Metal Ion Stimulation of Phospholipase D-Like Activity of Isolated Rat Intestinal Mitochondria M

Metal Ion Stimulation of Phospholipase D-Like Activity of Isolated Rat Intestinal Mitochondria M. Madesh and K.A. Balasubramanian* The Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College & Hospital, Vellore 632 004, India

ABSTRACT: Presence of phospholipase D-like (PLD) activity Under certain pathological conditions, such as oxidative in the intestinal mitochondria was identiﬁed using endogenous stress, increase in cytosolic Ca2+ occurs and this in turn in- phospholipids as substrate. The enzyme had a pH optimum of creases the Ca2+ sequestration by mitochondria (13,14). It has 6.5, did not show trans-phosphatidylation activity in the pres- been proposed that PA may act as a Ca2+ ionophore (15,16) ence of ethanol or butanol, and the product formed was phos- and facilitate the influx of Ca2+ through the plasma mem- phatidic acid (PA). This was confirmed by separation of reac- brane. Although presence of PLD activity has been shown in tion products by high-performance liquid chromatography and liver, skeletal muscle and brain mitochondria, very little is analysis of composition of the PA formed which gave phosphate/fatty acid ratio of 1:2. PLD-like activity was further con- known on the properties of mitochondrial enzyme (17–19). firmed by the formation of ethanolamine and choline as prod- Since mitochondria are important in calcium homeostasis and ucts of enzyme action. This activity was stimulated by various PA might play a role in this process, the present study looks metal ions; when stimulated by Mg2+ and Ba2+, it hydrolyzed at the possible presence of PLD-like activity and its proper- both phosphatidylcholine and phosphatidylethanolamine, and ties in the intestinal mitochondria. when stimulated by Ca2+, it preferentially hydrolyzed phosphatidylethanolamine. There was no requirement for sodium oleate for the PLD-like activity in mitochondria. These results MATERIALS AND METHODS suggest that intestinal mitochondria have an active PLD-like en- Various lipid standards, Hepes, fluorescamine, p-bro- zyme which differs in certain properties from phospholipase D mophenacylbromide (pBPB), chlorpromazine (CHLP), and from other tissues. bovine serum albumin (BSA) were all obtained from Sigma Lipids 32, 471–479 (1997). Chemical Company (St. Louis, MO). Standard phosphatidylethanol and phosphatidylbutanol were prepared using A number of studies have shown that phosphatidic acid (PA) egg phosphatidylcholine and cabbage PLD in presence of is involved in cell signaling (1,2). PA can either be formed by ethanol or butanol (20). All other chemicals used were of an- the action of phospholipase D (PLD) on phospholipids or by alytical grade. phosphorylation of diacylglycerol. PLD can be activated in Preparation of mitochondria. Rats weighing 150–200 g, response to various stimuli and phorbol esters activate PLD fasted overnight, were decapitated, the small intestine re- through protein kinase C (3,4). Protein kinase C activation of moved and washed with ice-cold saline. Mitochondria were PLD takes place by both phosphorylation-dependent and in- isolated from enterocytes as described by Masola and Evered dependent mechanisms (5,6). Lipopolysaccharide stimulates (21). Isolated mitochondrial fraction was suspended in cellular PLD activity, and this requires an increase in intra- EGTA-free medium containing 250 mM sucrose/5 mM Hepes cellular Ca2+ (7). Exposure to oxygen-derived free radicals pH 7.4 and stored in ice at protein concentration of 8–10 also stimulates cellular PLD activity (8,9). Using PLD from mg/mL. Isolated mitochondria were relatively pure as judged various sources, it has been shown that divalent metal ions by the mitochondrial marker enzyme succinic dehydrogenase. can activate this enzyme (10,11). Microsomal and lysosomal contamination in the mitochon- Calcium plays an important role in many cellular func- drial preparation were checked by assaying marker enzymes, tions, and intracellular calcium is stored mainly in endoplas- namely, glucose-6-phosphatase, acid phosphatase and aryl- mic reticulum and to a small extent in the mitochondria (12). sulfatase, and these activities were not detectable. Protein was measured using BSA as standard (22). Enzyme assay. Mitochondrial PLD-like activity was mea- *To whom correspondence should be addressed. sured using endogenous mitochondrial phospholipids as sub- Abbreviations: PA, phosphatidic acid; pBPB, p-bromophenacylbromide; strate. Mitochondria (1 mg protein per mL in 0.25 M sucrose, PLD, phospholipase D; DAG, diacylglycerol; CHLP, chlorpromazine; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatogra- 5 mM Hepes buffer pH 7.4) were incubated at 37°C for 30 phy; PE, phosphatidylethanolamine; PC, phosphatidylcholine. min. Following incubation, total lipids were extracted by

