposed in the rat brain (Keough and others, 1972). Diacylglycerols enriched in arachidonate and stereate are rapidly produced in brain shortly after decapitation (AveldaNo and Baz~n, 1975). Other reactions, such as the "back reaction" of choline (ethanolamine) phosphotransferase ~Kanoh and Ohno, 1976) could contribute to this complex pool. The molecular species of phosphatidylcholine and ethanolamine were separated by argentation chromatography after converting the phosphoglycerides into acetyldiglycerides (Parkes and Thompson, 1973, 1975). These non polar derivatives offer the advantages of giving far better and more reproducible resolution into molecular species than the intact phosphoglycerides, besides requiring lower activation temperatures. About eight groups of molecular species were identified in retina: saturates, monoenes, di-, tri-, tetra-, penta-, hexa-, and "supra"-enes. The saturates, containing exclusively saturated fatty acids, accounted for 18% of the phosphatidylcholines and i% of tile phosphatidylethanolamines (Table 2). It is evident from Table i that a great proportion of PC saturated species is dipalmitoyl-PC, i.e. 16:0 bound to positions 1 and 2 of the glycerol backbone, since palmitate accounts for an 80% of the fatty acids of this fraction. Other subspecies in the saturates may be 18:0-16:0 as well as other minor possibilities. In phosphatidylethanolamine, saturated molecular species are very low (Table 2), and their composition, (Table ]b), indicates that dipalmitoyl species are lower in PE than in PC on a percentual basis. Monoepes are formed by a monoenoic fatty acid (oleic, palmitoleic, etc) and a saturated one, giving rise to phospholipids containing one double bound per molecule. These species are important constituents of phosphatidylcholine, and are very low in PE (Table 2). The saturated fatty acids are palmitic and stearic, the former predominating in monoenoic-PCs and the latter in monoenoic-PEs (Table ib). Major subspecies in the dienoic fraction are monoenoic-monoenoic and saturated-dienoic fatty acid combinations. The tetraenoic species are predominantly formed by arachidonate-stesrate and arachidonate--palmitate pairs (Table ib), the former predominating in PE and the second being more important among tetraenoic PCs. Other combinations like 18:1-20:3 are also probable constituents of this fraction. Major subspecies of the hexaenoic fraction may be docosahexaenoate-stearate and docosahexaenoate-palmitate on the basis of the fatty acid composition shown in Table lb. Combinations like 18:1-22:5 may also occur in this fraction. Hexaenes are the predominant species of cattle retina phosphatidylethanolamines and the second in importance among chemical classes of PC (Table 2). Hexaenoic phosphatidylcholines predominate in the toad retina. Molecular species even more unsaturated than the hexaenes occurred in the phosphoglycerides of both toad (Avelda~o and Baz£n, 1977), and cattle retina. These species, which make up a 6% of the phosphatidylcholines, contribute to a 13% of the phosphatidylethanolamines in the mammalian retina. In the amphibian they form nearly half of the phosphatidylethanolamines (Table 2). Their fatty acid composition shows very high levels of docosahexaenoate (Table lb) as well as of other polyenoic acyl groups, whereas the content of saturated fatty acids is very low. This indicates that some of the species that form this fraction are dipolyunsaturated, some of which may contain docosahexaenoate in both positions of the glycerol backbone.
