Metabolism of phospholipids in the retina

Vision Res. Vol.22,pp.1539to 1548.1982 Printed in Great Britain

METABOLISM

0042-6989/82/121539-10$03.00/0 Pergamon PressLtd

OF PHOSPHOLIPIDS

IN THE RETINA

LSU Eye Center, Louisiana State University Medical Center School of Medicine, 136 South Roman Street, New Orleans, LA 70112, U.S.A.

INTRODUCTION

A striking difference between neural tissue gray matter and an equivalent volume of any tissue, such as liver, is the several hundredfold greater surface area of the former. Because the neural retina is indistinguishable from gray matter, the retina is considered an integral part of the central nervous system. Retinal cells also comprise an extensive area of folded membranes. However, visual cells contain even larger quantities of membranes in their photoreceptors. Phospholipids (PL) make up approximately half the dry weight of these membranes. PL are often viewed as providers of the highly fluid environment that rhodopsin requires to function. However. if this is their sole function, it becomes difficult to explain the diversity in PL molecules. Therefore, it is likely that PL play a number of roles in addition to controlling the fluidity properties of photoreceptor membranes. This article reviews recent work, mainly from our laboratory, on the composition and metabolism of PL, fatty acids (FA), and neutral glycerides in the vertebrate retina. DE NOVO BIOSYNTHESIS OF PHOSPH~LIPIDS

IN THE RETINA

Muscarinic cholinergic and a-adrenergic stimulation (Hokin-Neaverson, 1977; Michell, 1980; Hawthorne and Pickard, 1977) enhance the turnover of polar moieties of PL, primarily in phosphatidylinositol (PI) and phosphatidic acid (PA) (for references see Hokin-Neaverson, 1977). More recently, the functional implications of this lipid effect, in conjunction with Ca gating (Michell, 1980), presynaptic events during neurotransmission (Hawthorne and Pickard, 1977), and postsynaptic location (Fisher and Agranoff, 1981), have greatly stimulated activity in this field. It has also been suggested that phosphatidic acid is the Ahhreviations used: FA-fatty acids; numbers preceding the C indicate location of each double bond with the C at the carboxylic end number 1. Figures after C indicate chain length: number of double bonds; FFA-free fatty acids; PA---phosphatidic acid; DG-diacylgiyceroi; TG--triacyigIycero1; PC-phosphatidyicholine; PE-phosphatidyIet~nolamine; PI-phosphat~dytinositol; PL-phospholipids; PS-phosphatidylserine; ROS-rod outer segments; acid ; 20: 5-eicosapentaenoic MG-monoglyceride; 22:5-docosapentaenoic acid; 22:6-docosahexaenoic acid. Y K. 22.,12

”

Ca ionophore (Tyson et al., 1976) and that it may be the link between depolarization of nerve terminals and neurotransmitter release (Harris et a/., 1981; see Bazan, 1982 for other references). However, scant information is available on the de nova biosynthesis of PL in the central nervous system. In the retina an active 32P metabolism has been observed in PL (Dreyfus et al., 1971, 1973; Urban et al., 1973), including PI in rod outer segments (Hall et al., 1973). Although there has been some interest in the enzymes involved in the biosynthesis of retinal PL (Dreyfus et al., 197f; Swartz and Mitchell, 1973, 1974), it is only in the last few years that the de nova biosynthesis of glycerolipids has been evaluated. Tritiated glycerol was administered to the lymph system of the frog, after which autoradiography showed that membrane lipids were labeled during biogenesis in the visual cells (Bibb and Young, 1974a). In addition, during the renewal process, the label was displaced toward the apex of the photoreceptor outer segments as a function of post-injection time (Bibb and Young, 1974a). Both [‘“Cl and [2-3H]glycerol actively label retinal PL and glycerides in the sequence : PA-diglycerides(DG)-triglycerides(TG), during short incubation times using 95pM labeled glycerol (Bazan and Bazan, 1976; Giusto and Bazan, 1979a; Bazan et al., 1976). This concentration of glycerol was close to the K, value of brain glycerol kinase (Jenking and Hajra, 1976). A precursor-product relationship was also observed for PA-PI, and the labeling of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) followed the sequence: PA-DG (Bazan and Bazan, 1976; Giusto and Bazan, 1979a; Bazan et nl., 1976a). Figure 1 depicts the pathways followed by [2-%I]glycerol. Note that any [2-3H]glycerol entering glycolysis through dihydroxyacetone phosphate conversion loses its tritium label to cellular HZ0 (Benjamin and McKhann, 1973). Moreover, labeling occurs only in the glycerol backbone. Retinal PL contain a wide variety of molecular species, including disaturated and 22:6 types (Aveldano de Caldironi and Bazan, 1977, 1980: Miljanich et al., 1979). The dipolyunsaturated and 22:6 species have two 22:6 that are acylated to the same glycerol backbone and display an active metabolism, as seen with labeled glycerol (Aveldano de Caidironi and Bazan, 1977, 1980; Miljanich et al., 1979). In other tissues, saturated and less unsaturated fatty acyl chains are

1539

NICOLAS G. BAZAN

1540

‘H-

l-‘Ii- l_&

GI’ycrrol

DHA @~Glycolyrir

Glycerol-J-@ 1 -Fatty

acid

‘H-8 i -Fatty Phosphatidic acid

,,,_

acid

-Falty acid -8

m t

-Fatty ‘Ii-

acid

. Fatty acid

-Fatty

acid

-Fatty

acid

\ ‘H-

- @- lnositol B

1 -Fatty

‘l-l-

-Fatty

acid \ acid

-Fatty ‘H-

H -@-Choline

! -Fatty acid

Fig.

acid

-Fatty acid

I, Flow of [2-3H]glycerol

through the dr mmo biosynthetic route of glycerolipids.

acylated in the C, position and polyunsaturated fatty acyl chains are acylated in the C, position, Of the saturated fatty acids, palmitic acid was shown to be taken up from the incubation medium by the retina and acylated at varying rates in the different lipids. After 30 min of incubation all lipids showed less palmitate acylation than glycerol labeling (Bazan et ul.. 1976a: Ilincheta de Boschero and Bazan, 1982).

METABOLISM

OF PHOSPHATIDIC

PHOSPHATlDYLlNOSITOL,

ACID.

