phosphatidylinositol:myo-inositol exchange activity in intact nerve endings: substrate and cofactor...

Jorirnnl of Neurochemistty Raven Press, New York 0 1986 International Society for Neurochemistry

Phosphatidy1inositol:myo-Inositol Exchange Activity in Intact Nerve Endings: Substrate and Cofactor Dependence, Nucleotide Specificity, and Effect

on Synaptosomal Handling of myo-Inositol

Gerard Berry, John R. Yandrasitz, Victoria M. Cipriano, Shing Mei Hwang, and Stanton Segal

Division of Biochemical Development and M o l e c ~ l a r Diseases, Children's Hospital of Philndelphiu; und Departments of Pediatrics and Medicine, University of' Pennsylvania School of Medicine, Philadelphia, Pennsylvatiia, U . S . A .

Abstract: Micromolar concentrations of CMP produced a large increase in Mn'+-dependent phosphatidylino- sitokmyo-inositol exchange activity in isolated nerve endings or synaptosomes. The apparent K , for CMP was 2 k M , and that for myo-inositol was 38 k M . Only cytidine nucleotides were capable of enhancing activity, and this effect is probably specific for CMP, because the synaptosomal preparation rapidly converted CTP o r CDP to CMP. Manganese did not affect the uptake of myo-inositol into the synaptosomal cytosolic fraction or myo-inositol levels. Determinations of myo-inositol specific activity showed that the Mn2+-enhanced labeling of phosphatidylinositol was not accompanied by a decrease of label content in free myo-inositol. This lack of an effect on intrasynaptosomal myo-inositol and the dependence of exchange on cytidine nucleotides whereas cytidine itself was previously found to be without effect show that for the bulk of Mn2+-dependent exchange activity, it is the

myo-inositol in the incubation medium that is being directly incorporated into membrane-bound phosphatidylinositol. Because CMP dependence is the hallmark of exchange catalyzed by CDP-diacylglycero1:inositol phos- phat idyl t ransferase , th i s e n z y m e is likely t o be responsible for most of the exchange activity in synaptosomes. The strong affinity of this exchange system for CMP suggests that endogenous levels of this nucleotide might support Mn2+-dependent exchange in the absence of added nucleotide. Key Words: Synaptosomes-Phos- phatidylinosito1:myo-inositol exchange activity-myo- Inositol-Phosphatidylinositol-Cytidine nucleotide- Manganese. Berry G . et al. Phosphatidylinosito1:tnyo- inositol exchange activity in intact nerve endings: Sub- strate and cofactor dependence, nucleotide specificity, and effect on synaptosomal handling of myo-inositol. J . Neurochem. 46, 1073- I080 (1986).

The enzyme CDP-diacylg1ycerol:inositol phosphatidyl transferase (EC 2.7.8.11) will catalyze the formation of phosphatidylinositol (PI) and CMP from the substrates CDP-diacylglycerol (CDP- DG) and myo-inositol in the presence of a divalent metal cofactor, Mn2+ or Mg2+. This reaction is re- versible, and Mn2+ is preferred over Mg2+. When de novo synthesis of PI is enhanced in metabolically active intact tissue preparations such as isolated nerve endings or synaptosomes, there is increased incorporation of radiolabeled myo-inositol and phosphate into PI. Enhanced incorporation of ra-

diolabeled myo-inositol into PI may be seen, however, in the absence of enhanced labeling with radiolabeled phosphate and in the absence of de novo synthesis of PI. Such incorporation of myo-inositol into PI by exchange of the free and lipid-bound moeities has been demonstrated in various mam- malian tissues (Agranoff et al., 1958; Paulus and Kennedy, 1960; Hubscher, 1962; Thompson et al., 1963; Broekhuyse, 1971; Jungawala et al., 1971; Jungawala, 1973; Holub, 1974; Takenawa et al., 1977; Yandrasitz and Segal, 1979; Brammer and Carey, 1980; Tolbert et al., 1980; Bleasdale and

Received May 21, 1985; accepted October 8, 1985. Address correspondence and reprint requests to Dr. G . Berry

at Division of Biochemical Development and Molecular Dis- eases, Children's Hospital of Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104, U.S.A.

Abbreviations used: CDP-DG, CDP-diacylglycerol; HEPES. N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PI, phosphatidylinositol.

