Triacylglycerol lipase mediated release of arachidonic acid for prostaglandin synthesis in rabbit kidney medulla microsomes

F’rostaglandins Leukotrienes and Essential 0 Longman Group UK Ltd 1991

Fatty

Acids

(1991) 42. 251-256

Triacylglycerol Lipase Mediated Release of Arachidonic Acid for Prostaglandin Synthesis in Rabbit Kidney Medulla Microsomes Y. Fujimoto,

S. Shimada, T. Fujikawa, S. Sakuma and T. Fujita

Department of Hygienic Chemistry, Osaka University of Pharmaceutical Sciences, Matsubara, Osaka 580, Japan (Reprint requests to YF) ABSTRACT.

The effect of triarachidonin on the synthesis of prostaglandins in rabbit kidney medulla microsomes was examined. Medulla microsomes were incubated with triarachidonin in 0.1 M - Tris/HCl buffer (pH 7.0) containing reduced glutathione and hydroquinone and the formed prostaglandin EZ, prostaglandht F2@and prostaglandin D2 were measured by high-pressure liquid chromatography using 9-anthryldiiethane for derivatization. The addition of triarachidonin (l-10 PM) stimulated prostaglandht formation in a dose-dependent manner. Under our incubation conditions rabbit kidney medulla was found to produce prostaglandin E2 mainly. When arachidonic acid, instead of triarachidonin, was added to the incubation mixture of microsomes, the identical profile of prostaglandin products was obtained. When the pH of the reaction mixture was changed from 7.0 to 8.0, the rate of triarachidonin-induced prostaglandin Ez formation was approximately 60% of that observed at pH 7.0. Studies utilizing Ca2+ and EGTA revealed that triacylglycerol lipase of kidney medulla is independent of Ca2’. The addition of epinephrine made the stimulatory effect of triarachidonin on prostaglandin Ez formation more pronounced. These results suggest that epinephrine-activated triacylglycerol iipase is present in the renomedullary microsomes, and this enzyme activity is a potential mediator of release of arachidonic acid for prostaglandin synthesis in the kidney medulla.

triacylglycerol lipase and triacylglycerol as a possible candidate for providing free arachidonic acid to cyclooxygenase in kidney medulla (12); however, this study is still limited to using tissue slices. It has been reported that renomedullary triacylglycerol lipase activity is predominantly distributed in the particulate subcellular fractions of rabbit renal medulla (13). In addition, Coleman and Haynes (14) have shown that microsomal triacylglycerol liactivity pase may function to hydrolyze endogenously synthesized triacylglycerol. Since the enzymes for the conversion of the released arachidonic acid into prostaglandins are also present in the microsomal fraction of rabbit kidney medulla (Pi), it appears that the enzymatic modification of triacylglycerols and the synthesis of prostaglandins from the liberated fatty acids can be regarded as taking place in the microsomal fraction. The present paper reports about the effect of triarachidonin on the biosynthesis of prostaglandins in rabbit kidney medulla microsomes. Our data are discussed in view of the possible role of triacylglycerol lipase in providing arachidonic acid to cyclooxygenase in kidney medulla.

INTRODUCTION Mammalian tissues contain no significant amounts of preformed prostaglandins or their free precursor arachidonic acid. Thus prostaglandin formation requires the hydrolysis of esterified arachidonic acid from tissue lipids. Arachidonic acid is found in cells and tissues esterified mainly to phospholipids. Numerous studies from our (1, 2) and other (3-7) laboratories have implicated phospholipase A2 and phospholipase C as mediators of the release of arachidonic acid for prostaglandin synthesis in kidney, platelets, and other tissues. In inner medulla, triacylglycerols have also been suspected as potential sources of arachidonic acid for prostaglandin synthesis because the interstitial cells in this region of the kidney contain abundant triacylglycerols present in the form of lipid droplets and enriched in arachidonic acid (8-l 1). Recently, we have reported the presence of

Date received 19 September 1990 Date accepted 20 November 1990

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Prostaglandins Leukotrienes and Essential Fatty Acids