AND

PHOSPHATIDYLSERINE

PI display a highly active rate of biosynthesis both in the retina (Bazan, 1978; Bazan er ~1.. 1976a. 1976b, 1977). This has been seen in toad (Bazan and Bazan, 1976, 1977) and rat (Careaga and Bazan. 1981) retina. and using the bovine retina in vitro (Bazan rt al., 1976a, 1977). In all cases, very active de ~IOIYJsynthesis of PI, rather than 32P turnover, occurs in the retina. Cationic amphiphilic drugs enhance the synthesis of PI (Bazan, 1978 and this paper. In rir,o studies on 32P-injected frogs showed that PI from ROS was the most significantly labeled lipid (Hall et al.. 1973). In addition, when C3H]glycerol and [3H]inositol were used as precursors and evaluated in a number of the PL. the unique dynamics of PI became apparent in the ROS (Anderson ct al.. 1980a). The effect of light on retinal PL composition, extractability, and 32P-labeling has been surveyed (Dreyfus rt ul., 1971, 1973; Mason and Fager, 1973; Urban et ul., 1973). Light enhances the glycerol labeling of retinal lipids in the toad (Bazan and Bazan. 1977). PA and PI are the primary PL whose biosynthesis increased due to light stimulation (Bazan and Bazan. 1977). Similar data were obtained in the rat retina (Schmidt, 1982). PI metabolism was also stimuirl vice and in vitro

lated by light when labeled inositol and 32P were used as precursors (Schmidt, 1982). Moreover. when the incorporation of [3H]inositol into retinal PI was examined by autoradiography. horizontal cells appeared to be selectively labeled (Anderson and Hollyfield. 1981). It has yet to be ascertained whether these effects are due solely to the turnover of the polar moiety or if they also reflect some form of dc ~OIYJ synthesis. Phosphatidylserine (PS) is an acidic PL found in small quantities in membranes. Retinal PS contains large quantities of 22:6 (Anderson et (I/.. 1975; Anderson and Risk, 1974). In brain, PS liposomes have been shown to exert potent physiological actions (Bruni ct ul.. 1976). Serine incorporation into retinal PS has been studied (Mizuno, 1976) and the synthesis of this lipid has been shown to be greatly enhanced by propranolol and phentolamine (Bazan ct rrl.. 1976a. 1977: Bazan ct 01.. 1981b). The effects of these drugs are described in more detail below. The known pathway for biosynthesis of PS in the brain involves an energy-independent, Ca-dependent base exchange reaction (Wykle, 1977). L-serine plus PE produces PS plus ethanolamine. Although there is evidence indicating the presence of this pathway in the retina (Mizuno, 1976; Anderson and Kelleher, 1981), propranolol-stimulated PS synthesis follows a different route. Based on labeling profiles, the suggestion has been made that PS may be synthesized from retinal PA (Bazan ct u/.. 1976a). This route may be stimulated by propranolo1 or phentolamine (Bazan et c-11.. 1976a). Recently, it has been proposed that, in brain microsomes, a pyrophosphatidic acid intermediate is involved in the synthesis of PS from PA (Pullarkat c’t ul.. 19X1). It IS only in bacteria that a pathway involving CDP-DC; in the synthesis of PS has been well established. PA is the common precursor of all PL and glycerides in the retina, as well as in other tissues (Bazan t’t al., 198 Id). 22: 6 makes up more than 20’,,, of the PA acyl groups of bovine retinal microsomes (Gusto and Bazan, 1979b). An even greater amount has been found in the PA of photoreceptor membranes (Bazan et d.. 1982a). The reported values of 22:h from PA of photoreceptor membranes are the highest ever reported in any of the examined cellular membranes and raise questions about the metabolic origin and functional significance of this acyl group. PA metabolism in the vertebrate retina is characterized by the following: (a) a very active dr HOW synthesis both in kx~ (Careaga and Bazan, 1981 : Bazan and Bazan. 1976) and in vitro (Bazan cr rrl.. 1976a; Gusto and Bazan, 1979b; Bazan and Bazan, 1976; Bazan et al., 198la, 1981b); (b) a net synthesis measured by PA accumulation as a result of the short-term incubation of retinas with 0.5 mM propranolo1 (Ilincheta de Boschero et II/.. 1980 and this paper); (c) a relatively large pool size and active radioactive glycerol labeling of PA in ROS membranes (unpublished data); (d) a rapid interorganelle

Phospholipid metabolism in the retina distribution from the endoplasmic reticulum (Bazan et al., 1981b), resulting in a highly labeled PA pool in the cytosolic supernatant (Bazan et al., 1981b); and (e) a highly active labeling by 20:.5 and the elongation and desaturation products, 22:5 and 22:6. These observations have led to the suggestion that retinal PA, in addition to its role as an intermediate in lipid metabolism. may have functions of its own. COMPOSITtON DIGLYCERIDES

AND

METABOLISM

1541

PA

OF

AND TRIGLYCERIDES

The toad retina contains seven times as much DG as the brain (Aveldano and Bazan, 1972, 1973). Arachidonate makes up only 7% while 22:6 represents 42% (Aveldano and Bazan, 1972, 1973). In general, the FA composition of these DG resembles that of PL in the brain. Moreover, DG are metabolically active lipids (Sun and Horrocks, 1969, 1971). Rabbit and bovine retinas contain smaller amounts of 22:6 in their DG, although the FA patterns are again different from those seen in brain (Aveldano and Bazan, 1974a). In brain, brief ischemia or eIectroshock triggers a rapid production of DG (Banschbach and Geison, 1974; Aveldano and Bazan, 1975, 1979; Bazan, 1970). The distinctive profile of 22:6 in toad retina DG prompted the suggestion that this peculiarity may represent a metabolic differentiation of the retina that enables it to sustain the high 22:6 level seen in membrane PL, notably in ROS (Aveldano and Bazan, 1972, 1973). A variety of DG pools are present in the vertebrate

retina, and active labeling with palmitate (Ilincheta de Boschero and Bazan, 1982), arachidonate (Bazan and Bazan, 1973, and glycerol (Giusto and Bazan, 1979a) has been seen in ROS. It is thought that PI may give rise to DG through a phospholipase C, since the fatty acid patterns of these lipids are very similar (unpublished data). These studies, as well as others, suggest that the following DG pools may be present in the retina: (1) pools derived from PA and leading to PC or PE; (2) pools derived from degradation of PI; (3) pools derived from acylation of monoglyceride (MG); (4) pools derived from PA, leading to TG; and (5)

pools derived from degradation of PC. In non-neural tissues, TG play a central role as storage sites for FA. However, the retina (Giusto and

Bazan, 1979a) and brain (Sun and Horrocks, 1969, 1971; Bazan, 1970) contain very small pools of TG, indicating that in these tissues TG may not be an energy store. Radioactive arachidonate (Bazan et al., 1976b; Bazan and Bazan, 1975) and glycerol (Bazan et al., 1976a, 1976b, 1977; Giusto and Bazan, 1979a) are known to rapidly label retinal TG. Subcellular studies have disclosed that TG are present in all fractions, including ROS (Bazan et nl., 1981b). The function of this neutral lipid in the retina remains unclear, but it is possible that TG act as a storage site for certain acyl groups and excess glycerol back-bone when there is an excess flux of de noao synthesized glycerolipid.