1073

1074 G. BERRY ET AL.

Wallis, 1981 1. Nucleotide-dependent PI: myo-inositol exchange m a y b e ca ta lyzed by C D P - DG:inositol phosphatidyl t ransferase , resulting in both exchange of phosphatidyl moieties between PI and CDP-DG and exchange of f ree and lipid-bound myo-inositol (Hokin-Neaverson et al., 1977). In addition, the work of Takenawa et al. (1977) and Tak- enawa and Egawa (1980) suggests the existence of a nuc leo t ide- independent , Mn '+-dependent e n - zyme that catalyzes myo-inositol specific base exchange with PI in a manner analogous to that seen for phosphatidylethanolamine and ethanolamine or serine. We have recently reported on the ability of t he t ransferase p roduc t , CMP, in micromolar amounts t o enhance markedly PI:myo-inositol exchange activity in an intact synaptosomal preparation (Berry et al., 1983). Because CMP dependence is the hallmark of exchange catalyzed by CDP-DG inositol phosphatidyl transferase (Paulus and Ken- nedy, 1960; T h o m p s o n e t al . , 1063; Ste ine r and Lester, 1972; Hokin-Neaverson et al., 1977; Bleas- dale and Wallis, 1981). this enzyme may be responsible for the exchange activity in synaptosomes.

In this article, we report on the substrate and cofactor dependence and nucleotide specificity of synaptosomal CMP-dependent P1:myo-inositol exchange activity and the effect of enhanced exchange activity on synaptosomal handling of myo-inositol. A n increased a w a r e n e s s a n d knowledge of th i s myo-inositol exchange activity is important because this activity may contribute t o basal myo-inositol incorporation into PI and complicates, or may even negate, interpretations of PI turnover in studies where my0-[2-~H]inositol is used as a metabolic tracer.

MATERIALS AND METHODS

Preparation of synaptosomes and incubation media

For each experiment, synaptosomes were prepared from the cerebral hemispheres of four rats using differ- ential centrifugation techniques and a five-step Ficoll gradient as described previously (Warfield and Segal, 1974). All operations were performed in the cold. The basic incubation medium, a modified Krebs buffer, consisted of the following: 138 mM NaCI, 1 mM sodium phosphate, 1.3 mM CaCI,, 2 mM MgSO,, I p.M MnCI,, and 5 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) at pH 7.4. This buffer was equilibrated in an atmosphere of 95%' O2 and 5% CO, at 0°C. Malate ( I mM) and pyruvate (2 mM) were added to the synaptosomes suspended in the oxygenated buffer. and each flask was preincubated at 37°C for 10 min in a shaker bath before the initiation of any experiment.

Assessment of P1:myo-inositol exchange activity Each assay was started by pipetting I ml of the prein-

cubated synaptosomal suspension ( I mg protein) into a test tube that contained 6.7 p.Ci of myo-[2-3H]inositol (333 +Ci/pmol) and varying amounts of unlabeled myo-

inositol, MnCI,, and nucleotide. The final volume was I ml with - 1 mgiml of synaptosornal protein. For K,, determinations, incubations at 37°C in a shaker bath were stopped at 10 min by addition of 1 ml of a cold 20% trichloroacetic acid solution containing 20 mM myo-inositol to the synaptosomal suspensions followed by transfer to an ice bath. The suspension was pelleted by centrifugation at 40,000 g for 30 min, resuspended in 10 ml of a 20 m M myo-inositol solution, and repelleted by centrifugation. One milliliter of methanoliwater ( 1 : 1 voli vol) was used to resuspend the resultant pellet from which PI was extracted as described by Yandrasitz and Segal (1979), except that additional tnyo-inositol (20 mM) was added during partition steps lo minimize the presence of free my0-[2-~H]inositol in the chloroformlmethanol phase. The lipid extracts were transferred to glass scintillation vials, evaporated to dryness with a stream of N,, and counted for radioactivity in a Packard TRI-CARB liquid scintillation counter following addition of 10 ml of OCSiethanol (70:28 volivol). Counting efficiency was as- sessed using ['Hltoluene.

To correct for carry-over of free myo-[2-'H]inositol, identical amounts of myo-[2-'H]inositol were added to 1 ml of synaptosomal suspension after addition of I ml of 20% trichloroacetic acid with 20 mM myo-inositol. These samples were analyzed in parallel with the experimental samples and served as controls for the carry-over of free my0-[2-~H]inositoI. To determine whether all of the tritium counts in the Folch extract represent labeled PI, some of the extracts were split and either counted directly as above or subjected to HPLC analysis (Yandrasitz et al., 1981). Using this HPLC system, the tritium counts in the PI peak equaled the counts in the extract following correction for free myo-[2-'H]inositol carry-over. The polyphosphoinositides were lost from the Folch extract during partioning with 0.1 M HCI and therefore do not account for any tritium label. In subsequent experiments, however, using 0.6 M HCI at the partition step, P1-4- phosphate and PI-4,5-bisphosphate labeled with myo- [2-'H]inositol were recovered and accounted for -20% of the total tritium label in phospholipids. Protein content was determined by the method of Lowry et al. (1951), with bovine serum albumin as slhndard.