385
Retina Phosphoglycerides TABLE 2
Molecu!ar Species Composition of Retina Phosphoglycerides
Species Saturates Monoenes Dienes Tri- + Tetraenes Pentaenes Hexaenes Supraenes
Phosphatidylcholines
Phosphatidylethanolamines
Cattle (4)
Cattle (4)
18.3 28.5 4.7 11.9 3.4 26.9 6.3
+ ~ ~ + + ~ +
0.8 0.7 0.2 0.2 0.4 i.O 0.2
Toad (i) 7.7 30.1 4.9 12.3 2.8 38.2 4.3
1.0 3.9 2.7 12.O 3.2 63.9 13.0
+ + ~ + + + ~
O.i 0.4 0.3 0.5 0.5 2.0 1.9
Toad (i) 2.0 1.8 1.5 14.3 3.1 28.1 49.2
Results are expressed as weight %, and were obtained after resolving the acetyldiglycerides on silver ion-TIC and subjecting the me thyl esters derived from each species to GLC. Methyl-nonadecanoate was used as internal standard. In one case in which these species were separated, it was possible to find heptaenoic molecular species (mainly containing 18:1 and 22:6) all the way down to a species remaining at the origin of the AgNO3-TLC plates which contained 95% docosahexaenoate. The pattern of molecular species found in retina phosphoglycerides is therefore more complex than the one reported for most tissues, going from fully saturated to exceedingly unsaturated molecules. Since the latter are not present in other neural tissues in signigicant amounts, it is possible that these species of phosphoglycerides are highly concentrated in the outer segments of photoreceptors. Triacylglycerols containing docosahexaenoate in the three positions of the glycerol backbone were reported to occur in the tapetum lucidum of the sand trout (Nicol and others, 1972). However this is the first case, to our knowledge, that didocosahexaenoyl-phosphoglycerides are found in microsomes from a mammalian tissue. LABELING OF MOLECULAR SPECIES BY RADIOACTIVE GLYCEROL The polyenoic molecular species take up relatively more radioactivity from 14Cglycerol in the toad than in the cattle retina glycerolipids. This is in agreement with the higher proportion and higher unsaturation degree of phosphatidic acid and diglycerides in the toad retina. The distribution of radioactivity in phosphatidyleholines and -ethanolamines (Fig. i) is similar to the distribution of mass (Table 2) suggesting no large differences in specific activities of the species in both animals. This was not the case for phosphatidylinositol, at least in the cattle retina, since a predominant proportion of the radioactivity appeared in the fraction including penta-, hexa-, and supraenes. Taken together, these species com prise less than 15% of the total mass of the lipid, which indicates their very high biosynthetic rate. The "supraenoic" fraction is synthesized in retina at considerably high rates (Fig. i). These species are labeled in diacylglycerols at early incubation times, suggesting that supraenoic phosphoglycerides may be synthesized from the corresponding species of 1,2-diacylglycerols. These species also occur and are labeled from glycerol in phosphatidylinositol and phosphatidylserine (Figs. 4 and 5).
386
H. i. Aveld~no
de Caldironi
40
aud N. C. ii~z:in
Phosphatidylcholines
•~ l.O
I
Phospha tidylot hanolamines
g 2O
>6
r~ L. 0
6
5
4
3
2
1
0
1
0
Diacylgtycerots
40
2O t-
>6
o
Q_
40 20
6
~ 5
>6, 6,5 Fig.
4
3
2
itols
4
2,3
1,0
i.- Comparison of the distribution of radioactivity from labeled glycerol among molecular species of cattle ( ~ ) and toad ( • ) retina glycerolipids. The retinas were incubated for 30 min in the medium of Ames and Hastings (1956) in the presence of 5 ~Ci each of 14C_glycerol. The molecular species of PC, P E a n d ])G were resolved as acetyldiglycerides (Parkes and Thompson, 1973, 1975) and the Pls by spotting the intact phosphoglyceride (Holub and Kuksis, 1971 c).
The time-course of labeling of PCs and PEs shows that the polyenes are synthesized at relatively higher rates than the oligoenes (Fig. 2). This is also the case in diacylglycerols, where monoenes and saturates are produced at nearly linear rates while the uptake of radioactivity in the polyenes began to be decreased. In spite of the fact that tetraenes are the major fraction of diglycericies, they are not the most labeled, as observed in rat brain (MacDougall and others, 1975~. When retinas were incubated in the absence of labeled glycerol, after a preincubat ion with the precursor (Fig. 3) total diglyceride radioactivity rapidly dropped, indicating prelabelled diacylglycerols were being used for the biosynthesis of PE and PC. Again, the biosynthesis of polyenoic species of phosphoglycerides is fast er than that of oligoenes, which are still increasing at 40 min incubation. It has been shown in rat liver thac 2-1inoleyl-PC and 2-hexaeuos'l-PE ar~ the major products of the de novo pathway from 1,2-diacylglycerols (Kanoh and Ohno, 1976).