. /

Fig. 2. Comparison of the effect of propranolol and propranolol glycol on the synthesis of the retinal PA from [2-3H]glycero1. Bovine retina1 homogenates were prepared in IOmM HEPES (pH 7.3) after intact retinas were preincubated for 20min in an oxygenated glucose-containing medium. Aliquots were taken and incubation was performed in the presence of 630nmoles [2-3H(N)]g1ycerol. Controls (0), 0.5 mM propranolol glycol (A) and 0.5 mM

propranolol (0).

FREE ARACHIDONIC DOCOSAHEXAENOK

AND

ACIDS IN RETINA

The retina and other parts of the central nervous system contain a small FFA pool (Bazan, 1970, 1976). Although TG are very active metabolically in these tissues, the small pool size limits the contribution this neutral lipid can make to the FFA pool, as compared with the PL contribution (Bazan, 1970). Therefore, excitable membranes in the brain give rise to FFA at the onset of ischemia and after a single electroconvulsive shock (Bazan, 1970, 1976). Underlying these changes is the deacylation of membrane PL by phospholipase A2 or a sequence involving phospholipase C-DG lipase (Bazan, 1970, 1976). Arachidonic and stearic acids are released at faster rates undc- these conditions. A large proportion of highly unsaturated FFA was found during in vitro incubation of retinas (Aveldano de Caldironi

et al., 1981). Both phospholipase

(Swartz

NICOLAS G. BAZAN

1542

and Mitchell, 1973) and acyltransferase (Swartz and Mitchell, 1974) may contribute to acyl exchange reactions in the retina. Active arachidonate acid metabolism in the retina is described below. In the presence of high K’ concentrations, dibutirylcyclic AMP stimulates the production of retinal FFA (Aveldano de Caldironi rt cd.. 1981; Bazan et (II., 1981a). This agrees with the hypothesis that the deacylation of membrane PL is a regulatory step in the control of membrane permeability and that cyclic nucleotides regulate the enzymes involved in the production of FFA in neural tissue (Bazan, 1970, 1976; Aveldano de Caldironi et ul.. 198 I). A greatly increased free arachidonic acid was observed in the retina after anoxia and as a function of incubation time. Essentially all this FA was derived from PL. inasmuch as the arachidonate in TG remained unchanged (Bazan, 1976). When bovine retinas were incubated with radioactive arachidonic acid, a significant amount of labeling was seen in DG, TG, PC, and PI (Bazan and Bazan. 1975). In the retina. endogenous free 22:6 has been analyzed in rabbit, toad, and bovine retinas (Aveldano and Bazan, 1974a). In the bovine retina. a highly dynamic pool of 22:6 was found when bovine serum albumin was added to the incubation medium (Aveldano and Bazan, 1974b) as described below. FFA were also released when glucose was omitted (Bazan, 1976). Thus, overall. a similar anoxia-induced FFA release occurs in the retina and brain. Phospholipase A, seems to be activated under these conditions since there is an increase in lyso-PC and lyso-PE (Bazan, 1976; Giusto and Bazan. 1979a). When [‘4C]glycerol labeled retinas were exposed to anoxic conditions, an increase in labeled lyso-PL was observed. However. if only glucose was omitted from the much larger quantities of incubation medium, lyso-PL were observed, indicating an enhanced PL breakdown with increased turnover (Giusto and Bazan, 1979a). Retinas incubated in the presence of FFA-free bovine serum albumin showed an increased amount of FFA (Aveldano and Bazan. 1947b). In addition, it was determined that FFA were displaced from the tissue to the medium in greater proportions as the albumin concentration was increased. Most of the displaced FFA were highly unsaturated FA such as 22:6 and arachidonic acid. It is likely that what occurred was a phospholipase AZ activation followed by movement of the FA outward toward the medium. It has not yet been determined how much the PL of plasma membrane and of intracellular membranes contribute to this effect. METABOLISM

OF EICOSAPENTAENOATE

AND DOCOSAHEXAENOATE

Docosahexaenoate (22:6, II - 3) is found in particularly large quantities in the synaptic membranes of the CNS (Sun and Sun, 1976) and in ROS (Ander-

son rt ~1.. 1974; Anderson and Maude, 1970; Anderson et d., 1975; Anderson and Risk, 1974; Aveldano de Caldironi et al., 1981; Daemen. 1973; Farnsworth and Dratz, 1976; Stone rt cd., 1979). It is synthesized by a sequence of desaturation and elongation steps of CoA derivatives from the essential FA. linolenic acid (18:3. II - 3). However, essential FA-deficient diets are unable to remove 22:6 from the CNS (Anderson and Maude, 1972: Forrest and Futterman, 1972; Futterman et al., 1971; Tinoco et cd.. 1977). HOW this tenacious retention is brought about is not known. Twenty-two C atoms with six double bonds (22:6, II - 3) or another very similar chemical structure seem to be required for photoreceptor and synaptic functions. 22: 6 depletion observed during linolenic acid-deficiency is replaced by an increase in docosapentaenoic acid (22:5. II - 6) when supplemented with linoleic acid (IX:?, II - 6) (Tinoco et ul., 1977). This FA is the closest to 22:6, II - 3 that the retina can produce under these conditions. Losses of retinal 22:6 have been described, however. in experimental diabetes (Futterman and Kupfer, 1968; Futterman er ul.. 1968, 1969). Rats fed diets deficient in essential FA supplemented with linolenic acid showed more prominent u-waves than animals receiving diets supplemented with other FA (Benolken et al., 1973). Although the polyunsaturated acyl chains are thought to play an important role in rhodopsin function (Anderson, 1978; Daemen. 1973; Daemen and Grip, 1980), no evidence for this is available. Several years ago the late Sydney Futterman reported that [ “C]malonyl-CoA labeled polyenoic FA. including 22:6. in homogenates of dog retinas (Futterman et d., 1968, 1969). Very recently, retinoblastoma cells were found to produce all the derivatives of linolenic acid (Hyman and Spector, 1981) that are shown in Fig. 3. The last two steps in the biosynthesis of retinal 22:6 have been studied in the rat eye by means of intravitreal injections of [I-14C] 20:s (Bazan e’t d.. 1982b). 20:5 injected into the vitreous body is taken up by the retina and actively metabolized. The fate of this FA includes (a) acylation at different rates in individual PL and (b) elongation to 22:5 and further desaturation to 22:6. Desaturation and elongation products evolve in individual PL at different rates and in different time intervals after intraocular injection. PI exhibits the highest apparent rate of [l-‘4C]20:5 acylation, followed by PC and PA (Table 1). Three minutes after injection, the rates of [I-‘4C]20: 5 labeling in PS and PE were approximately six times lower than the rate for PA. The rate of [1-‘4C]22:5 labeling in PA, however, was similar to the rates of PI and PC. For the time periods studied, PC exhibited the highest rate of [1-‘4C]22:6 acylation of any of the PL, followed by PA (Table I). The large amounts of label found in PI and PC are indicative of deacylation-acylation reactions. In fact, polyunsaturated FA are thought to be introduced into PL through this cycle (Bazan and Giusto, 1980;

Phospholipid metabolism in the retina a -Linolenic

acid

( n-3 series 1

9,12,15-c,,:, A 6 Desaturosr I

6,9,

1543

(Bazan et al., 1982b; Giusto and Bazan, 1979b). In addition, these results show that there exists in uiuo a A - 4 desaturase and a possible direct desaturation of 22: 5 in retinal phospholipids.

