469

Biochimica et Biophysics Acta, 664 (1981) 469-474 Elsevier/North-Holland Biomedical Press

BBA 57796

SELECTIVE INDUCTION OF DE NOVO PROSTAGLANDIN BIOSYNTHESIS IN RABBIT KIDNEY CORTEX

MICHAL SCHWARTZMAN and AMIRAM RAZ *

Department of Biochemistry, The George S. Wise Center of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv (Israel)

(Received October 21st, 1980)

Key words: Prostaglandin synthesis; Arachidonic acid; Bradykinin stimulation; Acetylsalicylic acid; (Rabbit kidney cortex)

summary

Ureter-obstructed kidney develops during perfusion an enhanced responsive- ness to bradykinin-stimulated prostaglandin release. This enhanced prostaglan- din generation results from de novo synthesis of prostaglandin synthetase and acylhydrolase enzymes during the perfusion and is therefore unaffected by acetylsalicylic acid (aspirin) inhibition of prostaglandin synthesis prior to initia- tion of perfusion. Studies were carried out to identify the renal cellular site in which the newly synthesizing prostaglandin generating system is localized. Kidneys with or without aspirin pretreatment were perfused for either 1 or 5 h. Following perfusion, medullary and cortical slices were incubated and prosta- glandin E2 production measured. Medullary slices showed similar prostaglandin E2 biosynthetic activity in kidneys perfused for 1 or 5 h. Furthermore, medullary prostaglandin generation was inhibited (90-95%) by aspirin pre- treatment and did not increase during subsequent perfusion for 5 h. In con- trast, cortical slices from kidneys pretreated with aspirin regained their full activity after 5 h of perfusion, this regeneration being abolished by infusion of the protein synthesis inhibitor, cycloheximide. The same differences in activ- ities between medulla and cortex were also seen when microsomal fractions were compared. The perfusion-induced formation of prostaglandin synthetase activity is thus specifically localized in the cortex and can be detected in corti- cal microsomes. This cortical activity is unique in that endogenous arachidonic acid released from esterified lipids is converted to prostaglandins, whereas exogenous added arachidonic acid is not. It thus appears that the induced corti- cal acylhydrolase and prostaglandin synthetase activities are tightly coupled and that the true molecular form of precursor arachidonate for this prostaglan- din generating system is esterified and not free arachidonate.

-

* To whom correspondence should be addressad.

OOOS-2760/81/0000~000/$02.50 0 Elsevier/North-Holland Biomedical Press

470

Introduction

Peptide hormone-induced renal prostaglandin release involves a selective deacylation of esterified arachidonic acid in a tightly coupled process in which a major portion (25-50%) of the released arachidonate is converted to prosta- glandins [ 1,2]. In the ureter-obstructed kidney model employed in many of these studies, there is a time-dependent increase in prostaglandin release in response to bradykinin or angiotensin II stimulation. This enhanced prostaglan- din release is the result of de novo gradual synthesis of both a hormone-sensi- tive lipase and prostaglandin synthetase enzymes during the perfusion [ 2,3].

A major question still unresolved concerns the renal cellular site in which the newly synthesizing prostaglandin synthetase activity is located. In vitro studies on this question by Needleman et al. [2] were inconclusive, being unable to show distinct, perfusion-dependent, enhanced activity in cortical or medullary microsomes from the ureter-obstructed kidney. This report provides the first direct demonstration that the newly synthesized prostaglandin synthetase is found in the cortex of the perfused ureter-obstructed kidney.

Methods and Materials

(I) Isolated perfused kidney Male rabbits, 2.5-3.0 kg (local strain, New Zealand White-derived) were

used. Ureter-obstructed rabbit kidneys were prepared and perfused with Krebs- Henseleit buffer (pH 7.4, 37°C) at the rate of 15 ml/min as described pre- viously [ 11. The kidney effluent was observed to flow from both the renal vein and the ureter. Bradykinin was dissolved in saline (pH 7.5) and injected (0.5 c(g in 0.1 ml) into the perfusing media just as it enters the kidney. Kidney effluents (60 ml, 4 min collection) were collected immediately prior to (con- trol) and after hormone stimulation. Where indicated, cycloheximide was infused into the kidney at the rate of 50 fig/mm In the aspirin experiments, aspirin solution (in saline, pH 8.0) was administered to rabbits (20 mg/kg, intra- venously) 12 h prior to removal of kidneys for perfusion and was also infused into the kidneys at the beginning of the perfusion course (0.5 mg/ml final con- centration, 20 min).

