Phospholipase C activity in rat kidney Effect of deoxycholate on phosphatidylinositol turnover

65

Biochimica et Biophysics Acta, 7 12 (1982) 65-70 Elsevier Biomedical Press

BBA 51127

PHOSPHOLIPASE EFFECT NORMA Department

C ACTIVITY

OF DEOXYCHOLATE B. SPEZIALE

*, EMIR

of Pharmacology,

(Received May 21st, 1981) (Revised manuscript received

Key words: Phosphatidylinositol;

IN RAT KIDNEY ON PHOSPHATIDYLINOSITOL

H.S. SPEZIALE

*, ALICIA

TERRAGNO

TURNOVER

and NOBERTO

A. TERRAGNO

**

New York Medical College, Valhalla, NY 10595 (U.S.A.)

March

17th, 1982)

Diacylglycerol;

Phospholipase;

Arachidonate

metabolism

Rat renal cortical and medullary slices incorporate [ 14C]arachidonate into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerols. The percent distribution of [ “C]arachidonate among the various phospholipids is similar in renal cortex and medulla, although the total amount of radioactively labeled phospholipids is higher in the renal medulla. Subsequent incubation of prelabeled slices in the presence of deoxycholate induces a loss of radioactivity from [ 14C]phosphatidylinositol, with a concomitant increase in 1,2-[ “C]diacylglycerol. Neutral lipids are not affected. The degradation of phosphatidylinositol to [ 14C]diacylglycerol indicates the presence of phospholipase C activity. Renal medulla seems to be more sensitive to deoxycholate than the renal cortex. Deoxycholate also induces slightly the disappearance of some 14C radioactivity from phosphatidylethanolamine and phosphatidylcholine, which might reflect activation of phospholipase A,. The activity of the phospholipase C could constitute the first step in the sequence of reactions that leads to the release of arachidonic acid.

Introduction

of phospholipase C precedes, and hence induces, the subsequent release of arachidonic acid [S-8]. Phospholipase C activity induces the specific breakdown of phosphatidylinositol [8,9]. The increased turnover of phosphatidylinositol in response to appropriate stimulation is part of the so-called ‘phosphatidylinositol effect’, which is strongly implicated in the mobilization of Ca2+ within cells [9- 111. The presence of phospholipase C has been shown in several tissues [ 121, and in the present study we report its activity in rat renal tissue using the conversion of endogenous phosphatidylinositol to 1,2-diacylglycerol as an assay.

Stimulation of various cell types induces the release of arachidonic acid and its conversion to prostaglandins. The intracellular endogenous levels of the prostaglandins’ precursor is extremely low [l] since most of the arachidonic acid is esterified into membrane phospholipids and triacylglycerols. Phospholipase A, removes arachidonic acid from phospholipids, and several studies have suggested an important role for this enzyme in normal and pathological renal function [2-41. However, it has recently been shown in platelets that the activation * Fellows

of the Consejo National de Investigaciones Cientificas y Tecnicas (CONICET). Argentina. ** To whom correspondence should be addressed at (present address): Instituto de Farmacologia Clinica y Experimental (IFCE), Av. Tellier 2160, 1440 Buenos Aires, Argentina. OOOS-2760/82/0000-OOOO/sO2.75