Enzyme-mediated hydrolysis of cytidine nucleotides

For these experiments on time-dependent breakdown of cytidine nucleotides, each assay was started by pipetting 1 ml of the preincubated synaptosomal suspension into a test tube that contained 2 nCi of [S-'H]CTP (final concentration 10 p M ) and MnCI,, (final concentration 0.5 m M ) . The incubation at 37°C i n a shaker bath was stopped after 10 min by placing the test tube in an ice bath. For zero-time determinations, the mixed suspensions were immediately placed in the ice bath. The suspensions were then pelleted by centrifugation at 8,000 g for 10 min, and the supernatants were immediately placed in a -70°C freezer until analyzed. To determine whether the synaptosomal breakdown of added cytidine nucleotides was enzymatically mediated, identical assays were performed using boiled synaptosomal membranes. For these experiments, isolated synaptosomes were boiled for 5 min and then cooled in an ice bath, and the suspension was then centrifuged at 40,000 ,q for 20 min. The pellet

P1:myo-INOSITOL EXCHANGE ACTIVITY IN S YNAPTOSOMES 1075

was then resuspended in the modified Krebs buffer and incubated as described above.

Reverse-phase ion pair HPLC (Darwish and Prichard, 1981) was used to separate cytidine and the cytidine nucleotides. Each 0.5-ml injection onto the HPLC apparatus contained 0.45 ml of supernatant and 0.05 ml of a tetrabu- tylammonium phosphate solution with 10 nmol each of cytidine, CMP, CDP, and CTP for a final tetrabutylam- rnonium phosphate concentration of 10 mM. During each assay, the peaks were collected, evaporated to dryness with a stream of N2, and counted for radioactivity in a Packard TRI-CARB liquid scintillation counter following addition of 0CS:ethanol (70:28 vol/vol). In an additional experiment, 0.9 nCi of stock [S-3H]CTP was incubated at 37°C in water for 10 min and then analyzed with carrier cytidine nucleotides by HPLC, and in another experiment it was directly injected onto the HPLC apparatus with carriers to determine the extent of spontaneous breakdown of [S3H]CTP.

Effect of Mn2+ on myo-inositol uptake The uptake of myo-inositol by synaptosomes was de-

termined as previously described (Hwang et al., 1978; Warfield et al., 1978). In brief, synaptosomal suspensions were incubated at 37°C for 40 rnin in the modified Krebs buffer with 20 p M myo-[2-'H]inositol(170 pCi/mmol) and 1 p M or 0.5 mM MnCI,. Synaptosomes were pelleted by centrifugation and lysed with 10% trichloroacetic acid, and the radioisotopic label was then counted in the orig- inal supernatant, the pellet extract, and the membrane pellet. Uptake is expressed as pmol of myo-inositolimg of total synaptosomal protein and was corrected for label found when synaptosomes were pelleted immediately after addition of labeled myo-inositol. [c~rhoxy-'~C]Inulin was used to estimate the volume of trapped medium in the pellet.

Effect of Mn2+ on content and specific activity of synaptosomal myo-inositol

GLC was used to measure changes in synaptosomal myo-inositol content and specific activity following incubations with increased MnCI, concentrations. Synap- tosomes were incubated i n a modified Krebs buffer as described above at 37°C for 40 min with 6.7 pCi of myo- [2-3H]inositol (final concentration 20 pM) and 1 F M or 0.5 mM MnCI,. The incubation was stopped by addition of 15 ml of cold Krebs buffer to the synaptosomal suspension followed by centrifugation at 40,000 g for 20 min. For specific activity determinations, the pellet was washed with additional cold Krebs buffer and recentri- fuged. The nzyo-inositol was extracted with 10% trichloroacetic acid, derivatized with trimethylsilylating re- agents, and analyzed by GLC as previously described (Warfield and Segal, 1978). The peaks corresponding to derivatized myo-inositol were collected at the stream splitter, and radioactivity was determined by liquid scintillation counting. Peak areas were determined by elec- tronic integration (minigrator).

For myo-inositol content determinations, only unlabeled myo-inositol was present, and the wash of the synaptosomal pellets was omitted. A Somogyi filtrate (Som- ogyi, 194.5) was prepared after addition of 20 nmol each of the internal standards ribitol and a-methylmannoside. Results were calculated as nmol of myo-inositollmg of

protein. Duplicate samples were analyzed for each experiment.