12, 15-C,,:4

EFFECTS

OF CATIONIC

E I ongat ion I

8,

II)

14,

Il-c,,:,

A 5 Decaturare -

I 5,8,

II,

14,

17,-cz0,5

Elongation 1 7, IO, A I 4,7,

13, 16,

19,-Cee:5

4

Deroturase

IO,

13, 16, 19-C,,.6

( docosahrxoenoic

AMPHIPHILIC

DRUGS

acid

1

Fig. 3. Synthesis of 22:6 from a-linolenic acid. [l-‘“Cl 20:5 was used as a precursor as described in Fig. 5.

Giusto and Bazan, 1979a). Moreover, there was little acylation of I-acylglycero-3-P with arachidonoyl-CoA and 22:6-CoA compared to the acylation of alkylglycero-P in rat brain microsomes (Fleming and Hajra, 1977). A second pathway may involve the acylation of polyunsaturated FA during PA formation, subsequently producing glycerolipids containing these FA

At the 1975 Phospholipid Conference in Cortona, Italy, it was reported that propranolol and phentolamine redirected the de nova biosynthesis of glycerolipids in the bovine retina (Bazan et al., 1976a) in concentrations that antagonize the dopamine-sensitive adenylcyclase of the retina through p- and a-adrenergic receptors, respectively (Brown and Makman, 1972, 1973). Surprisingly, the findings led to the conclusion that adrenergic receptors were not involved in this mechanism, since both propranolol and phentolamine exerted identical metabolic effects on the lipids in the retina (Bazan et a/., 1976a). Propranolol, phentolamine, and other cationic amphiphilic drugs modify selectively the flux of radioactive glycerol via the biosynthetic pathway of retinal lipids (Bazan et a/., 1976a, 1977, 1981b; Ilincheta de Boschero and Bazan, 1982; Ilincheta de Boschero et al., 1980). PA, PI, and PS showed an accumulation of label; an inhibition of PC, PE, and TG synthesis was also observed at this time. It has been suggested that PA phosphohydrolase is inhibited by these cationic amphiphilic drugs and also exhibits several other drug-induced lipid metabolic effects. Since PS increases as PA does, the suggestion has been made that there is a pathway for the synthesis of PS in the retina

Table 1. Labeling rates of individual phospholipids of the retina after intravitreal injection of [l-r4C]eicosapentaenoic acid

Phospholipid

Time after injection (min)

20:5 22~5 22~6 (pm01 x min-’ x mg of protein r)

Phosphatidic acid

3 5 30

1.57 0.99

0.25 0.23

0.077 0.110 0.067

Phosphatidylserine

3 5 30

0.25 0.096

0.068 0.042

0.023 0.026 0.017

Phosphatidylinositol

3 5 30

4.47 3.63

0.24 0.35

0.032 0.098 0.022

Phosphatidylcholine

3 5 30

2.17 2.26

0.22 0.49

0.220 0.290 0.138

Phosphatidylethanolamine

3 5 30

0.30 0.34

0.096 0.160

0.036 0.022 0.018

Rates were calculated based upon the radioactivity found in after 3, 5 and 30min of injection. Values are means from two experiments. In each case, two to eight retinas from different rats 14 and 30 nmol of the labeled fatty acid were injected into each in Bazan er al. (1982b).

each fatty acyl group to four independent were pooled. Between eye. Other details are

NICOLASG. BAZAH

1544

Puise - chose k3B] Retinas

experiments

6t~eerotn~,~~e~ were

i

;

pqy=?e

0

IO

A

20

&

4

0

&

t 40

20

Preincubotion

Chose

4 60

of the label

Fig. 4. Outline of pulse-chase experiments. Bovine retinas were preincubated and labeled with [2-3H]glycerol [5 pCi, 99.52 Ci/mmol], and washed four times with fresh incubation medium. Preincubation and incubation times are given in minutes.

that stems from PA (Bazan er al., 1977, 1981b). There is a concomitant PA accumulation. Therefore, it is possible to measure net synthesis of this lipid during short incubation times. In addition, cationic amphiphilic drugs have been thought to elicit the following effects on lipid synthesis: (a) stimulation of PA synthesis (Bazan et al., 1976a; Ilincheta de Boschero et ul., 1980); (b) stimulation of PI synthesis (Bazan et al., 1977. 1976a; Bincheta de Boschero rt al., 1980); (c) modifications of DG metabolism (Bazan el ni., 1976a); fd) enhanced de~drboxylation of PS (Ilincheta de Boschero et ul., 1980); and (e) modifications of the choline labeling in PC (Bazan et ul.. 1976). Figure 2 shows that the entire structure of propranolo1 is required to stimulate PA synthesis; removal of the isopropylamine group of the drug produces an ineffective compound (Ilincheta de Boschero and Bazan, 1982). Propranololgiy~ol is known to be the major metabolite of propranolol in the neural tissue (Saelens et ui., 1974). The first propranoiol-induced lipid changes were seen by Hauscr and Eichberg (1975) using the pineal gland and 32P . Similar effects were seen in iris muscle (Abdel-Latif and Smith, 1976), liver (Brindley and

L

I

0

IO

Preincubation

1

20

I

0

I

Bowley, 1973, and lymphocytes (Michell et ul.. 1976) using fen~uoramine and its derivatives or propranolot. A pulse-chase experimental design was used to study the reversibility of drug-induced modifications in retinal glycerolipid metabolism and to determine if it was possible to measure net synthesis of PA (Figs 4 and 5). All drug-induced changes in the dr noro biosynthesis of glycerolipids were found to be partially reversible within the 60 min chase-of-the-label period, with the exception of PI (Ilincheta de Boschero M. E. and Bazan N. G.. unpublished data). Figure 6 presents data on the PA pool of retinas incubated according to the outline given in Figs. 4 and 5. Changes in the amount of [2-3H]glycerol and the endogeneous pool size of PA are given. The changes that occurred in the radiolabeled glycerol pool during chase of the label indicate a rapid loss of label (Fig. 6. open circles). However, when propranolo1 was present during this period, it prevented the loss of label for up to 30 min of chase, and increased the amount of label present in PA (Fig. 6, solid circles). After 60 min of chase, all groups exhibited a decrease in label.

1

I

I

20 Chose

I

40 of

the

I

60

label

500 ELM propronolol

Fig. 5.