Retina Phosphogiycerides
I
r
~ E
20 ~
I
D6 I
I
i
I
"~
o>6
Z
o ~'40 c
o cD
PE
PC
¢-
387
i
I
i
I
I
i
1
i
uO "1 62
x~
20
x3
10
30 rain
10 30 of i n c u b a t i o n
10
30
14 Fig. 2.- Incorporati6n of radioactivity from C-glycerol in cattle retina phosphatidylcholines, -ethanolamines, and diacylglycerols. The retinas were incubated for the specified intervals in the presence of the precursor (5 ~Ci/retina).
PC ]
6o
c~
I
S
DG
PE I
i
, ,-~----o
O~, 6 •
m "~
6
z~ 5
40 20
o
t
i
I
I
iX
E~
•~ 40 20
10
30
10 min
of
30
10
30
incubation
Fig. 3.- Labeling of phosphatidylcholines,-ethanolamines, and diacylglycerols after removing radioactive glycerol. Cattle retinas were incubated for 15 min with 5 ~Ci/retina of 14C-glycerol. After extensive washings, incubation continued in glycerolfree media.
388
M. I. Aveld~no de Caldironi and N. G. Baz~n
The data in figs. 3 and 4 suggest that hexaenoic phosphoglycerides may be synthesized de novo in the retina, as suggested by their high rates of labeling and by the fact that hexaenoic diglycerides are exhausted faster than other species upon withdrawal of glycerol. The same possibility is suggested from the high levels of docosahexaenoate in phosphatidic acid (Table 1 and Giusto and Baz~n, 1979b) The changes in diacylglycerols do not account for the changes in phosphatidylcholi nes and-ethanolamines, but it should be kept in mind that an important proportion of the diglycerides are used for the synthesis of triglycerides in the cattle ret ina. Preliminary data suggest that saturated species of TG concentrate most of the glycerol in our in vitro model. In separate experiments carried out with 3H-glycerol, the distribution of the label among phosphatidylinositols changed as a function of time after removal of the precursor (Fig. 4). After a maximum taking place at 5 min, the percentage of radioactivity in oligoenes and saturates shows a continuous drop, while that in the tetraenes is increased. This suggests that tetraenoic phosphatidylinositols may be synthesized in retina from oligoenoic species trough acyl-exchange reactions introducing arachidonate as shown in liver and brain (Holub and Kuksis, 1971a, 1971b; MacDougall et al., 1975).
40.. > O
30 20
0
10 oO~2..o_o>6
o
I
I
L_ 0
U
E
I
101-
I
I
I
I
I
Q I
3 I
I
I
,
0
20 40 60 0 20 40 60 min of inc ubation
Fig. 4.- Distribution of radioactivity among molec ular species of phosphatidylinositol, after removing radioactive3glycerol. The retinas were incubated with H-glycerol during IO min. Then it was washed out and incubation proceeded for an additional period of 40 min.
389
Retina Phosphoglycerides EFFECT OF PROPRANOLOL ON THE LABELING OF MOLECULAR SPECIES OF RETINA PHOSPHOLIPIDS
Short-term incubations of retinas with propranolol result in a stimulated synthesis of phosphatidylinositol and phosphatidylserine (Ilincheta and others, this bookS. Table 3 shows that the incubation for only iO min with the drug causes a drastic unbalance in the synthesis of molecular species of both lipids, saturated and oligoenoic spe cies being more stimulated than the polyenes. TABLE 3 Effect of Propranolol on the Percentage of Radioactivity appearing in the Saturated + Oligoenoic Phosphatidylinositols and Phosphatidylserines.