Bligh and Dyer’s method (23) and the PA was separated by by vol). This was identified after exposure to iodine vapor thin-layer chromatography (TLC) and quantitated by phos- (29). phate estimation. Extracted lipids were spotted on silica gel Lipid analysis. Neutral lipids were separated on silica gel G plates impregnated with 0.5 M oxalic acid and separated G plates using the solvent system hexane/diethyl ether/acetic using the solvent system chloroform/methanol/conc. HCl acid (80:20:1, by vol). Spots were identified by iodine expo- (85:13:0.5, by vol) (24). PA spots corresponding to standard sure, scraped, and eluted. Cholesterol (30), diglycerides, and were identiﬁed by iodine exposure, scraped, and eluted from triglycerides (31) were estimated as described. Free fatty the plates. PA was quantitated by phosphate estimation after acids were methylated and quantitated by gas chromatogra- acid digestion (25). PA formed was also separated and iden- phy after separation on a 5% EGSS-X column. Heptade- tified along with standard using two other solvent systems canoic acid was used as internal standard. Individual phos- (26,27). Formation of PA was further confirmed by separa- pholipids were separated on silica gel H plates using the sol- tion of extracted lipids on TLC followed by isolated PA spot vent system chloro-form/methanol/acetic acid/water separated by high-performance liquid chromatography (25:15:4:2, by vol) (32) and quantitated by phosphate esti- (HPLC) (28). A normal-phase silica 5-µm column was used mation after acid hydrolysis. Individual aminophospholipids with the solvent gradient of 1–9% water in hexane/2- were also quantitated after derivatization with fluorescamine propanol (3:4, vol/vol) at a flow rate of 1 mL/min and the and separation on silica gel H plates impregnated with eluents monitored at 220 nm. Transphosphatidylation was 3% magnesium acetate using the solvent system chloro- checked by inclusion of 300 mM ethanol or butanol in the form/methanol/NH4OH/water (60:40:5:2, by vol) (33). incubation mixture, and extracted lipids were separated on Eluted individual spots were quantitated using Shimadzu SF silica gel G plates using the upper phase of ethyl ace- 5000 spectrofluorometer (Tokyo, Japan) with excitation at tate/isooctane/acetic acid/water (13:2:3:10, by vol) (27). PA 395 nm and emission at 468 nm. formation in the presence of ethanol was further checked by Separation and analysis of ethanolamine. Following incu- HPLC. Metal ion stimulation of phosphatidylcholine degra- bation, enzyme activity was terminated with 10% trichloro- dation generated choline which was separated by TLC, using acetic acid. After cooling on ice, the mixture was transferred the solvent system methanol/0.6% NaCl/ammonia (50:50:5,

FIG. 2. Thin-layer chromatographic separation of phosphatidic acid (PA) and phosphatidylethanol. Silica gel G plate was developed using the upper phase of the solvent system ethylacetate/isooctane/acetic FIG. 1. Effect of various metal ions (at 0.1 and 1 mM final concentra- acid/water (13:2:3:10, by vol). Lane 1 contains PA and phos- tion) on phospholipase D activity of intestinal mitochondria. Each value phatidylethanol standard. Lane 2 represents Ca2+-activated mitochon- represents mean ± SEM of three separate estimations. *P < 0.05 versus drial PA formation in the absence of ethanol. Lane 3 represents PA for- control (CON) incubated mitochondria. mation by Ca2+ activation in presence of 200 mM ethanol.

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FIG. 3. High-performance liquid chromatography separation of phosphatidic acid formed in mitochondria; (A) standard phosphatidic acid and phosphatidylethanol; (B) unincubated mitochondria; (C) mitochondria incubated with 1 mM Ca2+; (D) mitochondria incubated with 1 mM Ca2+ + 200 mM ethanol. Total lipids were extracted. Phosphatidic acid separated by thin- layer chromatography was eluted and run on high-performance liquid chromatography as described in the text.

