Phosphatidylinositol Breakdown

Proc. Nati. Acad. Sci. USA Vol. 77, No. 6, pp. 3292-3296, June 1980 Biochemistry

Evidence for a role in stimulus-secretion coupling of prostaglandins derived from release of arachidonoyl residues as a result of phosphatidylinositol breakdown (phospholipid effect/cyclooxygenase inhibitors/nonsteroidal anti-inflammatory drugs/phosphatidic acid/ pancreatic enzyme secretion) PAUL J. MARSHALL, JOHN F. DIXON, AND LOWELL E. HOKIN* Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706 Communicated by David E. Green, March 20,1980

ABSTRACT That stimulation of secretion in exocrine and 5, 11, and 16). In recent years much attention has been paid to endocrine glands is associated with increased turnover of the role of prostaglandins (PGs) in modulating or regulating a phosphatidylinositol and phosphatidic acid has been known for many years. In the present work, mouse pancreases were pre- variety of physiological functions (17, 18). The formation of PGs labeled with [14C]arachidonic acid in the presence of the is controlled by the release of arachidonic acid from phospho- secretogogue carbamoylcholine. They were then incubated in lipids (19), and because PtdIns from brain, liver, and mouse media containing atropine and 1% albumin. The atropine pancreas consists largely of 1-stearoyl-2-arachidonoyl-sn-gly- causes the tissue to revert to the resting state, and the albumin cero-3-phosphoinositol (13, 20, 21), the release of arachidonic binds free [14C]arachidonic acid. The tissues were finally incubated in media containing no stimulant or the stimulant acid on stimulation of PtdIns breakdown followed by the for- caerulein, which is not blocked by atropine. Stimulation with mation of PGs, which would play a role in stimulus-secretion caerulein led to a 44% loss of [1-14C]arachidonic acid from coupling, is an attractive hypothesis. We have tested this hy- phosphatidylinositol. About half of this released arachidonic pothesis by studying the effects of: (i) nonsteroidal anti-in- acid ended up in phosphatidic acid. The remainder of the loss flammatory drugs (NSAID), which are cyclooxygenase inhib- could not be accounted for in any other lipid. No other phos- itors, in pholipids showed statistically significant changes on stimula- (ii) arachidonic acid the presence and absence of the tion. Several lines of evidence indicated that the missing ara- cyclooxygenase inhibitor indomethacin, and (iii) PGs on chidonic acid was converted to prostaglandins, which play a role secretogogue-stimulated amylase secretion in isolated mouse in stimulus-secretion coupling. Four nonsteroidal anti-in- pancreas incubated in vitro. We have also examined the change flammatory drugs inhibited secretogogue-induced amylase se- in steady-state level of [1-'4C]arachidonic acid in prelabeled cretion from pancreases, and their potencies paralleled their lipids on stimulation of pancreases with the secretogogue potencies in inhibiting cyclooxygenase, which converts arachidonic acid to prostaglandins. Amylase secretion was stimu- caerulein. The data indicate that arachidonic acid is released lated by arachidonic acid, and this stimulation was blocked by from Ptdlns on stimulation with caerulein and they suggest that the nonsteroidal anti-inflammatory drug indomethacin. Other part of this arachidonic acid forms a PG or PGs, which are in- fatty acids failed to elicit amylase secretion. At concentrations volved in stimulus-secretion coupling. of 3-10 nM, prostaglandins I2, E1, E2, D2, and F2. gave statistically significant stimulations of secretion. Other prostaglandins tested gave no significant stimulation. MATERIALS AND METHODS Amylase Secretion. Adult male mice (25 g) from an inbred In 1953 Hokin and Hokin (1) reported that cholinergic stimu- Swiss-Webster strain were used. The pancreases were removed lation of amylase secretion, paralleled by the secretion of other and incubated as described by Hokin-Neaverson (12). The pancreatic enzymes from pigeon pancreas slices (2), was asso- pancreas tissue was preincubated for 15 min at 37°C before the ciated with an 8-fold increase in the incorporation of [32P]or- addition of any drugs. When NSAID were tested, the pancre- thophosphate into the total phospholipids. This "phospholipid ases were transferred to fresh media containing the drugs and effect" was subsequently found in a variety-of exocrine and incubated for an additional 15 min. This pretreatment was endocrine glands as well as synaptic tissue on stimulation with found necessary to maximize their inhibitory actions. The appropriate agonists (see reviews 8-6). The phospholipid effect NSAID, free fatty acids, and PGs were prepared immediately primarily involved increased turnover of phosphatidylinositol before use and added in dimethyl sulfoxide (Me2SO). An equal (PtdIns) and phosphatidic acid (PtdA) (7). The increased volume of Me2SO was added to control vessels. The volume of turnover of phosphate was associated with an increased turn- Me2SO was usually 0.25% of that of the incubation medium and over of glycerol and inositol (8), and it was confined to the en- never exceeded 0.5%. These concentrations of Me2SO had no doplasmic reticulum, as demonstrated by combined differential effect on