Laboratory animals, chemicals, and radioisotopes Rats were obtained from Charles River Breeding Labs

(Wilmington, MA, U.S.A.). rnyo-[2-3H]Inositol was obtained from The Radiochemical Centre (Amersham, En- gland), and [5-'H]CTP and [~urboxy- '~C] inulin from New England Nuclear (Boston, MA, U.S.A.). N-(Trimethylsilyl)imidazole and column packings were from Applied Science (State College, PA, U.S.A.). Sol- vents were from Burdick and Jackson Laboratories (Mus- kegon, MI, U.S.A.) or J. T. Baker (Phillipsburg, NJ, U.S.A.). The sulfuric acid used in HPLC solvents was ULTREX grade from Baker. HEPES was obtained from Calbiochem (La Jolla, CA, U.S.A.). Tetrabutylam- monium phosphate was from Eastman Kodak Co. (Koch- ester, NY, U.S.A.). OCS was from Amersham (Arlington Heights, IL, U.S.A.). All other chemicals were reagent grade.

RESULTS Exchange activity dependence on substrate and cofactor concentrations

We previously demonstrated that maximal exchange activity requires intact synaptosomes (Berry et al., 1983). Because an intact synaptosomal preparation was used for characterization of this enzyme activity, a physiological buffer was required. Whenever possible, substrate concentrations were chosen that would not deviate greatly from the in vivo state. However, the normal level of CMP in the CNS interstitium is unknown, and the use of supplementary Mn2 + is always pharmacologic. Be- cause the phosphate concentration in our modified Krebs buffer is 1 mM, concentrations of MnCI, of > I mM were not used because of the possibility of complex formation.

The dependence of myo-[2-3Hlinositol incorporation into PI on Mn2+ concentrations is shown in Fig. 1. A myo-inositol concentration of 100 kA4 was

0 l o t

8 -

6 -

200 400 600 800 1000

p M Mn++

FIG. 1. Effect of Mnz+ on PImyo-inositol exchange activity. The rate of incorporation of my0-[2-~H]inositol into PI, performed as described in Materials and Methods, is plotted against the concentration of the metal cofactor Mn2+. The concentration of myo-inositol was 100 pM. Data are mean 2 SEM values for four determinations in two experiments.


chosen because it approaches CSF levels yet allows for a specific activity of myo-inositol in the incubation medium great enough to allow easy detection of incorporation into PI. Manganese increased incorporation of free my0-[2-~H]inositol into PI, and the magnitude of this stimulation was comparable to that seen in longer incubations where PI specific activity was measured using a more sensitive GLC method (Yandrasitz and Segal, 1979). Although the effect of Mn2+ on exchange activity appears to be leveling off at I mM, an apparent K , was not calculated because higher concentrations of Mn2+ were not tested. The incorporation of [33P]P, into PI is not significantly increased when Mn2+ is used to stimulate exchange activity (Yandrasitz and Segal, 1979). Incorporation of my0-[2-~H]inositol into polyphosphoinositides was not affected by Mn2+ or CMP (manuscript in preparation).

The dependence of my0-[2-~H]inositol incorporation into PI on myo-inositol concentration is shown in Fig. 2. An apparent K , of 38 pM in the presence of 0.5 mM Mn2+ and 1 pM CMP was obtained by Lineweaver-Burk analysis. The effect of CMP on n?yo-[2-3H]inositol incorporation into PI is shown in Fig. 3 . CMP produced a very large increase in the Mn2+-dependent myo-[2-3H]inositol incorporation into PI. The apparent K for CMP was 2 pM in the presence of 0.5 mM MI?+ and 100 p M myo-inositol, and concentrations of CMP as low as 0.5 p M resulted in a greater than fivefold stimulation. When synaptosomes were incubated in the standard Krebs buffer, which contains I p M Mn2+, 5 pM CMP alone was capable of increasing exchange activity (Table I) . In other experiments (data not shown), 50 p M CMP was about as effec- tive as 5 p M .

Nucleotide specificity To test whether the P1:myo-inositol exchange ac-

T

u L L 200 400 600 800 loo0

,uM rnyo-inositol

FIG. 2. Effect of myo-inositol concentration on PI:myo-inositol exchange activity. The rate of incorporation of myo- [2-3H]inositol into PI, performed as described in Materials and Methods, is plotted against the concentration of myo- inositol. The concentrations of CMP and Mn" were 1 and 500 pM, respectively. Data are mean i SEM values for four determinations in two experiments.

tivity was specifically dependent on CMP, 10-min incubations were performed in the presence of 0.5 mM MnC1, with 100 pM concentrations of various nucleotides. The results are shown in Table 1. Only the cytidine nucleotides were capable of enhancing exchange activity, and other nucleotides did not in- terfere with Mn2+-dependent exchange. Because of the analytical method currently used, myo- [2-3H]inositol labeling of PI without added MnCl, or CMP was not consistently above the zero-time control levels. With more sensitive GLC measurements of PI specific activity, labeling of PI in the basic incubation medium has been quantitated (Yandrasitz and Segal, 1979). At very high concentrations (1 mM), CTP, ATP, NAD+, and, to a lesser degree, CDP inhibited exchange activity (data not shown), perhaps because of the production of py- rophosphate, which will readily complex with Mn2+. Similar inhibitory effects of high concentrations of nucleotides on Mn'+ -dependent exchange activity have already been reported (Takenawa et al., 1977; Takenawa and Egawa, 1980).