Controls
(4)
Propranolol
(4)
Phosphatidylinositols
25.4 + 3.1
46.2+3.7
Phosphatidylserines
39.9 + 7.5
69.9+4.4
The retinas were incubated for iO min in the presence or absence of propranolol (0.5 mM). 3H-glycerol was added and in cubation proceeded for an additional period of IO minutes. Lipids were extracted and molecular species resolved on AgNO3-TLC, in the form of acetyldiglycerides. The radioactivities appearing in the polyenes on one hand and in the saturates + oligoenes on the other were added, and percentages calculated. The changes elicited by propranolol in the labeling of phosphatidylinositols, -se rines, -cholines and -ethanolamines in a microsomal preparation obtained after in cubating bovine retinas for 30 min in the presence of glycerol are shown in Fig. 5. The labeling of monoenoic and saturated PCs is inhibited 15 fold while the oth er species are inhibited about iO fold as compared to controls. The stimulation of these species in PI is about 7 fold, while the rest of the species undergo a 3 to 5 fold stimulation during this period. The highest changes in saturated and oligo enoic species appear in phosphatidylserine: they are increased about i0 times in the presence of other species stimulation of 2 fold. In contrast with phosphatidylcholine and triacylglycerol, the inhibition of the labeling of phosphatidylethanolamine by propranolol is weaker. Moreover, depend ing on the time of preincubation with the drug, no inhibition at all has occurred in some experiments. When the effect of propranolol was surveyed in phosphatidyl ethanolamines (Fig. 5), the polyenes were inhibited while the saturates and oligo enes were stimulated. This is a remarkable example of metabolic heterogeneity in a single phosphoglyceride and with a single precursor, and suggests a different metabolic origin of molecular classes of phosphatidylethanolamine. The inhibited species could mainly arise from diacylglycerols through ethanolamine phosphotransferase activity as it is probably the case for phosphatidylcholines through choline phosphotransferase. Due to the definite stimulation of the oligoenoic+saturated species in phosphatidylserine, which attain very high specific ac tivities if the mass involved is considered, a precursor-product relationship among
390
M. i. Aveld~no de Caidirotli and N. C. BazSll
both phosphoglycerides in retina is suggested, specifically involving these specie~ 3H-labeled serine is actively taken up by phosphatidylserine and phosphatidylethanolamine, perhaps through phosphatidylserine decarboxylation, the overall process being stimulated by propranolol (Ilincheta and others, this volume).
PHOSPHATIDYLCHOLINES
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Fig. 5.- Effect of propranolol on the labeling of molecular species of microsomal phosphoglycerides obtained from cattle retinas incubated 30 min with 3H-glycerol. The retinas were preincubated for 10 min before the addition of glycerol; the incubating media contained no CaCI2 and 0.5 mM ( • ), or no ( [] ) propranolol. Microsomes were prepared, lipids were extracted and molecular species resolved.
When an entire tissue like the retina is incuabated 30 min with an u b i q u i t o u s precursor like glycerol, besides assesing de novo synthesis the contribution from one or more of the following reactions to the f~nal distribution of radioactivity among species of phosphoglycerides may be expected: a) acyl-exchange reactions (any phospholipid); b) N-methylation (species of PE+PCs); c) reversion of the bio synthetic routes (radioactive PCs or PEs*labeled DG, which in turn could be recycled); d) decarboxylation (PCs+PEs); e) base-exchange reactions (PCs~-~PEs+~PSs); f) other reactions. The specificities of some of these reactions have been investigated in other tissues but the retina has still many answers to give, by using suitable precursors and pharmacological tools, on the regulation of the intermediary metabolism of its lipids.