MATERIALS AND METHODS Materials Arachidonic acid, triarachidonin, L-epinephrine, and prostaglandins E2, FZa and D2 were purchased from Sigma Chemical Co., St. Louis, MO, USA. GSH (reduced glutathione), hydroquinone and EGTA were obtained from Wako Pure Chemical Industries, Osaka, Japan. All other reagents were analytical grade. Preparation of microsomes Kidneys from rabbits (male, 2-2.5 kg) were removed as described elsewhere (l), and the medulla was homogenized in buffer (0.3 M sucrose 10 mM-Tri$I-ICl, pH 7.4; 5 ml/g of tissue). The microsomal fraction was prepared by the method of Erman and Raz (16). The medulla homogenate was centrifuged at 1500 g for 15 min in a Hitachi model 20PR refrigerated centrifuge. The supernatant was then centrifuged at 10 000 g for 15 min in the same centrifuge. To obtain a microsomal fraction, the supernatant was further centrifuged at 140 000 g for 60 min in a Beckman model TL-100 ultracentrifuge. The microsomal pellet was resuspended in 0.1 MTri@-ICl buffer, pH 7.0, to a protein concentration of 10 mg/ml. Incubation conditions and measurement of prostaglandin formation Medulla microsomes (0.5 mg of protein) were incubated with the indicated concentrations of triarachidonin or arachidonic acid in 1 ml 0.1 MTris/HCl buffer (pH 7.0) containing 160 PM-GSH Triarachidonin was and 45 PM-hydroquinone. firstly dissolved in dimethyl sulfoxide and then diluted 200-fold into the reaction mixture. Dimethyl sulfoxide at 0.5% (v/v) had no effect on prostaglandin production in medulla microsomes. Incubations were in air at 37°C for 20 min or 60 min with shaking and were terminated by the addition of 6 vol. of petroleum ether. The aqueous phase was then acidified (approx. pH 3) with 0.5 N HCl and extracted with 6 vol. of ethyl acetate. Prostaglandins in the extracted lipid wert: simultaneously determined by a high-pressure liquid chromatographic (h.p.1.c) method as described in our recent paper (17). This method produces excellent resolution of 6-keto prostaglandin F la, prostaglandin E2, prostaglandin Fza, prostaglandin 4, prostaglandin A2 and thromboxane B2 in the same sample. Briefly, prostaglandins were measured after esterification of prostaglandins with 9-anthryldiazomethane (ADAM) (18). Since ADAM contains many impurities which interfere with the h.p.1.c. determination, the purification of prostaglandins es-

terified with ADAM (PGs-ADAM) was attempted by use of a normal-phase silica cartridge (Sep-pak, Waters Associates). The cartridge was prepared by rinsing it with 10 ml of methanol followed by 20 ml of toluene/chloroform (1: 1, v/v). The sample was passed through the cartridge. The cartridge was washed with toluene/chloroform (1: 1, v/v, 10 ml) and the PGs-ADAM was then quantitatively eluted with acetonitrile/methanol (4:1, v/v, 10 ml). Peak heights were measured for the quantification of the PGs-ADAM relative to the standard derivatives prepared from authentic prostaglandins. By use of this method it was demonstrated that the major prostaglandins produced in our incubation of medulla microsomes were prostaglandin EZ, prostaglandin FZa and prostaglandin D2, while negligible amounts of 6-keto prostaglandin F1,, prostaglandin AZ or thromboxane B2 were found. Protein determination Protein was determined by the method of Lowry et al (19) using bovine serum albumin as standard.

RESULTS AND DISCUSSION Figure 1 illustrates the effects of various concentrations of triarachidonin on prostaglandin synthesis in rabbit kidney medulla microsomes. Incubations were performed in 0.1 M-Tris/HCl buffer supplemented with a mixture of compounds (GSH and hydroquinone) previously shown to stimulate overall acid conversion of arachidonic into prostaglandins by microsomes of rabbit medulla (15) and other tissues (20). Exogenous triarachidonin showed a dose-dependent stimulation of prostaglandin production at concentrations ranging from l-10 PM. At concentrations of lo25 PM the reaction rate was essentially unchanged. The stimulation was reflected in the synthesis of all three prostaglandins (prostaglandin E2, prostaglandin FZcuand prostaglandin 4). However the rate of formation of prostaglandin E2 was much greater than that for the other products. The time course of triarachidonin-induced increases in prostaglandin formation by rabbit kidney medulla microsomes is shown in Figure 2. The effect of triarachidonin (10 PM) was apparent within 10 min after addition to the incubation mixture and persisted for 60 min. The rate of formation of each of the prostaglandins was nearly linear during the first 20 min of incubation and then slowed down gradually with time. This time course closely resembles that of the isolated prostaglandin hydroperoxidase (21), where it is due to an irreversible self-deactivation of the enzyme during the synthesis of the endoperoxides (22).