Because the enhanced exchange activity seen with CTP or CDP could be due to the production of CMP in the incubation medium as CTP or CDP is hydrolyzed, the amount of CTP that breaks down during 10-min incubations was determined. The results (Table 2) indicate that almost all of the CTP had been hydrolyzed to CDP and CMP. CTP breakdown occurred rapidly, with the immediate product being CDP. Incubations up to 10 min showed further conversion of the CDP to CMP and of CMP to cytidine.

Because the rapid hydrolysis of CTP in the suspension of intact synaptosomes strongly suggested an enzymatically mediated process, we incubated [5-'HICTP with boiled synaptosomal membranes. The results (Table 2) show that the boiled synaptosomes will not support the breakdown of CTP. In other experiments, the commercially available [5-3H]CTP was incubated in Rater for 10 min or was injected directly onto the HPLC apparatus. As shown in Table 2, the tritium label found in cytidine, CMP, and CDP in the boiled synaptosomal membrane experiments was comparable to that found without exposure to synaptosomes. The recovery of the tritium-labeled cytidine moiety in these experiments was excellent. Although we cannot elim- inate the possibility of some enzymatically mediated hydrolysis of cytidine nucleotide during handling of specimens in the cold after incubation, the important point is that under conditions that result in myo-inositol exchange, there was breakdown of CTP or CDP to CMP.

Effect of Mn2+ on synaptosomal free myo-inositol The purpose of these sets of experiments was to

determine the effect of exchange activity on synaptosomal myo-inositol, o r more specifically to determine what pool of myo-inositol was being uti-

PI:myo-INOSITOL EXCHANGE ACTIVITY IN S YNAPTOSOMES 1077

FIG. 3. Effect of CMP on PI:myo-inositol exchange activity. The rate of incorporation of my0-[2-~H]inositol into PI, performed as described in Materials and Methods, is plotted against the concentration of CMP. The concentrations of myo-inositol and Mn2+ were 100 and 500 pM, respectively. Data are mean t SEM values for four determinations in two experiments.

; 20 -

P 1 8 . : g 16

- - 14 -

2 4 6 8 1 0 200 400 600 800 loo0

,uM CMP

lized for incorporation into PI during stimulation with Mn2+. Transport experiments were performed to determine the effect of Mn2+ on the amount of radioactivity in the precursor pool of myo-inositol. The myo-[2-3H]inositol labeling of whole synaptosomes in the presence of 0.5 mM Mn2+ increased 55% (Table 3). However, partition of the Mn2+- stimulated uptake showed almost all label to be in the trichloroacetic acid pellet representing synaptosomal membranes (403% increase), whereas uptake into the free synaptosomal pool was not changed significantly. Mn2+, therefore, did not increase labeling of PI with myo-[2-3H]inositol by stimulating the uptake of myo-[2-3H]inositol from the incubation medium. Tolbert et al. (1980) de-

TABLE 1. Effect o j nucleotides on synaptosomal Mn'+-dependent PI:myo-inositol exchange activity

rny0-12-~H]lnositol incorporation into PI

Mn2 + Nucleotide ( d p m h g proteinit0 min)

- 1 P M U 1 p M I rnM cytidine a 1 p M 5 p M C M P 282 t 42 0.5 mM - 0.5 mM 0. I rnM AMP 569 97 0.5 mM 0. I mM ATP 414 t 54 0.5 mM 0.1 mM GMP 791 t 116 0.5 mM 0.1 rnM NAD+ 609 t 116 0.5 mM 0.1 mM NADH 700 t 120 0.5 mM 0.1 mM NADP' 484 t 56 0.5 mM 0.1 mM NADPH 611 t 85 0.5 mM 0.1 rnM CMP 15,996 t 1,099 0.5 mM 0.1 mM CDP 14,486 t 1,102 0.5 mM 0. I mM CTP 11,443 t 846

663 t 104

Synaptosomes were incubated at 37°C for 10 min in a physiologic Krebs buffer containing 100 p M my0-[2-~H]inositol (45 pCi/pmol) with MnCI, and various nucleotides or cytidine in final concentrations as noted. The incubations were stopped by placing the test tubes containing the synaptosomal suspensions in an ice bath. Phospholipids were extracted from a synaptosomal pellet. The [Z7H]PI content was determined by liquid scintillation counting of the lipid extract. Data are expressed as dpmlmg of protein/lO min, where each mean i SEM value is the result of four determinations in two experiments, except for six determinations in three experiments with Mn2+ alone.