0 1982 Elsevier Biomedical

Materials [ “C]Arachidonic acid pCi/mol) was obtained Press

(specific activity: 52.8 from New England

66

Nuclear, Boston, Massachusetts; sodium deoxycholate and phospholipase A, from Crotalus adamanteus was from Sigma Chemical Company, St. Louis, MO; phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and 1,2-diacylglycerol were purchased from Supelco, Bellefonte, PA. 0.25 mm silica gel H plates were obtained from Applied Science Division, State College, PA; 0.25 mm silica gel G plates from Brinkmann Instruments, Inc., Westbury, NY; and X-ray film for autoradiography from Eastman Kodak Company. Rochester, NY. Methods Labeling of renal tissue slices. After decapitation of male Sprague Dawley rats (250-280 g), both kidneys were removed and maintained on ice-cold Kreb’s solution. Each kidney was cut in half through the pelvis along its longitudinal axis, and the renal cortex and medulla were separated by scissor and scalpel dissection. The renal medullary and cortical tissues were individually sliced (approx. 0.5 mm thick) using a Stadie-Riggs microtome. This procedure provides uniform samples of renal cortex and medulla [ 131. For each experiment, 100 mg of tissue slices were collected in 1.25 ml of cold Krebs-Ringer bicarbonate buffer containing 5.5 mM glucose. Samples were then incubated at 37°C for 1 h with 0.5 PCi of [ “C]arachidonic acid in a metabolic shaking bath. The solvent of the labeled arachidonic acid was first dried off, resuspended by sonication in 25 ~1 of the medium and then added to the incubating mixture. At the end of that incubation period, the slices were washed and centrifuged three times with ice-cold Krebs’ solution. Assay conditions and lipid extraction. Each tissue sample was then incubated in 1 ml of fresh Krebs’ solution for 30 min in a shaking water bath at 37°C with or without different concentrations of deoxycholate, as stated in the figures. Incubation was stopped by adding 4.0 ml of chloroform/methanol (1: 2, v/v) [ 141 and samples were homogenized in glass tubes with a teflon pestle at 3000-3500 rpm. Phases were split by adding 1.24 ml of chloroform and 1.25 ml of water. The lower

chloroform phase was removed flow of nitrogen at 25°C [15].

and dried under

a

Chromatographic lipid extraction. The dried lipids were redissolved in chloroform, applied onto activated silica gel H thin-layer chromatographic plates (0.25 mm thick) and developed in a solvent system consisting of chloroform/methanol/acetic acid/water (75 :45 : 12: 3, v/v) [IS] (system I). In this solvent system, phosphatidylinositol and phosphatidylserine (RF 0.47) migrate together. while phosphatidylcholine, phosphatidylethanolamine and sphingomyelin are well separated. Separation of phosphatidylinositol from phosphatidylserine was achieved by two-dimensional chromatography on 1% ammonium oxalate-impregnated silica gel H plates using the previous solvent system in the first dimension and chloroform/methanol/ 13.5 N ammonia/water (70:30:0.5:4, v/ v ) in the second dimension (system II) [16]. For the partial characterization of arachidonic acid, arachidonic acid metabolites and neutral lipids, the silica gel from the solvent front area (system I) was scraped and the lipids eluted and extracted using a mixture of chloroform/methanol/acetic acid/water (75 : 45 : 12: 8, v/v) [ 141. The silica gel was washed three times, the extracts dried, redissolved in chloroform and applied on silica gel G thin-layer chromatographic plates using two different solvent systems: 1, upper phase of a mixture of ethyl acetate/2,2,4_trimethylpentane/ acetic acid/water (9 : 5 : 2 : 10, v/v) (system III). (In this system the R, values for arachidonic acid and diacylglycerol were 0.87 and 0.91, respectively. The triacylglycerols ran with the solvent front ether/diethyl PI.): 2, mixture of petroleum ether/acetic acid (90: 10: 1, v/v). (The R, values for arachidonic acid and triacylglycerols were 0.07, 0.25 and 0.44, respectively [8] (system IV).) Further identification of diacylglycerol was achieved by elution of the corresponding zones of the plates developed in systems III and IV, and digestion at 65°C for 2 h in closed tubes containing 5% KOH/MeOH [6]. After the mixtures were neutralized with HCl, lipids were extracted with chloroform and rechromatographed using solvent system II in order to show the released fatty acid. The migration of the lipids was detected by I, vapors or autoradiography as appropriate. Specific

67

TABLE

areas corresponding to those of the standards were quantitated in a scraped and the 14C radioactivity liquid scintillation counter with toluene/4% Omnifluor mixture. In order to ascertain the position of incorporation of the fatty acid into the phospholipids, we isolated phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine by thin-layer chromatography (system I). After elution, the individual phospholipids were sonicated in 1 ml of Krebs’ solution containing 10 mM Ca*+, 2.5 mM deoxycholate and 5 units of phospholipase A,. The mixture was incubated at 37°C for 2 h, and lipids were then extracted as described above and separated by thin-layer chromatography on silica gel G using solvent system III. In this system, the phospholipids remain in the origin and the R, value for arachidonic acid is 0.87. The thin-layer chromatography plate was then analyzed by radiochromatograph scan.