(2) Analysis of kidney effluent Renal effluent samples were acidified to pH 3.5 with 2 M citric acid and

extracted twice with 2 vol. of ethyl acetate. The extracts were dried over Na#04, concentrated in vacua and dissolved in 2 ml of chloroform/methanol (2 : 1). The extracts were subjected to thin-layer chromatography and the prostaglandin Ez content determined by bioassay on rat stomach strip [4]. Recovery of prostaglandin E, was determined by addition to each effluent sample of [3H]prostaglandin Ez (4 - 104 cpm). The overall recovery of prosta- glandin Ez was 50-60%.

(3) Slices experiments At the end of perfusion experiments, the kidneys were removed and placed

on ice. Slices (approximately 1 mm thick) were prepared from medulla and

471

cortex. The slices were rinsed.with Tris-HCl buffer (0.1 M, pH 8.0) and incu- bated in the same buffer (0.2-0.3 g slices in 2 ml of buffer) with shaking at 37°C for 30 min. After incubation the medium was extracted and assayed for prostaglandin Ez content as described above.

(4) Microsomes experiments Slices after incubation were collected and homogenized in 5 vol. of Tris-HCI

buffer (0.1 M, pH 8.0). The tissue homogenate was centrifuged at 8000 X g for 15 min. The 8000 X g supematant was centrifuged at 140 000 X g for 60 min and the microsomal pellet resuspended in Tris-HCl buffer. Microsomes from medulla and cortex (l-l.5 mg protein) were incubated (final volume 1 ml) with or without arachidonic acid (2 E.cg) for 30 min at 37°C. The prostaglandin Ez content was assayed as described before. Protein concentration was deter- mined by the method of Lowry et al. [5] using bovine serum albumin as a standard.

(5) Materials Arachidonic acid was obtained from Nu-Check (Elysian, MN, U.S.A.). Cyclo-

heximide and aspirin were obtained from Sigma (St. Louis, MO, U.S.A.). [5 6 8 9 , 9 9 , 11,12,14,15(n)-3H]prostaglandin E2 (specific activity 120 Ci/mol) was purchased from New England Nuclear, (Boston, MA, U.S.A.). All other reagents and solvents were analytical grade.

Results

Three types of ureter-obstructed kidney preparation were perfused for either 1 h or 5 h as outlined in Fig. 1 and the prostaglandin Ez release under basal conditions and after bradykinin stimulation was determined. In the perfused kidneys which received no additional treatments (control kidneys) hourly stimulation with a fixed bolus dose of bradykinin produced a progressive increase in prostaglandin Ez production. During the 5 h perfusion period an approximate lo-fold increase in bradykinin-stimulated prostaglandin E2 release was observed. Intravenous administration of aspirin solution 12 h prior to the experiment together with infusion of aspirin solution to the perfused kidney at the start of an experiment produced an initial 85-95s inhibition of basal and hormone-stimulated prostaglandin Ez release. However, 90-120 min after cessation of aspirin infusion, a progressive rise in prostaglandin E, generation was observed which paralleled that of control kidneys. This rise was prevented if cycloheximide was infused throughout the perfusion course. This result is essentially the same as that of Morrison et al. [6] and fully supports their con- clusions that: (1) the progressive rise in hormone-stimulated prostaglandin Ez release is dependent on the time course of the perfusion and represents new prostaglandin-synthesizing activity which therefore cannot be inhibited by aspirin administration prior to the perfusion; (2) the new prostaglandin-syn- thesizing activity requires new enzyme protein synthesis since it is inhibited by cycloheximide administration,

In order to assess the relative prostaglandin biosynthetic activities of the renal cortex vs. medulla, we prepared cortical and medullary slices from the