Experimental design followed to study the effect of propranolol during labeling with [2-‘H]gly. cerol. Basic outline is as in Fig. 4.

Phospholipid

0

204060

0

20

40

60

min

metabolism in the retina

’

m-2

Fig. 6. [2-3H]glycerol labeling and pool size of PA in bovine retinas. Time (min) corresponds to the chase of the label in Figs 4 and 5. Top boxes are without propranolol in the preincubation period; without (0) and with (0) propranolo1 in the chase of the label period. Bottom boxes are with propranoiol during preincubation; without (A) and with (A) propranolol during chase of the label.

although retinas exposed to propranolol during this period exhibited a smaller decrease than retinas not exposed. Previous studies (Bazan et al., i976a; Giusto and Bazan, 1979a) support the concept that PA loses label by further conversion to DG, PI, and perhaps, PS (Bazan et al., 1976a). The synthesis of PI and PS is greatly enhanced by propranolol treatment of the retina (Bazan et al., 1976a, 1981b). This enhancement indicates that the effect of propranolol seen in Fig. 6 (solid circles) reflects (a) the inhibition of PA phosphohydrolase yielding DG from PA, and (b) the stimulation of PA de nova synthesis. However, the decrease in labeling seen at 60 min may be an indication that the [3H]-containing pool of PA is being channeled toward PI or, alternatively, is overcoming the drug-induced inhibition of PA phosphohydrolase. The labeling and content of the PA pool were examined when propranolol was present during the preincubation-labeling period (Fig. 6). Larger increases in [2-3H]glycerol labeling and pool size of PA were seen in retinas preincubated with propranolol compared with retinas preincubated in the absence of the drug. A rapid loss of radioactivity was seen if the drug was not added during the chase of label. A similar trend occurred if the drug was present, although at 60min there was twice as much [2-3H]glycerol as there was in retinas not exposed to propranolol (Fig. 6, right; triangles vs open circles at Omin). The pool size of PA did not change in the absence of the drug during the incubation period. However, propranolol promoted a further increase in the size of the labeled pool that was finally about three times the size of the control (solid triangles vs open circles). Moreover, a phospholipidosis involving myeloid, membranous structures rich in PL has been described in the retina and other tissues during chronic treatment with chloroquine (Smith and Berson, 1971; Martin et al., 1978). It is possible that an impaired

1545

synthesis of PL could result in phospholipidosis (Michell et al., 1976; Bazan et al., 1976a, 1977). Amphiphilic cations such as propranolol may (a) alter the lipidic organization of the membrane by interacting with charged lipids and by physical interaction of the hydrophobic moiety with acyl chains of PL (Seydel and Wassermann, 1976); (b) directly interact with membrane proteins, some of which may be enzymes engaged in the metabolism of glycerolipids; and (c) disrupt specific PL pools composed of annular structures around proteins. As a result of this nonspecific binding to endoplasmic reticulum, disruptions in function may arise. It is likely that other membranes also establish interactions with amphiphilic cations. Membrane functions other than lipid synthesis may be affected. These nonspecific modes of action have some features in common with the action of local anesthetics, i.e. they both interact with membranes and consequently increase membrane fluidity (Singer, 1977). It is still not known whether the effect of propranolol on retinal lipid synthesis is connected directly to the membrane anesthetic effect of this drug or is a separate action. Propranolol also may exert some of its effects by ionizing membrane-bound calcium (Porzig, 1975). Among the many questions that have yet to be explored is the role that vitamin E plays in photoreceptor membranes and retinal synaptic membranes. Vitamin E deficiency has been shown to lead to retinal degeneration in dogs (Riis et al., 1981). In addition, the role that lipid peroxidation plays in retinal functioning (Shvedova et al., 1978) appears to support the concept that there is a need to maintain protective mechanisms for the double bonds of FA. Moreover, during continuous light exposure, there is a selective decrease in 22:6 (Joel et al., 1980; Wiegand et cd., 1981) as well as Vitamin E (Joel er a!., 1981) in the retina of the rat. The dynamics of PL during the renewal of photoreceptor membranes (Young, 1976) remains to be thoroughly examined. Autoradiographic data have shown that labeled FA (Bibb and Young, 1974b) and glycerol (Bibb and Young, 1974a) are dispersed diffusely throughout visual cell outer segments. This pattern is in contrast with the patterns of labeled amino acids (Bok and Young, 1972; Young, 1976) which display band-like migratory patterns from the base of the outer segment to the apex. It is likely that PL metabolism differs along the ROS. The oldest membranes, near the apex. may be shed by the localized activation of phospholipases that helps to break down the rod tip. The base of the rod, the youngest membranes, may have a different PL metabolic status, as these membranes may be concerned with completing their assembly. It is also possible that the bulk of the PL may be engaged in rhodopsin functioning. In other tissues, certain protein kinases are modulated by a DG derived from PL breakdown (Takai et al., 1979), and in the retina, there is a unique conservation mechanism to maintain 22:6

NIWLAS

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even when the diet is deficient in essential FA over a relatively long period of time (Anderson and Maude, 1972; Tinoco rr (I/., 1977; Futterman et al., 1971). When this diet is continued, the electroretinogram is affected and precursors of 22: 6 specifically restore the u-wave (Wheeler et (I/., 1975). It is conceivable that the retina and perhaps also the pigment epithelium participate in intercellular and biochemical pathways that operate to prevent loss of this acyl group of membrane lipids. In addition, very little information is available on the fate of 22:6 in the retina. There are several other aspects of lipid metabolism in the retina that need to be explored. Although there is a relatively high content of arachidonate in retinal lipids, little is known about the biochemistry of the prostaglandins and other derivatives from this or from other essential FA in the retina. It is also time to correlate lipid metabolism with cellular function. cellular structure, and the pathogenesis of retinal diseases. The elegant biochemical and autoradiographic work on PI metabolism done in the laboratories of Drs Anderson and Hollyfield (1981) is a good example of what is needed. Insofar as the pathogenesis of retinal diseases is concerned, work in experimental models of degenerative and other diseases will help define the involvement of lipid metabolism in the alterations of photoreceptor membranes. Ack,lu~~ledUenlerlts-This work was supported