amylase secretion. After the pretreatment period the centrifugation (9) and radioautography with [3H]myo-inositol tissues were transferred to fresh media containing the stimulant (10). The phospholipid effect involves a fall in steady-state level with or without inhibitory drugs. Unless otherwise indicated, of PtdIns and a rise in steady-state level of PtdA (11-15). the final incubation was 30 min. Aliquots of the incubation Although the phospholipid effect was reported over 25 years ago, its physiological significance is still not understood (see refs. Abbreviations: PtdIns, phosphatidylinositol; PtdA, phosphatidic acid; PtdSer, phosphatidylserine; PtdEtn, phosphatidylethanolamine; The publication costs of this article were defrayed in part by page PtdCho, phosphatidylcholine; Me2SO, dimethyl sulfoxide; NSAID, charge payment. This article must therefore be hereby marked "ad- nonsteroidal anti-inflammatory drugs; CbmCho, carbamoylcholine; vertisement" in accordance with 18 U. S. C. §1734 solely to indicate PG, prostaglandin; PZ, pancreozymin. this fact. * To whom reprint requests should be addressed. 3292 Biochemistry: Marshall et al. Proc. Natl. Acad. Sci. USA 77 (1980) 3293 media were then assayed for amylase by the method of Rick and Stegbauer (22). The activity of amylase in the medium was calculated as nmol of maltose released per mg wet weight of original pancreas per min at 250C. In the figures, the data are expressed either as percent inhibition of the secretogogue- stimulated secretion or percent of the control tissue incubated without any pharmacologically active compounds. All concentrations of added substances are those in the final incubation 40 medium. [1-"4CArachidonic Acid Released from Prelabeled PtdIns. [1-14C}Arachidonic acid in toluene (Amersham) was dried 40 - under N2 and taken up in Me2SO, and 20-1. aliquots were An//~~~~ added to 2 ml of incubation medium. Each 2 ml of incubation medium contained 0.44 ,uCi (1 Ci = 3.7 X 1010 becquerels) of 20/ [1-14C]arachidonic acid (specific activity, 55.5 mci/mmol). The concentration of arachid6nic acid during incubation was 4 MAM. Pancreases were incubated and the radioactivity in lipids was 10 9 8 7 6 5 4 determined as described in Table 3. -log[indomethacin] (M) Materials. Pancreozymin (PZ), caerulein, and fatty acid-free FIG. 2. Log dose-response curves for indomethacin inhibition bovine serum albumin were obtained from Calbiochem, and of hormone-stimulated amylase release. The data points for PZ (0) Me2SO was obtained from Aldrich. Unless otherwise indicated, and caerulein (C) are averages of two experiments. all other chemicals were purchased from Sigma. PZ also stimulates pancreatic enzyme secretion by binding to a receptor different from that for muscarinic drugs, as evi- RESULTS denced by the lack of blockage by the muscarinic antagonist Effects of NSAID Cn Secretogogue-Stimulated Secretion atropine (24). However, indomethacin inhibited this secretion of Amylase. Carbamoylcholine (CbmCho), a muscarinic ago- and the secretion elicited by caerulein, which is a decapeptide nist, stimulates enzyme secretion in the exocrine pancreas; the analogue of PZ (Fig. 2). We used concentrations of PZ (100 concentration of CbmCho required to elicit maximal secretion ,gg/ml) or caerulein (0.5 Mg/ml) found to elicit maximal amy- was found to be approximately 1 uM (not shown). The mean lase secretion (not shown). Indomethacin appeared to maxi- and SD stimulation of amylase secretion in nine experiments mally block PZ- or caerulein-induced secretion at a concen- were 230 + 30% (range, 180-50%). Pretreatment of pancreases tration of 10 MM. with increasing concentrations of NSAID, which are cycloox- Effects of Arachidonic Acid on Amylase Secretion in the ygenase inhibitors, caused a marked decrease in CbmCho- Presence and Absence of Indomethacin. Arachidonic acid stimulated amylase secretion (Fig. 1). Indomethacin was the is the substrate in the synthesis of PGs, and this synthesis is most potent inhibitor tested and was followed by phenylbuta- blocked by cyclooxygenase inhibitors such as indomethacin (23). zone, aspirin, and salicylic acid in that order. At concentrations Amylase secretion was stimulated by arachidonic acid (Fig. 3). tested (10-10 to 10-3 M), CbmCho could not overcome the in- Maximum amylase secretion was obtained at 100 ,M arachi- hibition by 10-6 M indomethacin (not shown). Basal amylase donic acid, which is comparable to the concentration (added levels were unaffected by treatment with indomethacin or with exogenously) required for platelet aggregation (25). Indo- any of the other NSAID (not shown). The inhibition of amylase methacin blocked arachidonic acid-stimulated secretion, as was secretion by NSAID in our system parallels the inhibitory ac- the case for secretogogue-stimulated secretion. Maximal inhi- tions of these drugs on cyclooxygenase with respect to the ef- bition was seen between 10-5 and 10-6 M indomethacin (Fig. fective concentrations and order of efficacy of the different 4). When other fatty acids or the non-PG precursor methyl drugs as reported by others (23). arachidonate were added at concentrations comparable to those of arachidonic acid, no amylase secretion was elicited (Table 1). 80 220