1 3 PERFUSION TIME &h

5

Fig. 1. Prostaglandin E2 release from the perfused ureter-obstructed kidney during hourly stimulation with hradykinin. Kidney effluents (60 ml, 4 min collection) were collected prior to and after bradykinin injection (0.5 l(g in 0.1 ml saline) and prostaghmdin E2 content was determined as described In Methods and Materials. Aspirin (ASA.&),-20 mg/kg, was administered 12 h before and at the beginning of the per- fusion experiment (0.5 mg/mI final concentration). Cycloheximide (0, 60 /.tg/mI final concentration) was infused Into the kidney throughout the experiment. Values given (mean *S.E., n = 3) represent net prosta- glandin E2 release by bradykinin stimulation and were calculated by subtracting the values for prosta- glandin E2 release immediately prior to bradykinin administration from the values obtained after hor- mone administration. a, control.

various kidney preparations after perfusion for 1 h or 5 h. In medullary slices from control kidneys, prostaglandin E2 generation after 1 and 5 h of perfusion were not significantly different (Table I). Aspirin pretreatment abolished 95- 100% of prostaglandin E2-synthesizing activity at 1 h and 85-9O%lat 5 h of perfusion. Although the medullary biosynthetic capacity in aspirin-treated kidneys after 5 h of perfusion was higher than after 1 h of perfusion it apparently

TABLE I

PROSTAGLANDIN E2 BIOSYNTHESIS IN MEDULLARY AND CORTICAL SLICES FROM KIDNEYS PERFUSED FOR 1 OR 6 h

Control kidneys, kidneys pretreated with aspirin and kidney pretreated with aspirin and infused with cycloheximide were perfused as described in the legend to Fig. 1. Following 1 or 5 h of perfusion, medul- Iary and cortical slices were prepared and incubated (30 min. 37’C with shaking). ProstagIandin E2 released into the incubation medium was determined. Values are means fS.E. (n = 3).

Aspirin Perfusion Medulla cortex pretreatment time of kidney (h) Prostaglandin E2 % inhibition ProstagIandIn E2 56 inhibition

production, by aspirin production by aspirin @g/g tissue) @g/g tissue)

1 3.2 + 0.5 - 0.48 + 0.08 -

+ 1 <0.05 100 <O.Ol 100 - 5 3.6 r 0.4 - 0.43 f 0.05 - + 5 0.38 f 0.12 90 0.41 f 0.06 4.7 + 5* 0.36 2 0.1 90.5 0.02 + 0.005 95.4

* Aspirin-pretreated kidneys perfused with cycloheximide throughout the perfusion course.

473

TABLE II

MICROSOMAL PROSTAGLANDIN E9 BIOSYNTHESIS IN RENAL CORTEX AND MEDULLA

Cortex and medulla microsomes were prepared from control and aspirin-pretreated ureter-obstructed kid- neys after 5 h of perfusion. Incubations were carried out in the presence or absence of arachidonic acid (2 ng) for 30 min at 37OC and the prostaglandin Eg content determined. The values are the mean +S.E. (n = 3). In incubations containing arachidonic acid, % inhibition by aspirin was calculated for the addi- tional amount of prostaglandin E2 generated from added arachidonic acid.

Aspirin pretreatment of kidney

Addition to Medulla microsomes Cortex microsomes microsomes incubation Prostaglandin E2 96 inhibition ProstagIandin E2 % inhibition

production by aspirin production by aspirin (ng/mg protein) (nglmg protein)

- + -

+

52t 9 - 34 + 5 -

- 15+ 5 71.2 37 i 2 0 Arachidonic 126 * 14 - 52 + 3 -

acid (2 pg) Arachidonic 32+ 2 77.0 43 f4 66.7 acid (2 pg)

did not represent new enzyme synthesis since it was not abolished by cyclohexi- mide. In contrast, cortical slices pretreated with aspirin regained their full activity after 5 h of perfusion, this regeneration being completely abolished by cycloheximide administration. These results (Table I) demonstrate that the cortex, but not the medulla, is the cellular site which contains the perfusion- induced, cycloheximide-sensitive new activity for prostaglandin generation.

We next attempted to demonstrate similar differences in prostaglandin activ- ities in subcellular microsomal fractions from cortex and medulla. The results (Table II) indicated similar differences in prostaglandin E2 generation between microsomal fractions of cortex and medulla from kidneys after 5 h of perfu- sion. The activity of medullary microsomes from kidneys pretreated with aspirin was inhibited by 72-77%, whereas the activity of cortical microsomes was un- affected by aspirin pretreatment. However, when we measured prostaglandin generation from exogenous added arachidonate, cortical microsomes from aspirin-treated kidneys showed 67-72s inhibition, similar to the inhibition ob- served with medullary microsomes.