Small incorporation not detectable above background.

scribed similar effects in isolated hepatocytes treated with Mn2+.

Because it was still possible that Mn2+ treatment could affect the specific activity of the myo-inositol precursor pool by increasing synaptosomal myo- inositol efflux, we measured myo-inositol levels by GLC. After incubation for 40 min with 0.5 mM MnCl,, there was no significant change in the chem- ical amount of synaptosomal myo-inositol com- pared with incubations in the standard buffer: 10.06 k 2.38 versus 10.05 ? 1.48 nmol/mg of protein (mean 2 SEM, n = 3). Because there was no loss of myo-inositol, these studies also show that high concentrations of MnCI, did not affect synaptosomal integrity. In addition, we have previously shown no impairment in L33P]P, incorporation into PI when synaptosomes are incubated with acetylcholine and Mn2+ (Yandrasitz and Segal, 1979); this response clearly requires intact synaptosomal structures.

Synaptosomal levels of PI (15 n m o l h g of protein) are comparable to myo-inositol levels (Warfield and Segal, 1978) and are not affected by Mn'+ treatment (Yandrasitz and Segal, 1979). If Mn2+ were to affect primarily exchange of lipid-bound myo-inositol with intrasynaptosomal myo-inositol, the increased labeling of PI should be accompanied by a corresponding decrease in the specific activity of the free myo-inositol pool. For this reason, we used GLC to measure the specific activity of myo-inositol after incubations for 40 min with 1 p M or 0.5 mM MnCI,. Synaptosomal my0-[2-~H]inositol specific activity was not decreased, but in fact was increased 38 i 2% (mean ? SEM, n = 4) in the presence of 0.5 mM Mn2+. This increase contrasts with the many- fold increase in synaptosomal [2-3H]PI after a similar incubation with 0.5 mM MnCl,. Taken together, these experiments show that the intrasynaptosomal pool of myo-inositol is not primarily responsible for the Mn2+-stimulated labeling of PI.

Localization of activity Although electron microscopic analysis of the

synaptosomal preparation shows the bulk of material to be synaptosomes, small amounts of membrane fragments, vesicular elements, e tc . , are

J . Neurochem., Vol . 46, No. 4 , I986


TABLE 2. Time-dependent hydrolysis o ~ [ S - ~ H ] C T P by intact synaptosomes and boiled synaptosomal membranes

Cytidine CMP CDP CTP Total

Intact synaptosomes 15 s 117 i 30 641 2 50 1,520 i 5 1 121 -I- 19 2,400 10 min 264 -t 54 1,107 i 32 936 i 27 89 i 30 2,400

15 s 136 i 20 60 i 28 231 i 106 1,739 2 62 2,166 10 min 127 i 19 57 i 33 193 i 96 1,687 i 69 2,064

No synaptosomes 104 5 26 20 i 9 49 ? 7 1,865 i IS8 2,038

Synaptosomes or boiled synaptosomal membranes were incubated at 37°C for 10 min in a physiologic Krebs buffer containing 10 k.M [S-3H]CTP (0.2 PCiipmol) and 0.5 mM MnCI,. The incubations were stopped by placing the test tubes in an ice bath. For IS-s determinations, the warmed suspensions were immediately placed in an ice bath after mixing. Following centrifugation, the supernatants were immediately placed in a ~ 70°C freezer until HPLC analysis. During each chromatographic analysis, the cytidine, CMP, CDP, and CTP peaks were collected, and radioactivity was counted by scintillation spectrometry for 3H content. In three experiments, the stock [5-’H]CTP was incubated in water at 37°C for 10 min and then analyzed for breakdown, or it was injected directly onto the chromatograph and analyzed (no synaptosomes). Data are expressed as dpm where each mean 2 SEM value is the result of four determinations in two experiments, except for the three experiments without synaptosomes.

Boiled synaptosomal membranes

present as contaminants. The preparation is metabolically active and capable of energy-dependent active uptake of various substrates (Yandrasitz et al., 1979). To determine whether the small amount of contaminating membranes is responsible for the exchange activity, we incubated synaptosomes with 0.5 mM MnCI2, 10 p M CMP, and myo-[2-3H]inositol for 10 min and then subjected the elements in the synaptosomal incubation to a second five-step Fi- coll gradient centrifugation. Although the bulk of material was present in the synaptosomal layers, there were also myelin fragments, vesicles, etc., in the upper layers. After each band was exposed to hypotonic lysis and tritium counts were determined in the resullant membranes, 90% of the label was found in the synaptosomal bands. These studies suggest that the bulk of Mn2+-dependent myo- [2-3H]inositol incorporation was into synaptosomal PI and is due to the transferase enzyme acting as an ectoenzyme.