in part by a Research Manpower Award from Research to Prevent Blindness, Inc., New York City and by a grant from Fight for Sight, New York City. I would like to thank my colleagues Norma M. Giusto, Marta T. Aveldano de Caldironi, M. Monica Careaga, Monica G. Ilincheta de Boschero, Victor L. Marcheselli. and Haydee E. P. Bazan for the several years of enjoyable work, part of which is discussed here. REFERENCES Abdel-Latif A. A. and Smith J. P. (1976) Effects of dl-propranolol on the synthesis of glycerolipids by rabbit iris muscle. Biochem. Pharmac. 25, 1697-l 704. Ames A. III and Hastings B. (1956) Studies on water and electrolytes in the nervous tissue. I. Rabbit retina: methods and interpretation of data. J. Neurophysiol. 19, 201 -212. Anderson R. E. and Kelleher P. A. (1981) Biosynthesis of retinal phospholipids by base exchange reactions. Evpl Eyr Res. 32. 729-736. Anderson R. E. and Maude M. B. (1970) Phospholipids of bovine rod outer segments. Biochrrnistry 9, 3624. Anderson R. E. and Maude M. B. (1972) Lipids of ocular tissues, VIII. The effects of essential fatty acid deficiency on the phospholipids of rat retina. Archs Biochrrn. Biopbys. 151, 270-276. Anderson R. W., Maude M. B. and Kelleher P. A. (19XOa) Metabolism of phosphatidylinositol in the frog retina. Biochim. hiophgs. Acfa 620, 236-246. Anderson R. E., Maude M. B., Kelleher P. A.. Maida T. M. and Basinger S. F. (1980b) Metabolism of phosphatidylcholine in the frog retina. Biochim hioph.r.\. Acta 620, 212-226. Anderson R. E., Kelleher P. A. and Maude M. B. (1980~) Metabolism of phosphatidylethanolamine in the frog retina. Biochim. hiophys. Acta 620, 2277235. Anderson R. E.. Kelleher P. A.. Maude M. B. and Maida T.

G. BAZAN M. (1980d) Synthesis and turnover of lipid and protein components of frog retinal rod outer segments. Neurochemistry

1, 39-42.

Anderson R. E. and Risk M. (1974) Lipids of ocular tissues. IX. The phospholipids of frog photoreceptor membranes. Vision Res. 14, 129 131. Anderson R. E. and Hollyfield J. G. (1981) Light stimulates the incorporation of inositol into phosphatidylinositol in the retina. Biocbim. hiophys. Actu 665, 619-622. Aveldano de Caldironi M. I. and Bazan N. G. (1972) High content of docosahexaenoate and of total diacylglycerol in retina. Biochem. biophys. Rrs. Cornmun. 48, 689-694. Aveldano de Caldironi M. I. and Bazan N. G. (1973) Fatty acid composition and level of diacylglycerols and phosphoglycerides in brain and retina. Bkhim. hiophvs. Actu 296, l-9. Aveldano de Caldironi M. I. and Bazan N. G. (1974a) Free fatty acids. diacyl- and triacylglycerols and total phospholipids in vertebrate retina: Comparison with brain. choroid and plasma. J. ,Yeurochrm. 23, 112771135. Aveldano de Caldironi M. I. and Bazan N. G. (1974b) Displacement into incubation medium by albumin of highly unsaturated retina free fatty acids arising from membrane lipids. FEBS Lett. 40, 53 56. Aveldano de Caldironi M. I. and Bazan N. G. (1975) Rapid production of diacylglycerols enriched in arachidonate and stearate during early brain ischemia. .1. Neurochm. 25, 9 19 -920. Aveldano de Caldironi M. I. and Bazan N. G. (1977) Acyl groups, molecular species and labeling by “C-glycerol and ‘H-arachidonic acid of vertebrate retina glycerolipids. .Adc. cp. Med. Biol. 72. 387-404. Aveldano de Caldironi M. I. and Baran N. CT. (1979) Alpha-methyl-p-tyrosine inhibits the production of free arachidonic acid and diacylglycerols in the brain after a single electroconvulsive shock. Ycwocharn. Rus. 4, 113 221. Aveldano de Caldirom M. 1. and Bazan N. G. (19X0) Composttion and biosynthesis of molecular species of retina phosphoglycerides. .Ilr,uro~ht,/,11str~ I, 3X1-392. Aveldano de Caldironi M. I.. Giusto N. M. and Bazan N. G. (19X 1) Polyunsaturated fatty acids of the retina. Proq. L/p. Rrs. 20, 49 57. Baker R. R. and Thompson W. (1972) Positional distribution and turnover of fatty acids in phosphatidic acid. phosphoinositides. phosphatidylcholine and phosphatidylethanolamine in rat brain in i’ir~. Biochim. biophys. -Ictu 270. 489 503.

Banschbach M. W. and Geison R. 1.. (1974) Post-mortem increase in rat cerebral hemisphere diglyceride pool size. J. ,Vrttrochrrn. 23, 875 X77. Bazan N. Cr. (1970) Effects of ischemia and electroconvulsivc shock on free fatty acid pool in the brain. Biochim. biophn. 4c’ttr 218, 1 IO. Bazan N. G. (1976) Free arachidonic acid and other lipids in the nervous system during early ischemia and after electroshock. A&. e\-p, &fed. Hiol. 72, 317 -335. Bazan N. G. (1978) Metabolism of phosphatidylinositol in the retma. In Cycfitols untl Pko.sphoi,losititles (Edited by Wells M. and Eisemberg F.). pp. 563-56X. Academic Press. New York. Bazan N. G. (1982) Metabolism of phosphatidic acid. In Hundbook q/ !Vwrochrntistry (Edited by Lajtha A.). Vol. 3. Plenum Press. New York. To be published. Bazan H. F. P. and Bazan N. G. (1975) Incorporation of (ZH)-arachidonic acid into cattle retina lipids: High uptake in triacylglycerols. diacylglycerols. phosphatidyland phosphatidylinositol. Life Sci. 17. choline 1671~ 167X. Bazan H. E. P. and Bazan N. G. (1976) Phospholipid composition and (i4C)-glycerol incorporation into glycerolipids of toad retina and brain. J. Nwrochrrrz. 27, 1051~1057