1

DISTRIBUTION THE TISSUE

OF

[‘4C]ARACHIDONIC

This table shows the distribution of medullary and cortical slices. Tissue incubated and lipids were extracted in Methods. Results are expressed radioactivity in the tissue. The

ACID

INTO

[‘4C]arachidonic acid in rat slices (100 mg, w/w) were and separated as described as percentage of the total radioactivity uptake for

medullary and cortical slices was 50 and 30%. respectively, of the total [‘4C]arachidonic acid added to the medium. Values for phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine are % of total phospholipids.

Total phospholipids Phosphatidylcholine Phosphatidylinositol Phosphatidylethanolamine Triacylglycerol Free arachidonic acid

Medulla

Cortex

35 59 19 22

20 62 I5 23 20 60

31 34

Results [ 14C] Arachidonate incorporation into rat renal cortex and medulla phospholipids. The uptake of [ “C]arachidonate by cortical and medullary slices after 1 h incubation varied between 30% (cortex) and 50% (medulla) of the total radioactivity present in the medium. In medullary slices, most of the radioactivity was incorporated into phospholipids. In the cortex, the largest percentage remained as free fatty acid. However, the percentage of distribution of radioactivity among the various phospholipids was similar in both cortex and medulla. The highest percentage of radioactivity was incorporated in phosphatidylcholine, followed by phosphatidylethanolamine and phosphatidylinositol (Table I), whereas sphingomyelin and phosphatidylserine were not labeled. No radioactivity was associated within the lysophospholipids when identified using systems I and II. After 2 h, the isolated radioactively labeled phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were incubated with C. adamanteus phospholipase A, in the presence of 2.5 mM deoxycholate, and most of the radioactivity originally associated with the phospholipids was recovered in the zone corresponding to the

*AA

Fig. 1. Radio-thin-layer chromatography showing the effect of phosphatidylcholine. Prephospholipase A a on prelabeled labeled phosphatidylcholine was incubated in Krebs’ solution for 2 h at 37’C, in the presence of 10 mM Ca*+. 2.5 mM deoxycholate and 5 units of phospholipase A,. (A) Phosphatidylcholine spot from system I, rechromatographed in system III. (B) Phosphatidylcholine spot after treatment with C. adamanteus phospholipase A *. PL. phospholipid; AA, arachidonic acid.

301 B

OCA

E xi20 ”

-

I5

30

45

60

TIME. MIN

PC

PE

PI

Fig. 2. Effect of deoxycholate on the degradation of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine from rat meduilary and cortical slices. Tissue slices (100 mg, w/w) prelabeled with [‘4C]arachidonic acid were incubated in Krebs’ solution in the absence (0.0 mm; q, 30 min) and in the presence of deoxycholate (5 mM deoxycholate for 30 min. W). PC, phosphatidylcholine; Pl, phosphatidyhnositol; PE, phosphatidylethanola~ne.

migration of the standard (Fig. 1).

free arachidonic

acid

~eoxycholate-induced changes in radioactive phospho~ipids. Incubation of prelabeled slices with 5 mM deoxycholate for 30 min induced a drastic decrease in radioactivity from phosphatidylinositol in medullary slices (51.058, P < 0.05) (Fig. 2A, B). Formation of diacyiglycerol follows phosphati~~~ino~ito~ degradation. Degradation of phosphatidylinositol to diacylglycerol indicated the presence of phospholipase C activity. The expres-