Triacylglycerol lipase hydrolysis in rabbit kidney medulla

253

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1

5

I

I

10

25

10

20

40

60

Time (min)

Concentration CUM) Fig. 1

Effects of triarachidonin on prostaglandin synthesis by rabbit kidney medulla microsomes. Microsomes (0.5 mg of protein) were incubated for 20 min at 37°C in 1.0 ml of 0.1 M-Tris/HCl buffer (pH 7.0) containing 160 PM-GSH and 45 PM-hydroquinone in the presence of different concentrations of triarachidonin. Each point represents the mean for five experiments (S.E.M. values were less than 5%). a, prostaglandin Ez; A, prostaglandin FzU;n, prostaglandin D,

Fig. 2 Time course of triarachidonin-induced increases in prostaglandin formation by rabbit kidney medulla microsomes. Microsomes (0.5 mg of protein) were incubated for 60 min at 37°C in 1.0 ml of 0.1 M-Tris/HCI buffer (pH 7.0) containing 160 PM-GSH and 45 PM-hydroquinone in the absence (0, A, 0) and the presence of 10 PM-triarachidonin (a, A, n). Each point represents the mean for four experiments (S.E.M. values were less than 5%). o or 0, prostaglandin E,; A or A, prostaglandin F,,; 0 or H, prostaglandin D,.

Next, the effect of triarachidonin on prostaglandin synthesis by rabbit kidney medulla microsomes was compared to the effect of arachidonic acid under similar experimental conditions. In preliminary experiments, we found that prostaglandin formation is linearly proportional to the concentration of arachidonic acid in the range of l-10 PM, and arachidonic acid produced a 6-fold greater increase in prostaglandin formation than that seen in response to triarachidonin at the same concentration. When the concentration of arachidonic acid

was 1.5 PM, the formation of prostaglandins was increased to a level comparable with that obtained with 10 PM-triarachidonin. As shown in Table 1, microsomes incubated in the presence of arachidonic acid (1.5 PM) or triarachidonin (10 PM) produced virtually identical profiles of prostaglandin products. In both cases, the major prostaglandin produced in our incubation was prostaglandin Ez (64%), and lesser amounts of prostaglandin Ffa (2021%) and prostaglandin D2 (15-16%) were made. Thus it seems likely that the enhancement of pros-

Table 1 Comparison

of the effects of arachidonic acid and triarachidonin on prostaglandin synthesis by rabbit kidney medulla microsomes Substrate

E,

None Arachidonic acid (1.5 PM) Triarachidonin (10 FM)

32.9 + 1.6 181.0 Y!Y 8.9 181.6 + 7.2

Prostaglandins formed (ng) F Za Dz 20.5 f 1.0 60.3 ?I 2.9 57.3 + 2.8

13.9 + 0.7 42.4 + 1.6 44.3 + 2.1

Total 67.3 283.7 283.2

Microsomes (0.5 mg of protein) were incubated for 20 min at 37°C in 1.0 ml of 0.1 M-Tris/HCl buffer (pH 7.0) containing 160 PM-GSH and 45 PM-hydroquinone in the absence and the presence of exogenous 1.5 PM-arachidonic acid or 10 PM-triarachidonin. Values are means + S.E.M. (n = 5)