Phospholipid

metabolism

Bazan H. E. P. and Bazan N. G. (1977) Effects of temperature, ionic environment and light flashes on the glycerolipid neosynthesis in the toad retina. Adu. exp. Med. Biol. 83, 489-495. Bazan H. E. P. and Bazan N. G. (1982) Lipid synthesis in retinas. In Methods in Enzymology. Visual Pigments and Purple Membranes (Edited by Packer L.). 81 Part H: 788-794. Academic Press, New York. Bazan N. G. and Giusto N. W. (1980) Docosahexaenoyl chains are introduced in phosphatidic acid during de nova synthesis in retinal microsomes. In Control of Membrane Fluidity (Edited by Kates M. and Kuksis A.), pp. 223-236. Humana Press, New Jersey. Bazan N. G., Aveldano de Caldironi M. I. and Rodriguez de Turco E. B. (1981a) Rapid release of free arachidonic acid in the central nervous system due to stimulation. Prog. Lip. Res. 20, 523-529. Bazan N. G., Aveldano de Caldironi M. I., Pascual de Bazan H. E. and Giusto N. M. (1976b) Metabolism of retina acylglycerides and arachidonic acid. In Lipid (Edited by Paoletti R., Porcellati G. and Jacini G.), Vol. 1, pp. 89-97. Raven Press, New York. Bazan N. G., Ilincheta de Boschero M. G. and Giusto N. M. (1977) Neobiosynthesis of phosphatidylinositol and of other glycerolipids in the entire cattle retina. Adu. exp. Med. Biol. 83, 377-388. Bazan N. G., Ilincheta de Boschero M. G., Giusto N. M. and Bazan H. E. P. (1976a) De nouo glycerolipid biosynthesis in the toad and cattle retina. Redirecting of the pathway by propranolol and phentolamine. Adv. exp. Med. Eiol. 72, 139-148. Bazan N. G., Di Fazio de Escalante M. S., Careaga M. M., Bazan H. E. P. and Giusto N. M. (1982a) Phosphatidie acid of photoreceptor membranes: High content of 22:6 and active [2-3H]glycerol metabolism. Biochim. biophys. Acta. To be published. Bazan H. E. P., Careaga M. M., Sprecher H. and Bazan N. G. (1982b) Chain elongation and desaturation of eicosapentaenoate to docosahexaenoate and phospholipid labeling in the rat retina in GDo. Biochim. biophys. Acta. 712, 123-128. Bazan H. E. P., Careaga M. M. and Bazan N. G. (1981b) Propranolol increases the biosynthesis of phosphatidic acid, phosphatidylinositol and phosphatidylserine in the toad retina. Studies in the entire retina and subcellular fractions. Biochim. biophys. Acta 666, 63-71. Bazan H. E. P., Marcheselli V. L., Careaga M. M. and Bazan N. G. (1981~) Biosynthesis of essential phospholipids in the retina. In New Trends in Nutrition, Lipid Research and Cardiovascular Diseases, pp. 17-23. Liss, New York. Benjamin J. A. and McKhann G. M. (1973) [2-3HJglycerol as a precursor of phospholipids in rat brain : evidence for lack of recycling. J. Neurochem. 20, 111 I. Benolken R. M., Anderson R. E. and Wheeler T. G. (1973) Membrane fatty acids associated with the electrical response in visual excitation. Science 182, 1253-1254. Bibb C. and Young R. W. (1974a) Renewal of glycerol in the visual cells and pigment epithelium of the frog retina. J. Cell Biol. 62, 378: _ Bibb C. and Young R. W. (1974b) Renewal of fatty acids in the membranes of visual cell outer segments. J. Cell Biol. 61, 327. Bok D. and Young R. W. (1972) The renewal of diffusely distributed protein in the outer segments of rods and cones. Vision Res. 12, 161. Brindley D. N. and Bowley M. (1975) Drugs affecting the synthesis of glycerides and phosphoplipids by rat liver. Biochem. J. 148, 461. Brown J. H. and Makman M. H. (1972) Stimulation of dopamine of adenylate cyclase in retinal homogenates and of adenosine 3’-5’-cyclic monophosphate formation in intact retina. Proc. natn Acad. Sci., U.S.A. 69, 539.

in the retina

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Bruni A., Toffano G., Leon A. and Boarato E. (1976) Pharmacological effects of phosphatidylserine liposomes. Nature 260, 331. Careaga M. M. and Bazan H. E. P. (1981) The rat retina is a useful in uivo model to study membrane lipid synthesis. Rates of biosynthesis of neutral glycerides. Neurochem. Rex 6, 1169-1178. Daemen F. J. M. (1973) Vertebrate rod outer segment membranes. Biochim. biophys. Acta 300, 255. Dreyfus H., Urban P. F., Neskovic N. and Mandel P. (1971) Distribution des phosphatides et marquage du phosphore lipidique et du phosphate mineral de retines de rat et de veau. Biochimica 53, 567-569. Dreyfus H., Virmaux N., Urban P. F. and Mandel P. (1973) Effet de la stimulation lumineuse sur I’extraction des phospholipides des segments externes des batonnets retiniens par divers detergents. J. Physiol., Paris 66, 79-85. Farnsworth C. C. and Dratz E. A. (1976) Oxidative damage of rod outer segment membranes and the role of vitamin E. Biochim. biophys. Acta 443, 556. Fisher S. K. and Agranoff B. W. (1981) Enhancement of the muscarinic synaptosomal phospholipid labeling effect by the ionophoie A23187. J.Neu;ochek. 37, 968-977. Fleming P. J. and Haira A. K. (1977) I-Alkvl-sn-glvcero-3phosihate acyl-Cok acyltransferase in rat bra% microsomes. J. biol. Chem. 252, 1663-1672. Forrest G. L. and Futterman S. (1972) Age-related changes in the retinal capillaries and the fatty acid composition of retinal tissue of normal and essential fatty acid-deficient rats. Invest. Uphthal. 11, 760. Futterman S., Downer J. L. and Hendrickson A. (1971) Effect of essential fatty acid deficiency on the fatty acid composition, morphology, and electroretinographic response of the retina. Inuest. Ophthal. 10. 151. Fuiterman S. and Kupfer C. (1568) The fatty acid composition of the retinal vasculature of normal and diabetic human eyes. Invest. Ophrhal. 7, 105. Futterman S., Rollins M. H. and Vacano E. (1968) The effect of alloxan diabetes on polyenoic fatty acid synthesis by retinal tissue. Biochim. biophys. Acta 164, 433. Futterman S., Sturtevant R. and Kupfer C. (1969) Effect of alloxan diabetes on the fattv acid comuosition of the retina, Invest. Ophthal. 8, 542: Giusto N. M. and Bazan N. G. fl979al Phosoholioids and acylglycerols biosynthesis and 14Cb2 prdductibn from [‘%Z] glycerol in the bovine retina: The effects of incubation time, oxygen and glucose. Expl Eye Res. 29, 155-168. Giusto N. M. and Bazan N. G. (1979b) Phosphatidic acid of retinal microsomes contains a high proportion of docosahexaenoate. Biochem. biophys. Res. Commun. 91, 791-794. Hall M. O., Basinger S. F. and Bok D. (1973) Studies on the assembly of rod outer segment disc membranes. In Biochemistry and Physiology oj” Visual Pigments (Edited by Langer H.), p. 319. Harris R. A., Schmidt J., Hitzemann B. A. and Hitzemann R. J. (1981) Phosphatidate as a molecular link between depolarization and neurotransmitter release in the brain. Science 212, 129&1291. Hawthorne J. N. and Pickard M. R. (1977) Metabolism of phosphatidic acid and phosphatidylinositol in relation to transmitter release from svnaotosomes. Adu. exe. Med. Biol. 83, 419427. . I Hauser G. and Eichberg J. (1975) Identification of cytidine diphosphatediglyceride in the pineal gland of the rat and its accumulation in the presence of d[-propranolol. J. biol. Chem. 250, 105. Hokin-Neaverson M. R. (1977) Metabolism and role of phosphatidylinositol in acetylcholine-stimulated membrane function. Adv. exp. Med. Biol. 83, 429.