60I - 200 0 180 .4 40 10 0 Q 140 20[- 120 -

I II 100 8 7 6 5 4 3 2 -log[inhibitor] (M) 80 e 8 7 6 5 4 3 FIG. 1. Log dose-response curves for inhibition by cyclooxygenase inhibitors ofamylase secretion stimulated by 1MM CbmCho. The -log[arachidonic acid] (M) curves for indomethacin (v), phenylbutazone (0), aspirin (A), and FIG. 3. Log dose-response curves for arachidonic acid-stimulated salicylic acid (0) were determined from points that represent the amylase release. The data points are the averages~of three experiments mean (and -SD) of triplicate experiments. ± SD. 3294 Biochemistry: Marshall et al. Proc. Natl. Acad. Sci. USA 77 (1980)

Table 1. Effects of free fatty acids on amylase secretion Specific activity of amylase, nmol of maltose/mg of tissue per min Fatty acid added 0.1 mM 1 mM None 5.0 0.7 4.9 0.7 Arachidonic acid 9.7 ± 1.1 10.2 ± 1.8 Methyl arachidonate 6.4 ± 0.5 5.7 ± 0.6 Linoleic acid 5.6 ± 1.1 5.8 ± 0.8 Linolenic acid 5.8 i 1.3 4.8 ± 0.8 Oleic acid 5.8 0.7 4.8 + 0.6 Stearic acid 5.2 i 1.0 5.2 + 0.8 The results of triplicate experiments are expressed as the mean amylase activity ± SD. When maximally stimulating concentrations of CbmCho and arachidonic acid were added together, secretion was essentially the same as with CbmCho alone (not shown). Indomethacin (1 ,MM) inhibited amylase secretion stimulated by either com- pound added separately or together. These data suggest that the stimulation by CbmCho operates through the pathway involving arachidonic acid. Effects of PGs on Amylase Secretion in Mouse Pancreas. PGs were tested for secretagogue activity at various concen- -log[indomethacin] (M) trations between 10-12 and 10-6 M (Table 2). Those that FIG. 4. Log dose-response curves for indomethacin inhibition stimulated secretion maximally at 3-10 nM [PGE2, PGE1, and of arachidonic acid-stimulated amylase release. The data points are PGI2 (prostacyclin)] gave stimulations similar to the stimulation averages of two experiments using 100 ,uM arachidoinic acid. with the maximally effective concentrations of CbmCho (1 ,M) or arachidonic acid (100MuM). At 3 nM but not 10 nM, PGF2L, finally stimulated (or not stimulated) with caerulein, whose stimulated amylase secretion. At 3 and 10 nM, PGD2 stimulated action is not blocked by atropine (see details m Table 3). There amylase secretion submaximally. Although it cannot be con- was a 44% loss in radioactivity in PtdIns, and this was accom- cluded which is the endogenously produced PG from this ex- panied by a 5.6-fold increase in radioactivity in PtdA (Table periment, it is interesting to note that the PGs appear to be the 3). The loss in radioactivity in PtdIns was approximately twice most potent stimulants for amylase secretion known. the gain in radioactivity in PtdA, suggesting a net loss of ara- Net Release of Arachidonic Acid from Ptdlns on Stimu- chidonic acid. In fact, by summing the radioactivity in PtdIns lation of Prelabeled Mouse Pancreases with Caerulein. and PtdA in each pancreas, a statistically significant loss of Stimulation of secretion in mouse pancreas by cholinergic arachidonic acid from PtdA + PtdIns could be demonstrated agents is associated with a net decrease in the steady-state level (Table 3). No statistically significant changes in radioactivity of PtdIns and a net increase in the steady-state level of PtdA that in phosphatidylserine (PtdSer), phosphatidylcholine (PtdCho), appears to be almost stoichiometric with the decrease in PtdIns or phosphatidylethanolamine (PtdEtn) were seen. These data (11-14). Blocking of secretogogue action with atropine causes provide rather strong evidence that the arachidonic acid, which returns of PtdIns and PtdA to prestimulated levels; if radioactive is recruited for PG formation, is derived from PtdIns break- glycerol or inositol is present from the beginning, a rise in ra- down. dioactivity in PtdIns is seen on adding atropine. Restimulation with PZ, which is not blocked by atropine, causes a loss of ra- DISCUSSION dioactivity in PtdIns. Therefore, in order to prelabel the PtdIns, It is shown here that stimulation of pancreases with caerulein the pancreases were incubated with CbmCho and [1-14C]ara- causes a 44% loss of arachidonoyl residues from prelabeled chidonic acid, quenched with atropine and fatty acid-free al- PtdIns. Although half of the arachidonoyl residues lost end up bumin (to bind residual arachidonic acid in the medium), and in PtdA, the other half cannot be accounted for by any other