Lipids, Vol. 32, no. 5 (1997) 474 M. MADESH AND K.A. BALASUBRAMANIAN

TABLE 1 Composition of Isolated Phosphatidic Acida 2+ Co C30 +Ca Phosphate (nmole) 10.50 20.98 60.70 Fatty acids (nmole) 16:0 10.00 18.75 57.45 18:0 and 18:1 5.70 10.10 28.45 18:2 6.26 8.45 15.20 20:4 3.10 5.40 19.00 Total fatty acid 25.56 42.70 120.05 Ratio of phosphate to fatty acid 1:2.24 1:2.03 1:1.97 a Co: Control unincubated mitochondria; C30: mitochondria incubated for 30 min without any acti- vator. For calcium activation, mitochondria were incubated with 1 mM Ca2+ for 30 min.

to a test tube, shaken on a vortex mixer for a minute, left for presence of Mg2+ + ethanol and 53.9 ± 1.9 in presence of Mg2+ 15 min and then centrifuged. The sediment was washed twice alone). Figure 2 shows the TLC separation of PA and phos- with 2 mL of 3% trichloroacetic acid and the supernatants phatidylethanol after Ca2+ activation of mitochondrial PLD- pooled together. Trichloroacetic acid from the supernatant like activity which showed the absence of phosphatidylethanol was removed by repeated extraction with diethyl ether, and formation in the presence of ethanol. This was further con- the solution was dried with nitrogen. The samples were dis- firmed by HPLC separation of PA formed in the absence and solved in 150 µL of 0.2 M boric acid pH 9.0, and 50 µL of presence of ethanol. Control mitochondria had very little PA ﬂuorescamine (2 mg in 0.25 mL acetone) was added. The re- which was increased when incubated with Ca2+. In the pres- action with ﬂuorescamine was allowed to proceed for at least ence of ethanol and Ca2+, all the product was recovered as PA, 5 min to hydrolyze the excess reagent. After derivatization, as evidenced by HPLC separation (Fig. 3). Table 1 shows the the samples were separated by TLC on silica gel G plates phosphate content and fatty acid composition of PA isolated (solvent system, chloroform/methanol/conc. ammonia/water (60:40:5:2.5, by vol). Fluorescamine-labeled ethanolamine was seen under ultraviolet light. This was scraped and eluted with 3.5 mL of methanol/4% ammonia (95:5, vol/vol) and quantitated using a spectrofluorometer with an excitation at 395 nm and emission at 468 nm. Standard ethanolamine was also derivatized with fluorescamine, and the fluorescence response of standard derivatized ethanolamine was linear in the concentration range of 25 to 1000 µM. Statistical analysis. The values represent mean ± SEM of three separate estimations, and Mann-Whitney U-test was done to compare the changes.

RESULTS The PLD-like activity in the isolated intestinal mitochondria was determined by measuring the PA formation from endogenous mitochondrial phospholipids. Figure 1 shows the effect of various divalent metal ions (Ca2+, Mg2+, Ba2+, Co2+, Mn2+, and Zn2+) at two different concentrations (0.1 and 1 mM) on PA formation. As shown, Ca2+, Mg2+ and Co2+ activated at both concentrations, whereas with Ba2+, Mn2+ and Zn2+, maximum activation was seen at 0.1 mM concentration. Mitochondrial PLD-like enzyme did not show transphosphatidylation activity. Inclusion of either 200 and 300 mM ethanol or butanol in the incubation medium did not result in the formation of phosphatidylethanol or phosphatidylbutanol when stimulated by metal ions. Under these conditions, all the product formed was 2+ FIG. 4. Effect of different pH on phospholipase D activity of intestinal recovered as PA (60.7 ± 1.5 nmoles PA in presence of Ca + mitochondria in the presence and absence of 1 mM Mg2+. Each value 2+ ethanol, 61.8 ± 2.4 in presence of Ca alone, 55.4 ± 1.7 in represents mean ± SEM of three separate estimations.