254

Prostagfandins Leukotrienes and Essential Fatty Acids Table 2 Effects of pH on triarachidonin-induced rabbit kidney medulla microsomes

prostaglandin

E, formation by

Treatment

None

Prostaglandin E, formed (ng) + 1.5 PM-arachidonic acid + 10 PM-triarachidonin

pH 7.0 pH 8.0

32.0 f 1.4 33.3 + 1.5

180.5 f 8.7 191.5 k 4.2

182.7 f 6.3 110.4 f 5.4

The conditions of the reaction were identical with those given in the legend of Table 1, except that the pH was varied as indicated. Values are means f S.E.M. (n = 5)

taglandin formation elicited by triarachidonin can be ascribed to increased availability of free arachidonic acid by triacylglycerol lipase, which in turn suggests the presence of triacylglycerol lipase in the medullary microsomes. Table 2 shows the influence of pH on triarachidonin-induced prostaglandin E2 formation by rabbit kidney medulla microsomes. Arachidonic acid (1.5 PM)-induced prostaglandin E2 formation kept the same level when the pH of the. reaction mixture was changed from 7.0 to 8.0. By contrast, at pH 8.0 the rate of triarachidonin (10 PM)induced prostaglandin E2 formation was approximately 60% of that observed at pH 7.0. These results indicate that microsomal triacylglycerol lipase activity is inhibited in the higher pH. The previous study demonstrated that renal medullary phospholipase A2 activity had a pH optimum of 9.0 (7). It can be conceived that, at variance with phospholipase A*, the pH optimum of microsomal triacylglycerol lipase is lower than 7.0. This speculation is, at least partially, supported by the observation by Liston and Nasjletti (13), who showed that triacylglycerol lipase activity in rabbit renomedullary crude homogenates was maximal at pH 5.6-6.0. Prostaglandin E2 formation could not be observed at pH 6.0 in our study, because the optimum ‘pH for prostaglandin synthetase in rabbit kidney medulla microsomes was found to be pH 7.5-8.5 and at pH 6.0 prostaglandin synthetase activity was only 25% that of maximal (15). Furthermore, we examined the effects of Ca*+ and EGTA on triarachidonin-induced prostaglandin

& formation by rabbit kidney medulla microsomes (Table 3). Ca*+ enhanced the production of basal prostaglandin E2 2.6-fold at a concentration of 1 mM. The stimulation by Ca*+ of basal prostaglandin E2 formation appears to be mediated via stimulation of a Ca*+-dependent phospholipase A2 (23-25) that cleaves arachidonic acid from phospholipids, primarily phosphatidylcholine. Ca*+ had no influence on arachidonic acid- or triarachidonininduced prostaglandin E2 formation. Moreover, prostaglandin E2 formation induced by arachidonic acid or triarachidonin was not inhibited by the addition of EGTA (1 mM). These results reveal that Ca*+ dependence does not seem to be a feature of triacylglycerol lipase activity. This contrasts with phospholipase A2 activity which requires Ca*+ for optimal activity. Triarachidonin in the presence of EGTA stimulated the formation of prostaglandin E2 at a high level as compared with triarachidonin alone (triarachidonin-induced prostaglandin E2 formation of 150.2 ng was enhanced by treatment with EGTA to 215.3 ng). We did not analyse this finding in detail, but the most feasible explanation is that removing available endogenous tissue Ca*+ by EGTA may result in new protein-lipid interactions. So, it seems possible that the breakdown of triacylglycerols by triacylglycerol lipase occurs rapidly in the presence of EGTA. Epinephrine was shown to stimulate the biosynthesis of prostaglandins in rabbit-kidney (26). We have reported that there is an epinephrine-activated lipase system in rabbit kidney cortex (27). In the same study, we have suggested that epinephrine is

Table 3 Effects of Ca’+ and EGTA on triarachidonin-induced kidney medulla microsomes Treatment None Ca2+ (1 mM) EGTA (1 mM)

None 32.4 f 1.5 84.3 + 1.5 27.2 + 1.3

prostaglandin E, formation by rabbit

Prostaglandin E, formed (ng) +lO /LM +1.5 /.LM Arachidonic acid Difference* Triarachidonin 179.5 + 8.9 231.8 f 11.2 175.2 + 8.5

147.1 147.5 148.0

182.6 + 9.0 233.2 + 10.8 242.5 + 12.0

Difference’ 150.2 148.9 215.3

The conditions of the reaction were identical with those given in the legend of Table 1, except that the addition of Ca*+ or EGTA was as indicated. Values are the means + S.E.M. (n = 5). Effects of Ca2+ and EGTA on arachidonic acid- or triarachidonin-induced prostaglandin E, formation were determined as the difference in the amounts of prostaglandin E, formed in the presence and absence of arachidonic acid (*) or triarachidonin (‘)