DISCUSSION

The dependence of P1:myo-inositol exchange activity on Mn2+ and myo-inositol concentrations in

TABLE 3. Ejfect of Mn” on uptake ojrny0-[2-~H]- inositol by synaptosomes

Control ( I K M Mn”) 0.5 mM Mn’+ Increase (‘36)

Soluble 540 t 2 0 ( l 0 ) 570 t 30 (10) 6% Membranes 90 t 10 (4) 450 i 60 (4) 403%

Total 610 i 40 (4) 940 i 120 (4) 55%

The uptake of inyo-[2-’H]inositol by synaptosomes, performed as de- xribed in Material5 and Methods, is expressed as pmol of myo-inositoli mg of proreid40 inin for the aoluble extract, the pellet containing membranes, and wholc synaptosomes. Data are mean ? SEM values (no. of determinations).

the intact synaptosomes is very similar to that reported for other tissues, when slices or homoge- nates were used for study. Our data are consis- tent with concentrations of 1-2 mM Mn2+ being required for maximal exchange activity in other tissues (Thompson et al., 1963; Takenawa et al., 1977; Bleasdale and Wallis, 1981). For partially purified enzyme from rat liver microsomes, Tak- enawa and Egawa (1980) reported maximal exchange activity at 0.5 mM Mn2+. The apparent Km for myo-inositol of 38 p M is in the same range (10- 40 pkl) as that previously reported in other tissues (Holub, 1974; Takenawa and Egawa, 1980; Bleas- dale and Wallis, 1981; Gibson and Brammer, 1981). In contrast, when synthesis of PI was measured as CDP-DG-dependent myo-[2-3H]inositol incorporation into PI, a higher apparent Km for myo-inositol (0.1-2.5 mM) was noted (Agranoff et a] . , 1958; Paulus and Kennedy, 1960; Takenawa et al., 1977; Wootton and Kinsella, 1977; Gibson and Brammer, 1981). In our experiments, there is a marked dependence of synaptosomal P1:myo-inositol exchange activity on CMP concentration, and the apparent K , of 2 F M points out the strong affinity of the transferase enzyme for CMP. Bleasdale and Wallis (1981) also showed exchange activity to be CMP dependent in rabbit lung microsomes, with an apparent Km of 0.4 mM for CMP in the presence of 1.4 mM myo-inositol and 3.8 mM MnCl,. In their system, however, no exchange activity was de- tected when Mn2+ alone was used as the stimulant.

A nucleotide-independent P1:myo-inositol exchange enzyme has been reported in rat liver; this exchange activity, however, has never been isolated free of transferase activity (Takenawa and Egawa, 1980). In addition, Takenawa et al. (1977) did not report on the ability of CMP to stimulate their par-

PI:myo-INOSITOL EXCHANGE ACTIVITY IN S YNAPTOSOMES 1079

tially purified nucleotide-independent exchange enzyme. In other studies, CMP stimulated exchange activity whenever it was tested (Thompson et al., 1963; Hokin-Neaverson et al., 1977; Bleasdale and Wallis, 1981) except when the enzyme activity was already maximal (Paulus and Kennedy, 1960). In this preparation, addition of Mn2+ without nucleotide markedly stimulated myo-[2-’Hlinositol incorporation into PI (Yandrasitz and Segal, 1979). How- ever, the current studies (Fig. 3) show that this Mn2+ -stimulated incorporation is only a small fraction of that obtained by adding CMP in micromolar concentrations. Because the synaptosomal preparation causes breakdown of CMP to cytidine, the apparent K , of CMP for PImyo-inositol exchange activity may actually be in the submicromolar range. Nanomolar concentrations of CMP, which might be available from endogenous sources (Lolley et al., 1961), could support exchange. Al- though it remains to be determined whether P1:myo-inositol exchange activity is always due to the action of CDP-dig1yceride:inositol phosphatidyl transferase, it is incumbent on investigators de- scribing nucleotide-independent exchange to show that the preparation is actually free of CDP-DG or CMP.

Only cytidine nucleotides were capable of stimulating the P1:myo-inositol exchange activity. We have previously shown that this effect is not due to the nucleoside cytidine, because even at a concentration of 1 mM, i t will not substitute for CMP (Berry et al., 1983). Because the symptosomal preparation readily converts CTP to CMP, the apparent stimulatory effect of CTP and CDP may be due to the generation of CMP. Similarly, breakdown of CTP and CDP to CMP has been reported in the rabbit lung microsomal preparation of Bleasdale and Wallis (1981), which also shows CMP-dependent exchange activity.