Fig. 4. Time-course of phospholipase C activity in slices of rat medulla (A) and cortex (B). The formation of diacylglycerol (0) and degradation of phosphatidylinositol (A) are expressed in cpm/lOO mg of tissue, W.W. All assays contained 5 mM deoxycholate.

sion of this activity required the addition of deoxycholate, as has been described previously in different tissues [ 161. Treatment of renal slices with increasing concentrations of deoxycholate also produced degradation of phosphatidylinositol and a comparable increase of [ “C]diacylglycerol. Medullary slices were more sensitive to lower concentrations of deoxycholate (Fig. 3A) than cortex slices (Fig. 3B). The increasing production of [ t4C]diacylglycerol at various time periods corresponded with the breakdown of radioactive phosphatidylinositol. The time-course of degradation of phosphatidylinositol and the correspondent formation of 1,2-diacylglycerol is shown in Fig. 4A and B. The endogenous substrate, in the presence of 5 mM deoxycholate, might we11 be the rate-limiting component of this reaction. The reaction is then not linear and might follow first-order kinetics, as has been established clearly for brain homogenates [9].

B

A

Discussion i-r.

x )

05

12

25

DEOXYCHOLATE, nM

50

OEOXYCHOLATE,

mM

Fig. 3. Effect of deoxycholate on phosphatidylinositol (A) diacylglycerol (a) on slices of rat medulla (A) and cortex Slices with prelabeled [“C]arachidonic acid were incubated 30 min in Krebs’ solution with various concentrations of oxycholate.

and (B). for de-

The incorporation of [ “C]arachidonic acid into phospholipids was significantly lower in cortical than in medullary slices. These data suggest that the acylation-deacylation mechanism could be less efficient in the cortex, and agree with the fact that prostaglandin synthesis is predominantly localized in the medulla [17], The distribution of 14C radioactivity showed that phosphatidylcholine comprised most of the labeling of the phosphohpid fraction in medulla and cortex; lesser amounts were found in phosphatidylethanolamine and

69

phosphatidylinositol; in all these phosphohpids, most of the f’4C]arachidonic acid seemed to be incorporated in the 2-position (Fig. 1). These results are consistent with those reported after infusion of [‘4C]arachidonic acid in rabbit perfused kidney [ 181. However, in rabbit [ 131 and rat [2] kidney slices, the uptake of [t4C]arachidonic acid was relatively inefficient 12,131. Our results show an efficient and reproducible pattern of incorporation of [ “C]arachidonic acid into rat renal cortical and medullary slices. Since in most tissues arachidonic acid is bound to the 2-position of membrane phosphohpids, it has been suggested that the deacylation reaction must be catalyzed by phospholipase A, [I]. In order to express phospholipase activity. we used deoxycholate, an anionic detergent, which has been shown to induce similar activity in several other tissues [ 161. Under our experimental conditions, treatment with deoxycholate produced an effective degradation of phosphatidylinositol. Breakdown of phosphatidylinositol, with a concomitant increase of diacylglycerol, constituted the expression of phospholipase C activity. Our assays were performed using Krebs’ solution, which contains 1 mM Ca*+, as an incubation medium. This concentration of Ca’+ might also activate phospholipase A,, as has recently been shown in platelets [ 161. Thus, the slight drop of radioactivity from phosphatidylethanolamine and phosphatidylcholine could be due to phospholipase A, activity (Fig. 2A and B). Deoxycholate (5 mM) also induces a 90% decrease in the incorporation of [ “C]arachidonic acid into phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and triacylglycerols (Speziale, E., Speziale, N., Terragno, A. and Terragno, N.A., unpublished data). This effect might reflect inhibition of acyltransferase activity and may explain why, despite the presence of free arachidonic acid in our system, it was not incorporated into the phospholipids by the acylation mechanism after activation of phospholipases in the presence of deoxycholate. The presence of phosphoIjpases C and A, in renal cortex and medulla could imply that the arachidonic acid release, after stimulation of rat kidney slices, involves the sequential activation of phospholipase C followed by phospholipase AZ, as was described in platelets [ 161. Another alternative