Triacylglycerol lipase hydrolysis in rabbit kidney medulla Table 4 Effects of epinephrine medulla microsomes Treatment None Epinephrine 10 /.LM 50 PM 100 PM

on triarachidonin-induced

prostaglandin E, formation by rabbit kidney

Prostaglandin E, formed (ng) +lO PM +lS PM Arachidonic acid Difference* Triarachidonin

Difference’

31.5 + 1.4

178.8 + 8.7

147.3

181.5 +

9.0

150.0

32.9 f 1.4 31.3 f 1.2 33.7 f 1.6

185.3 + 8.9 188.8 + 7.3 200.2 f 9.2

152.4 157.5 166.5

246.5 + 10.5 318.8 + 14.7 377.5 f 18.3

213.6 287.5 343.8

None

255

The conditions of the reaction were identical with those given in the legend of Table 1, except that the addition of epinephrine was as indicated. Values are the means + S.E.M. (n = 5). Effects of epinephrine on arachidonic acid- or triarachidonin-induced prostaglandin E, formation were determined as the difference in the amounts of orostaglandin E, formed in the presence and absence of arachidonic acid (*) or triarachidonin (‘)

connected closely with triacylglycerol breakdown. On the other hand, stimulation of prostaglandin synthesis by epinephrine may simply reflect its property as a cofactor for the cyclooxygenation of arachidonic acid, as shown by studies using microsomes prepared from kidney medulla (15). As shown in Table 4, prostaglandin Ez formation induced by arachidonic acid was slightly enhanced by epinephrine (10-100 PM) even in the presence of the activators GSH and hydroquinone. On the other hand, epinephrine was able to stimulate triarachidonin-induced prostaglandin E2 formation markedly. The effect of epinephrine was concentration-dependent. Our data were interpreted as indicating that epinephrine stimulated triacylglycerol lipase activity in the renomedullary microsomes and that the increased lipolysis and the resulting increased availability of free arachidonic acid was the main cause for the stimulation of triarachidonin-induced prostaglandin Ez formation by epinephrine. The results of this work suggest that epinephrineactivated triacylglycerol lipase is present in the renomedullary microsomes, and this enzyme activity is a potential mediator of release of arachidonic acid for prostaglandin synthesis in the kidney medulla. Also, a study of some properties of triacylglycerol lipase was performed. Under normal physiological conditions phospholipids seem to be a major source of substrate for prostaglandin biosynthesis. Direct deacylation of phospholipids by phospholipase AZ has been recognized as a favorable pathway, since this represents the simplest mode of release of arachidonic acid (24, 28). However, the difference in H optimum and the difference in response to Ca R between triacylglycerol lipase and phospholipase A2 may support an important role for medullary triacylglycerol lipase in mediating the release of arachidonic acid for prostaglandin synthesis in certain states. Glucocorticoids are widely used for the treatment of nephrotic syndrome. Although glucocorticoids in-

duce phospholipase inhibitory proteins, which bring about reduction of prostaglandin synthesis in renomedullary interstitial cells and other cell types (29-31), the urinary excretion of prostaglandin E2 and prostaglandin Fzu increases in rats receiving glucocorticoids (32, 33). An intriguing possibility is that in glucocorticoid-treated rats, the arachidonic acid for renal prostaglandin synthesis is supplied through a pathway that does not involve phospholipase. In this regard, the triacylglycerols of the renal medulla are rich in arachidonic acid and, conceivably, may be a source of unesterified arachidonic acid. Erman et al (34) have shown that chronic treatment with dexamethasone increases the level of unesterified arachidonic acid in the renal medulla of rats, associated with reduction of triacylglycerols. Thus it is possible that in kidney medulla the ‘triacylglycerol lipase pathway’ for synthesis of prostaglandins is insignificant under normal physiological conditions but that when ‘phospholipase pathway’ is blocked by glucocorticoids in certain pathological states, triacylglycerol lipase activity is an important mediator of release of arachidonic acid for prostaglandin synthesis. References 1. Fujimoto Y, Akamatsu N, Hattori A, Fujita T.

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