Several lines of evidence point to the fact that the P1:myo-inositol exchange activity in synaptosomes is directly accessible to extrasynaptosomal substrate, and we have previously suggested that this activity may reside on the plasma membrane (Berry et al., 1983). Measurement of the uptake of free myo-inositol by synaptosomes rules out the possibility that Mn2+ acts by greatly increasing the specific activity of the synaptosomal cytosolic myo- inositol pool. Essentially all the increased labeling of synaptosomes was found in membranes. Because the synaptosomal pools of free and lipid-bound myo-inositol are comparable (Warfield and Segal, 1978), if intrasynaptosomal myo-inositol were used to supply the large increase in lipid label, one would expect a corresponding decrease in the specific activity of the former. This was not found; in fact, the specific activity of intrasynaptosomal free myo-inositol was increased somewhat by Mn2i treatment. Given the fact that Mn2+ did not affect uptake of

tritium label into the cytosolic fraction, this result was unexpected. One possibility is that the increased specific activity in synaptosomal free myo- inositol is balanced by a decrease in the specific activity of inositol phosphates (not measured) suf- ficient to result in no net change in the label of the total water-soluble inositol pool as measured by the uptake experiments. It may be significant that Mn2+ is a potent inhibitor of brain myo-inositol-l- phosphatase (EC 3.1.3.25; Hallcher and Sherman, 1980), which converts myo-inositol-1-phosphate to free myo-inositol.

We previously reported that hypotonic lysis of synaptosomes to expose internal membranes did not increase CMP and Mn2+-dependent exchange activity of the preparation and, in fact, produced a relative decrease in the specific activity of exchange (Berry et al., 1983). Cytidine, which should enter synaptosomes by nucleoside transport (Bender et al., 1981), also did not significantly enhance exchange activity. Therefore, CMP, which should be excluded from synaptosomes, must be an extrasynaptosomal enzyme substrate. Although the data clearly show that it is the myo-inositol and CMP in the incubation medium that is directly interacting with transferase enzyme, it remains to be estab- lished absolutely whether the exchange activity actually resides in synaptosomal plasma membranes rather than in contaminating membranes. The finding that almost all of the labeled myo-inositol in membranes was in the synaptosomal bands, when the synaptosomal preparation incubated with Mn2+, CMP, and myo-[2-’H]inositol was subjected to a second centrifugation on a Ficoll gradient, sup- ports the former location. Our data suggest that for the bulk of measured activity, it is PI in the synaptosomal plasma membrane that undergoes exchange with extrasynaptosomal mTyo-inositol.

Of interest are the studies of Tolbert et al. (1980) and Prpic et al. (1982) on the effect of Mn2+ on hepatocyte uptake of myo-inositol and incorporation into PI. Hepatocytes, like synaptosomes, do not possess an energy- and Nai-dependent transport system for active uptake of myo-inositol (Chen and Vu, 1979; Prpic et al., 1982; Warfield et al., 1978). The findings of Tolbert et al. (1980) were comparable to ours in that Mn2+ markedly stimulated myo-inositol incorporation into PI and that the enhanced uptake of myo-[2-3H]inositol was largely confined to the hepatocyte membranes. In contrast, Prpic et al. (1982) showed much less of a stimulating effect of Mn2+ on myo-inositol incorporation into PI, a smaller uptake of myo-[2-3H]inositol into hepatocyte membranes, and a decreased uptake of myo-[2-’H]inositol in the soluble fraction. The latter findings are just what one would expect if Mn2+ had no overall effect on hepatocyte uptake of myo- inositol and simply enhanced internal P1:myo-inositol exchange activity, causing a release of lipid-

J . Nrirrochern., Vol. 46, N o . 4 . 1986

G. UERR Y ET A L .

bound unlabclcd rrtyo-inositol. Thesc vcry dift'ercnt and contrasting results cannot he easily cxplained and may be due to differcnces in the hepatocyte preparation s t hem se I ve s .

A large fraction of basal Inyo-inositol incorporation into P1 in synaptosomes and perhaps in other tissues may be due to exchange activity, because endogenous levels of CMP might support exchange even in the absence of added nucleotide given the marked affinity of the transferase enzyme for CMP. Failure to take this contribution of exchange into account will lead to erroneous conclusions re- garding PI turnover in studies where 1nyo- [2 -~H]- inositol is used to probe PI metabolism.

The physiologic significance of this P1:myo-inositol exchange system is unknown. Because it is external myo-inositol that is being utilized and trapped in membrane phospholipid, this system could function in a limited transport capacity if exchange were vectorial in nature.

Acknowledgment: This work was supported by grants HD08536 and HD00427 from the National Insti tutes of Health (Bethesda. MD, U.S.A.). We would like t o thank Karen Gunn, Joan Kubinski, and Ann Marie Wolfe for assistance in preparation of this manuscript .

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phosphatidylinositol:myo-inositol exchange activity in intact nerve endings: substrate and cofactor...

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