Retina Phosphoglycerides
391
REFERENCES Ames, A.lll, and B. Hastings (1956). Studies on water and electrolytes in nervous tissue, l.Rabbit retina: Methods and interpretation of data, J. Neurophysiol., 19, 201-212. Aveldafio, M.l.,and N.G. Baz~n (1974). Free fatty acids, diacyl- and triacylglycerols and total phospholipids in vertebrate retina: comparison with brain, choroid and plasma. J. Neurochem., 23, 1127-1135. Avelda~o, M.I., and N.G. Baz~n (1975). Rapid production of diacylglycerols enriched in arachidonate and stearate during early brain ischemia. J. Neurochem., 25, 919920. Avelda~o de Caldironi, M.I.,and N.G. Baz~n (1977). Acyl groups, molecular species and labeling by 14C-glycerol and 3H-arachidonic acid of vertebrate retina glycerolipids. In N.G. Baz~n, R.R. Brenner, and N.M. Giusto (Eds) Function and Biosynthesis of Lipids. Plenum Press, N.Y. pp. 397-404. Baz~n, N.G., M.I. Avelda~o~ H.E. Pascual de Baz~n, and N.M. Giusto (1976a). Metabolism of retina acylglycerides and arachidonic acid. In R. Paoletti, G. Porcelatti,and G. Jacini (Eds). Lipids., Raven Press,N.Y. pp. 89-97. Baz~n, N.G., M.G. Ilincheta de Boschero, N.M. Giusto, and H.E. Pascual de Baz~n (1976b). De novo glycerolipid biosynthesis in the toad and cattle retina. Redirecting of the pathway by propranolol and ph~ntolamine. In G. Porcellati, L. Amaducci, and C. Galli (Eds.). Function and metabolism of phospholipids in the central and peripheral nervous systems. Plenum Press, N.Y., pp. 139-148. Baz~n, N.G.,and C.D. Joel (1970). Gradient-thickness thin layer chromatography for the isolation and analysis of trace amounts of free fatty acids in large lipid samples. J. Lipi~ Res., ii, 42-47. Daemen, F.J.M. (1973). Vertebrate rod outer segment membranes. Biochem. Biophys. Acta, 300: 255-288. Giusto, N.M., and N.G. Baz~n (1979a). Phospholipids and acylglycerols biosynthesis 14 14 and CO 2 production from ( C)-glycerol in the bovine retina: the effects of incubation time, oxygen and glucose. Exp. Eye Res., 29, 155-168. Giusto, N.M., and N.G. Baz~n (1979b). Phosphatidic acid of retinal microsomes contains a high proportion of docosahexaenoate. Biochem. Biophys. Res. Commun., (in press). Holub, B.J.,and A. Kuksis (1971a). Structural and metabolic interrelationships among gl~cerop|~ha~des ofratliver in vivo. Can.J.Biochem., 49; 1347-1356. Holub, B.J.,and A. Kuksis (1971b). Differential distribution of orthophosphate 32p and glycerol- 14C among molecular species of phosphatidylinositols of rat liver in vivo. J. Lipid Res., 12, 699-705. Holub, B.J., and A. Kuksis (1971c) Resolution of intact phosphatidilinositols by argentation thin-layer-chromatography. J. Lipid Res., 12, 510-512. Kanoh, H., and K. Ohno (1976). The role of 1,2-diacyiglycerol: CDP-choline (ethanolamine) phosphotransferase in forming phospholipid species in rat liver. In P. Paoletti, G. Porcellati, and G. Jacini (Eds). Raven Press, N.Y., pp. 39-48. Keough, K.M.W., G. MacDonald, and W. Thompson (1972). A possible relation between phosphoinositides and the diglyceride pool in rat brain. Biochim. Biophys.Acta, 270, 337-347. MacDonald, G., R.R. Baker, and W. Thompson (1975). Selective synthesis of molecular classes of phosphatidic acid, diacylglycerol and phosphatidylinositol in rat brain J, Neurochem., 24, 655-661. Nicol, J.A.C.~ H.J. Arnott, G.R. Mizzuno, E.C, Ellison, and J.R. Chipault (1972). Oeurrence of glyceryl tridocosahexaenoate in the eye of the sand trout Cynoscion arenarius. Lipids, 7, 171-177. Parkes, J.G.,and W. Thompson (1973). Structural and metabolic relation between molecular classes of phosphatidylcholine in mitochondria and endoplasmic reticulum of guinea pig liver. J.biol. Chem. 245, 6655-6662. Parkes, J.G.,and W. Thompson (1975). Phosphatidilethanolamine in liver mitochondria and endoplasmic reticulum. Molecular species distribution and turnover.
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Can. J. Biochem., 53, 698-705. Pascual de Baz~n, H.E.-~and N.G. Baz~n (1976). Phospholipid composition and (14C)~ glycerol incorporation into glycerolipids of toad retina and brain, J. Neurochem., 27, 1051-1057, Rodrlguez de Turco, E.,and N.G. Baz~n (1977). Simple preparative and analytical thin-layer chromatographic method for the rapid isolation of phosphatidic acid from tissue lipid extracts. J_. Chromatosr., 137, 194-197, Rouser, G., S. Fleischer, and A° Yamamoto (1970)-. Two dimensional thin layer chroma. tographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids, 5, 494~496.