NICOLAS G. BAZAN

1548

Hyman B. T. and Spector A. A. (1981) Accumulation of N-3 polyunsaturated fatty acids by cultured human Y79 retinoblastoma cells. J. Neurochem. 37, 6C-67. Ilincheta de Boschero M. G. and Bazan N. G. (1982) Selective modifications in the de nouo biosynthesis of retinal phospholipids and glycerides by propranolol or phentolamine. Biochem. Pharmacol. 31, 1049-1055. Ilincheta de Boschero M. G., Giusto N. M. and Bazan N. G. (1980) Biosynthesis of membrane lipids in the retina: Subcellular distribution and propranolol action on phosphatidic acid, phosphatidylserine and phosphatidylethanolamine. Neurochemistry 1, 17-28. Jenking B. T. and Hajra A. K. (1976) Glycerol kinase and dihydroxyacetone kinase in rat brain. J. Neurochem. 26, 377. Joel C. D., Briggs S. J., Kahlow M. A., Sievert T. J. and Tsang G. K. W. (1980) Prolonged exposure to light causes loss of docosahexaenoic acid in retina. Truns. Am. Sot. Neurochem. 11, 212. Joel C.. Briggs S., Gaal D., Hannan J., Kahlow M., Stein M.. Tarver A. and Yip A. (1981) Light causes early loss of retinal tocopherol in uiuo. Intwt.-Ophthal. Visu;l Sci. (Suppl.) 20, 166. Martin L. J., Bergen R. L. and Dobrow H. R. (1978) Delayed onset chloroquine retinopathy : case report. . Ann..Ophthal. 10, 123. Mason W. T. and Fager R. S. (1973) Effect of light exposure on phospholipid extraction from squid photoreceptors. Comp. Biochem. Physiol. 47B, 347. Michell R. H. (1980) Muscarinic acetvlcholine receotors. In Cell&r Receptok for Hormones &d Neurotransmitters (Edited by Schulster D. and Levitzki A.), pp. 353-368. Wiley, London. Michell R. H., Allan D., Bowley M. and Brindley D. N. (1976) A possible metabolic explanation for druginduced phospholipidosis. J. Pharm. Phurmuc. 28, 331-332.

Miljanich G., Sklar L., White D. and Dratz E. (1979) Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane. Biochim. hiophys.

Acta 552, 294.

Mizuno A. (1976) Incorporation amine into the phospholipids

of serine and ethanolin rabbit retina. J. Bio-

them. 80, 45.

Porzig H. (1975) Comparative study of the effects of propranolol and tetracaine on cation movements in resealed human red cell ghosts. J. Physiol. 249, 27. Pullarkat R. K., Sbaschnig-Alger M. and Reha H. (1981) Biosynthesis of phosphatidylserine in rat brain microsomes. Biochim. hiophvs. Acta 663, 117-123. Riis R.. Sheffy B., Loew E., Kern T. and Smith J. (1981) Vitamin E deficiency retinopathy in dogs. Am. J. Vet. Res. 42, 74. Rodriguez de Turco E. B. and Bazan N. G. (1977) Simple preparative and analytical thin-layer chromatographic method for the rapid isolation of phosphatidic acid from tissue lipid extracts. J. Chromatog. 137, 194-197. Saelens D. A., Walle T., Privitera P. J., Knapp D. R. and GafIney T. (1974) Central nervous system effects and

metabolic disposition of a glycol metabolite of propranolol. J. Pharmac. exp. Ther. 188, 86. Schmidt S. Y. (1982) Light effects on synthesis and phosphorylation of phosphatidylinositol in rat retinas. Inrest. Ophthal.

visual Sci., Suppl. 22, 66.

Seydel J. K. and Wassermann 0. (1976) NMR-studies on the molecular basis of drug-induced phospholidosis-II. Interaction between several amphiphilic drugs and phospholipids. Biochem. Phurmucol. 25, 2357. Shvedova A. A., Sidorov A. S., Novikov K. N., Galushchenko I. V. and Kagan V. E. (1978) Lipid peroxidation and electric activity of the retina. Vision Rrs. 19, 49. Singer M. A. (1977) Interaction of dibucaine and propranolol with phospholipid bilayer membranes-effect of alterations in fatty acyl composition. Biochem. Phrrrmuc~. 26, 51.

Smith R. S. and Berson E. L. (1971) Acute toxic effects of chloroquine on the cat retina: Ultrastructural changes. lnresr. Ophthul. 10, 237. Sun A. Y. and Sun G. Y. (1976) Functional roles of phospholipids of synaptosomal membrane. Adr. CJ.Yp.Med. Biol. 72, 169-197.

Sun G. and Horrocks L. A. (1971) The incorporation of (“‘C) palmitic acid into the lipids of mouse brain in riw. J. Neurochem.

18, 1963.

Sun G. Y. and Horrocks L. A. (1969) The metabolism of palmitic acid in the phospholipids. neutral glycerides and galactolipids of mouse brain. J. Neurochrm. 16, 184. Swartz J. G. and Mitchell J. E. (1973) Phospholipidase A activity of retina and pigment epithelium. Biochemislq 12, 5273.

Swartz J. G. and Mitchell J. E. (1974) Acyl transfer reactions of retina. Biochemistry 13, 5053. Takai Y., Kishimoto A., Iwasa Y.. Kawahara Y., Mori T.. Nishizuka Y., Tamaura A. and Fuji T. (1979) A role of membranes in the activation of a new multifunctional protein kinase system. J. Biochrm. 86, 575. Tinoco J.. Miljanich P. and Medwadowski B. (1977) Depletion of docosahexaenoic acid in retinal lipids of rats fed a linolenic acid-deficient. linoleic acid-containing diet. Biochim. hiophys. Actu 486, 575. Tyson C. A., Zande H. V. and Green D. E. (1976) Phospholipids as ionophores. J. hiol. Chern. 251, 13261332. Urban P. F.. Dreyfus H.. Neskovic N. and Mandel P. (1973) Phospholipid metabolism in light and dark adapted excised retina. J. ,Veurochrm. 20, 325. Wheeler T. G.. Benolken R. M. and Anderson R. E. (1975) Specificity of fatty acid precursors for the electrical rcsponse to illumination. Science 188. 1312. Wiegand R. D.. Giusto N. M.. Andrews L. D.. Monaco W. A. and Anderson R. E. (1981) Chemical changes in albino rat rod outer segments following constant illumination. Int,esr. Ophrhtrl. risutrl Sci. (Suppl.) 20, 4. Wykle R. L. (1977) Brain. In Lipid Mrttrholisrn in Mart~mtr/s (Edited by Snyder F.). pp. 317 366. Plenum Press. New York. Young R. W. (1976) Visual cells and the concept of renewal. Inrest. Ophrhal. 9, 70@725.