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TABLE 2 Neutral Lipid Composition (nmole/mg protein) of Intestinal Mitochondria Incubated with Metal Ionsa Control Incubated in presence of Lipids Control incubated Ca2+ Mg2+ Ba2+ Total cholesterol 139 ± 11.2 142 ± 9.9 133 ± 7.4 136 ± 12.4 141 ± 2.5 Triglyceride 94 ± 6.6 86 ± 5.3 93 ± 5.3 94 ± 2.3 88 ± 4.1 Diglyceride 59 ± 4.5 59 ± 3.6 56 ± 4.7 59 ± 6.2 58 ± 5.1 Free fatty acids 101 ± 12.0 116 ± 9.7 114 ± 10.5 112 ± 14.9 113 ± 2.8 aEach value represents mean + SEM of three separate estimations. Ca2+ (1 mM), Mg2+ (1 mM), and Ba2+ (0.1 mM) (final concentration) were used.

from the reaction mixture, and the ratio of phosphate to fatty Ca2+ + DAG: 61 ± 2.6; Mg2+ alone: 54 ± 1.9; and Mg2+ + acid was found to be 1:2, which confirms that the product DAG: 55 ± 2.3 nmoles/mg protein). Levels of DAG also re- formed was indeed PA. To rule out the possibility of PA forma- mained the same in control and metal ion-treated samples (data tion from diacylglycerol (DAG) by DAG kinase, isolated mi- not shown). Mitochondrial PLD-like enzyme showed a pH op- tochondria were incubated with 400 µM DAG (exogenous) and timum of 6.5, and this remained the same even when activated 1 mM ATP at 37°C for 30 min with and without metal ions. by metal ion (Fig. 4). The possible influence of endogenous Following this, lipids were extracted; DAG and PA were quan- metal ions on mitochondrial PLD activity was checked in the titated. The addition of exogenous DAG did not alter the PA absence and presence of 2, 5, and 10 mM EDTA. Unincubated formation in the presence of metal ions (Ca2+ alone: 59.8 ± 2; mitochondria had a PA content of 12.58 ± 1.36 nmoles/mg protein, which increased to 23.06 ± 3.16 nmoles after incubation for 30 min without any metal ions. Inclusion of various concentrations of EDTA did not alter the PA content (data not shown). PLD is known to act on both phosphatidylcholine (PC) and phosphatidylethanolamine (PE). To check the endogenous substrate for mitochondrial enzyme, mitochondrial lipids were analyzed after metal ion activation of PLD-like activity. As can be seen from Table 2, no significant change was seen in neutral lipids. Phospholipid separation by TLC indicated that in the case of Ca2+ activation, PLD-like enzyme preferentially used PE as substrate, whereas with Mg2+ and Ba2+, both PC and PE were hydrolyzed. Figure 5(A and B) shows PC and lyso PC content of mitochondria after metal ion activation of the enzyme. As can be seen, with both Mg2+ and Ba2+ stimulation, PC content decreased whereas Ca2+ stimulation did not alter the PC content. Moreover there was no significant change in the content of lyso PC after metal-ion activation. PC hydrolysis by PLD- like enzyme in the presence of Mg2+ and Ba2+ was further confirmed by the formation of choline which was separated and identified by TLC and is shown in Figure 6. Ca2+ stimulation of the enzyme did not produce choline, whereas Mg2+ and Ba2+ stimulation resulted in the formation of choline; and the maximum amount of choline formation oc- curred with Ba2+ stimulation of enzyme. Amino phospholipids were quantitated by fluorescamine derivatization followed by TLC separation. As shown in Figure 7(A and B), Ca2+, Mg2+, or Ba2+ stimulation decreased the PE content, and there was no significant change in the level of lyso PE following metal ion activation. Ethanolamine, the other FIG. 5. Phosphatidylcholine (PC) and lyso PC (LPC) content of mito- product of PLD degradation of PE, was separated by TLC chondria incubated with Ca2+ (1 mM), Mg2+ (1 mM), and Ba2+ (0.1 after fluorescamine derivatization and is shown in Figure 8. mM). C represents unincubated mitochondria, and C represents mi- o 30 Quantitation of ethanolamine formed showed that all three tochondria incubated for 30 min without metal ions.

Lipids, Vol. 32, no. 5 (1997) 476 M. MADESH AND K.A. BALASUBRAMANIAN

FIG. 6. Choline formation in response to Ca2+, Mg2+, and Ba2+ by mitochondrial phospholipase D. The methodology is described in the text. 1. Standard choline; 2. control unincubated mitochondria; 3. control mitochondria incubated for 30 min; 4. incubated with Ca2+; 5. incubated with Mg2+; 6. incubated with Ba2+.