Table 2. Effects of prostaglandins on amylase secretion Specific activity of amylase, nmol of maltose/mg of tissue per min Drug 3 nM P value 10 nM P value Control 6.5 + 1.0 PGE2 11.4 ± 1.4 <0.001 10.3 ± 1.9 <0.025 PGE1 9.9 1.0 <0.005 10.0 + 1.8 <0.025 PGI2 9.8 ± 0.7 <0.001 9.1 ± 0.8 <0.005 PGF2a 10.3 ± 2.2 <0.025 7.6 + 0.7 NS PGD2 8.9 ± 1.1 <0.005 8.1 ± 0.9 <0.05 Thromboxane B2 7.3 + 0.3 NS 7.2 ± 1.8 NS (15S)-Hydroxy-9a,lla-(epoxymethano)- 5Z,13E-dienoic acid 7.4 + 0.3 NS 6.3 ± 0.7 NS Arachidonic acid (100 gM) 11.0 ± 1.7 <0.001 CbmCho (1 uM) 15.0 ± 1.7 <0.001 All PGs were tested on individual pancreases on the same day and the experiments were repeated three or more times. The results are expressed as the mean amylase activity ± SD. P is for comparison with control; NS, not significant. Biochemistry: Marshall et al. Proc. Natl. Acad. Sci. USA 77 (1980) 3295

Table 3. Net release of radioactive arachidonic acid from PtdIns on stimulation of prelabeled mouse pancreases with caerulein Radioactivity, cpm/100 mg tissue Lipid Unstimulated Stimulated Increment P value PtdIns 11,370 4 1,030 6,330 + 458 -5,040 <0.001 PtdA 505 k 23 2,850 225 +2,340 <0.001 PtdA + PtdIns 11,900 I 1,060 9,180 h 479 -2,710 <0.025 >0.0125 PtdSer 4,270 I 506 3,860 4 350 -410 NS PtdCho 108,100 + 4,240 98,900 + 4,260 -9,200 NS PtdEtn 17,500 i 980 18,100 4 832 600 NS All mouse pancreases were incubated for 30 min in control incubation media as described under Materials and Methods with the further addition of [1-14C]arachidonic acid and 1 mM CbmCho. They were then transferred to control incubation media containing fatty acid-free bovine serum albumin at 10 mg/ml and 100 AM atropine and were incubated for a further 30 min. They were again transferred to control incubation media without (unstimulated) or with (stimulated) caerulein at 2 Ag/ml and incubated for 60 min. Atropine (100 AM) was present in all vessels during this incubation. The pancreases were then removed and frozen in centrifuge tubes by submersion in a dry ice/ethanol bath and stored at -70'C until work-up. Lipids were extracted by the method of Bligh and Dyer (26), with final pro- portions of 0.2 g of tissue (final average wet weight), 1.6 ml of H20, 2 ml of methanol, and 2 ml of chloroform. After removal ofthe first chloroform extract, the water/methanol phase and solid interface were acidified with HCl (final concentration 0.1 M) and reextracted with 2 ml of chloroform. The individual lipids were separated by two-dimensional thin-layer chromatography on 20 X 20 Adsorbsil-5 Prekotes (Applied Science Laboratories) that had been washed with 0.4 M boric acid (the wash was necessary to separate the PtdIns from the PtdSer). The solvent in the first dimension was chloroform/methanol/ammonia/H20, 70:30:2:3, vol/vol (27) and in the second dimension, chloroform/acetone/methanol/acetic acid/H20, 30:40:10:10:5, vol/vol (28). Enough extract was applied to give a total of 40,000 dpm. Twenty micrograms each ofPtdIns (Serdary Research Laboratories, London, Ontario), PtdSer, PtdA, and arachidonic acid were added as carriers. Iodine-stained spots were scraped directly into 10 ml of Aquasol (New England Nuclear) for measurement of radioactivity. Counting efficiency was 85.5%. The arachidonic acid spot and the solvent front spot (both not shown) were also routinely counted (they showed much wider variation in values). The recovery oftotal radioactivity applied to the plates averaged 85%. All counts were normalized to an average total radioactivity in the Bligh and Dyer extract from 30 pancreases of 229,200 cpm/100 mg (15 unstimulated, 233,100 and 15 stimulated, 225,400). The values are from eight unstimulated and eight stimulated pancreases.