pathway for arachidonic acid release could be by the combined activities of phospholipase C and diacylglycerol lipase. Diacylglycerol lipase would then produce the deacylation of the arachidonic acid bound to the 2-position of the diacylglycerol formed by the action of phospholipase C on phosphatidylinositol [5,6,19]. We have not yet found evidence for the presence of the diacylglyceroldeacylating enzyme in renal tissue. The presence of phospholipase C activity in rat renal cortex and medulla, and its possible relationship to arachidonic acid release, opens new areas with regard to physiological stimulation of renal tissue. We have reported previously that kinins and angiotensin II stimulate renal prostaglandin release [20-223. The possible mechanism of action of these hormones which may affect the phospholipase C activity in the renal cortex and medulla is now under investigation and might open new possibilities in the further understanding of the arachidonic acid release mechanism in the kidney. Acknowledgements

The authors wish to thank Dr. Eva Hirsh and Mrs. Sally McGiff for editing and Miss Lillian Delgado for typing the manuscript. We are deeply indebted to Dr. Eduardo Lapetina from the Burroughs Wellcome Research Laboratory for his advice and helpful discussions throughout the development of this work, and to Dr. John C. McGiff. This work was supported by USPHS-NIH grants, Nos. HL25406 and HL24811; and American Heart Association grant, No. 77-894. References I Kunze, H. and Vogt, W. (1971) Ann. N.Y. Acad. Sci. 180, 123-125 2 Limas, C. and H65-H72

Limas,

C.J. (1979)

3 Baggio, B., Favaro, S.. Piccoli, Prostagiandins 18, 83-92

Am.

J. Physiol.

A. and Borsatti,

236,

A. (1979)

4 Morrison, A.R., Pascoe, W. and Needleman, P. (1980) J. Bioi. Chem. 255, 20-22 5 Beli, R.L., Kennerly, D.A., Stanford, N. and Majerus, W.P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76. 3238-3241 6 Rittenhouse-Simmons, S. (1979) J. Clin. invest. 63, 580-587 7 Lapetina, E. and Cuatrecasas, P. (1979) B&him. Biophys. Acta 573, 394-402

70

8 Billah, M.M., Lapetina, E.G. and Cuatrecasas, P. (1979) Biochem. Biophys. Res. Commun. 90. 92-98 9 Lapetina, E. and Michell, R.H. (1973) Biochem. J. 131, 433-442 10 Lapetina, l-10

E.G. and

Michell,

R.H.

(1973)

11 Michell, R.H. (1975) Biochim. Biophys. 12 Hostetler, K.Y. and Hall, L.B. (1980) Res. Commun. 96, 388-392 13 Crowshaw. K. (1973) Prostaglandins 3. 14 Bligh, E.G. and Dyer, W.J. (1959) Can. 37, 911-919 15 Skipski, V.P., Peterson. them. J. 90, 374-378

FEBS

Lett.

31,

Acta 415. 81-147 Biochem. Biophys. 607-620 J. B&hem.

R.F. and Barclay,

Physiol.

M. (1964)

Bio-

16 Billah, M.M., Lapetina, E.G. and Cuatrecasas. Biol. Chem. 255, 10227- 1023 I

P. (1980) J.

17 McGiff, J.C., Crowshaw, K. and Itskovitz. H.D. (1974) Fed. Proc. 33, 39-47 18 Isakson, P.C., Raz, A., Denny, S.E.. Wyche, A. and Needleman, P. (1977) Prostaglandins 14, 853-871 19 Rittenhouse-Simmons, S. (1980) J. Biol. Chem. 255, 22592262 20 Terragno, N.A., Lonigro, A.J., Malik, K.U. and McGiff, J.C. (1972) Experientia 28, 437-438 21 McGiff, J.C., Terragno. N.A., Malik, K.U. and Lonigro. A.J. (1972) Circ. Res. 31, 36-43 22 McGiff, J.C.. Crowshaw, K.. Terragno, N.A. and Lonigro, A.J. (1970) Circ. Res. 26, 27, I-121~1-131