metal ions stimulated the formation, and Ca2+ stimulation tected. Moreover the PA content remained the same whether of the enzyme resulted in the maximum formation of ethanol was present or not. PA formation by mitochondrial ethanolamine (Fig. 9). PLD-like activity was also checked PLD was conﬁrmed by HPLC which gave an identical reten- in presence of phospholipase A2 inhibitors, namely, CHLP tion time to that of the authentic standard. Furthermore, and pBPB. CHLP and pBPB as such stimulated PLD activ- analysis of the PA formed showed a phosphate to fatty acid ity and this activity was further enhanced by the presence of ratio of 1:2 which indicated that PA is the reaction product. Ca2+ or Mg2+ (data not shown). That PLD-like enzyme indeed was responsible for PA formation was further confirmed by identifying the other products of reaction, namely, choline and ethanolamine. It may be that DISCUSSION either the intestinal mitochondrial PLD-like enzyme does not This study has demonstrated the presence of an active PLD- catalyze transphosphatidylation or this enzyme does not have like activity in the intestinal mitochondria and, using endoge- the transphosphatidylation activity, which was shown to be nous lipids as substrate, some of the properties of this enzyme different from PLD activity in rat brain membranes (34). were studied. Although PLD activity has been shown in Metal ions have been shown to activate or inhibit PLD ac- whole cells and subcellular membranes (1,2), very little is tivity depending on the source of the enzyme (8,35–37). In- known on mitochondrial PLD activity (18,19). PLD activity testinal mitochondrial PLD-like enzyme was activated by var- is usually assayed by PA formation or by measurement of ious metal ions, and it was concentration-dependent. In- phosphatidylethanol, which is the transphosphatidylation creased PA formation in mitochondria in the presence of product of PA in the presence of alcohol. In the case of in- metal ions was not due to DAG kinase activity since incuba- testinal mitochondrial PLD, no phosphatidylethanol formation of mitochondria in presence of DAG and ATP did not in- tion could be detected using endogenous substrate, as con- crease the PA formation. Moreover, all the DAG added was firmed by both TLC and HPLC separation. This was further recovered. Earlier, Rider and Baquet (38) reported that DAG confirmed using mitochondrial enzyme and egg PC as sub- exogenously added to liver microsomes caused 8–10-fold strate (data not shown). It was found that even in the presence stimulation of inorganic phosphate incorporation in the PA. of metal ions, phosphatidylethanol formation could not be de- Kanoh and Akesson (39) also observed a similar stimulation

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of the DAG kinase reaction by exogenous DAG. Since mitochondrial PA formation was not influenced by exogenously added DAG, this suggests that PA formation in mitochondria in the presence of metal ions is due to PLD-like activity rather than DAG kinase activity. It has been suggested that PA formed by the action of PLD may be hydrolyzed by the phosphatidate phosphatase to DAG, and this may make the quantitation of the PLD product, PA, difficult. In the case of intestinal mitochondria, it appears that the PA formed is stable, and this may be due to the absence of PA phosphatase activity. This was further supported by our observation that metal ion activation did not increase the level of DAG in mitochondria. Maximal PLD-like activity was obtained at pH 6.5, and activation by metal ions did not alter the pH optimum. This is similar to an earlier re- port on brain plasma membrane PLD activity (10), but differs from a neutral PLD from rat brain synaptosomal membranes (11). Analysis of mitochondrial lipids suggested that both PC and PE were used as substrate when activated by Mg2+ or Ba2+, whereas PE was preferentially used when activated by Ca2+. There are reports on PE preferring PLD activity (1). The basal

FIG. 7. Phosphatidylethanolamine (PE) and lyso PE content of intestinal mitochondria after incubation with Ca2+ (1 mM), Mg2+ (1 mM), and Ba2+ (0.1 mM). PE and lyso PE were derivatized with ﬂuorescamine and separated by thin-layer chromatography and quantitated by spectroﬂu- orimetry as described in the text. Each value represents mean ± SEM of

three separate estimations. Co represents unincubated mitochondria and C30 represents mitochondria incubated for 30 min without metal ions. *P < 0.05 for metal ions treated versus control mitochondria (incubated).

FIG. 8. Ethanolamine formation in response to Ca2+, Mg2+, and Ba2+ by mitochondrial phospholipase D. Ethanolamine isolation, fluorescamine derivatization, and separation are described in the text. Lane 1 represents mixture of standard amino acids, glutathione, spermine, and ethanolamine. Lane 2 represents only ethanolamine. Lane 3, unincubated control. Lane 4, control incubated for 30 min. Lane 5, mitochondria incubated with Ca2+. Lane 6, mitochondria incubated with Mg2+. Lane 7, mitochondria incubated with Ba2+.

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