Discussion

The isolated ureter-obstructed kidney develops during perfusion a highly enhanced capacity for hormone-stimulated release of arachidonic acid and sub- sequent prostaglandin generation. Indirect evidence suggested that these enhanced activities of both acylhydrolase and prostaglandin endoperoxide synthetase are localized in the cortex of the ureter-obstructed kidney [2]. Experiments designed to demonstrate this directly were, however, inconclusive. Needleman and coworkers found that despite the lO--20-fold higher lipolysis and prostaglandin generation in the perfused ureter-obstructed kidney as com- pared to the contralateral unobstructed kidney, the cortical microsomal prosta- glandin biosynthetic activity assayed with added [14C]arachidonate was the same in the two kidney preparations. Furthermore, the large difference in

prostaglandin generation activity between kidneys after short (1 h) and long (5 h) perfusion (Ref. 2, Fig. 1 and Table I) was undetectable when the micro- somal preparations from the two kidneys were compared [Z] .

We have recently shown the existence of two functionally distinct pools of esterified arachidonate in whole rabbit kidney [ 71. One pool is a hormone- sensitive pool from which a selective release of arachidonate is obtained in response to bradykinin or angiotensin II stimulation, In this pool, the apparent prostaglandin precursor is esterified arachidonate (and not the free acid) since ~achidonate released from this pool is tightly coupled to its conversion to prostaglandin Ez. Furthermore, exogenous free arachidonic acid infused into the kidney is not readily incorporated into this pool. The second pool of esteri- fied arachidonic acid is not responsive to hormone stimulation. Exogenous arachidonate infused into the kidney is readily incorporated into this pool. The results presented here demonstrate that cortical slices and cortical microsomes from 5-h-perfused aspirin-treated kidneys contain enhanced prostaglandin bio- synthetic activity which is absent at 1 h of perfusion. This enhanced activity is restricted to the cortex and is not found in medulla slices or microsomes. Significantly, the enhanced cortical activity can be demonstrated only when measuring endogenous prostaglandin E, formation, i.e., from endogenous esterified ara~hidonic acid which was released from cellular lipids. Treatment of the kidneys with aspirin prior to perfusion does not affect this activity, demon- strating that it is a newly synthesized enzyme activity generated during the per- fusion and inhibited by cycloheximide (Table II). Measurement of prostaglan- din biosynthetic capacity from added free arachidonate apparently reflects a different prostaglandin synthetase activity which is not induced during perfu- sion and therefore is inhibited by pretreatment with aspirin (Table II). These results are therefore in agreement with and provide an explanation for the pre- vious results of Needleman et al. [2] who, when using exogenous arachidonate as substrate, were also unable to demonstrate enhanced perfusion-dependent microsomal activity in either cortex or medulla of the ureter-obstructed kidney. The data in this report provide additional support for our previous con- clusion that the perfusion~induced hormone-sensitive lipase and pros~gl~din synthetase enzyme systems, now shown to be localized in the renal cortex, are tightly coupled and require esterified arachidonic acid and not free arachi- donate as the preferred substrate.

Acknowledgements

The skillful technical assistance of Miss Dvora Edehnan and Mr. Eliezer Libelman is gratefully acknowledged. This work was supported by a grant from the US-Israel Binational Science Foundation (BSF), Jerusalem, Israel.

References

Schwartzman, M. and Raz, A. (1979) Biochim. Biophys. Acta 572.363-369 Needleman, P., Wyehes, A., Bronson, S.D., Holmberg, S. and Morrison, A.R. (1979) J. Biol. Chem. 254.9172-9777 Morrison, A.R.. Pascoe, N. and Needleman, P. (1980) J. Biol. Chem. 265. 20-22 Eckenfels, A. and Vane, J.R. (1972) Brit. J. Pharmacol. 45, 461462 Lowry, O.H., Rosebrougb, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193. 265-275 Morrison, A.R., Moritz, H. and Needleman, P. (1978) J. Biol. Chem. 253, 8210-8212 Schwextzman, M., Libexman, E. and Raz, A. (1981) J. Biol. Chem. 266‘2329-2333