phospholipid, or by arachidonic acid or neutral lipids (latter some evidence that diglyceride is an intermediate in the data not shown). breakdown of PtdIns and possibly PtdCho (29-31), and it has Several lines of evidence support the hypothesis that ara- been suggested that increased concentrations of diglyceride chidonic acid released from PtdIns was converted to PGs that would lead to increased phosphatidic acid synthesis via the play a role in stimulus-secretion coupling in the exocrine enzyme diglyceride kinase (31). It is tempting to speculate that pancreas. First, the stimulation of secretion of amylase by the synthesis of PtdA on stimulation of breakdown of PtdIns various secretogogues (CbmCho, PZ, or caerulein) is inhibited serves to regulate the steady-state concentrations of arachidonic by NSAID, which are cyclooxygenase inhibitors. The order of acid available for prostaglandin synthesis. efficacy and the concentrations of these various drugs for in- A hypothesis purported to explain the physiological signifi- hibiting the two systems are essentially the same (23). Second, cance of the phospholipid effect, which has recently received arachidonic acid, which is acted on by cyclooxygenase to form considerable attention (see review 32), is that the breakdown PGs, stimulates amylase secretion, and this stimulation is of PtdIns opens Ca2+ gates in the membrane. The data pre- abolished by the cyclooxygenase inhibitor indomethacin. sented here would argue against "calcium gating" as the im- Amylase secretion is not obtained when other fatty acids, such mediate consequence of the phospholipid effect. Because Ca2+ as stearic acid, oleic acid, linoleic acid, linolenic acid, or the ester does appear to be involved in stimulus-secretion coupling in methyl arachidonate, are added in concentrations similar to the glands in which a phospholipid effect has been observed (32), concentration of arachidonic acid. When arachidonic acid and it is possible that PGs could be responsible for the movements CbmCho are combined, the resulting secretion is not signifi- of Ca2+, which in turn directly stimulate secretion, or, alter- cantly greater than with CbmCho alone, suggesting that the two natively, PGs and Ca2+ could be acting in a concerted compounds do not stimulate secretion by independent mech- manner. anisms. Third, addition of PGE2, PGEI, PGI2, PGF2a, or PGD2 at 3-10 nM stimulates amylase secretion almost as much as Note Added in Proof. After incubation of prelabeled pancreases for maximum concentrations of CbmCho. The other PGs tested 15 min in the presence of caerulein at 1 ,ug/ml, a radioactive peak gave no statistically significant stimulations. Calculations show cochromatographed on thin-layer chromatography with authentic that the amount of missing arachidonic residues in lipids on PGE2. This peak contained 5500 dpm per pancreas (the control value stimulation of secretion could form 210 nmol PGs per kg of was 2500), which compares with 5700 dpm lost from PtdA + tissue, which is considerably in excess of the maximally stim- PtdIns. ulating concentrations of the active PGs. It is not yet known which PG or PGs are actually formed endogenously from We thank Dr. Robert U. Simpson for his valuable assistance. We are arachidonic acid. grateful to Dr. John E. Pike, The Upjohn Company, for a generous gift It is interesting that breakdown of PtdIns, in addition to re- of prostaglandins used in this report. This investigation was supported leasing arachidonic acid, leads to a marked increase in the by grants from the National Institutes of Health (HL 16318) and from synthesis of PtdA. In thrombin-stimulated platelets, there is the National Science Foundation (PCM 76-20602). 3296 Biochemistry: Marshall et al. Proc. Nati. Acad. Sci. USA 77 (1980)

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