Triacylglycerol lipase-mediated release of arachidonic acid for renal medullary prostaglandin synthesis

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 261, No. 2, March, pp. 368-374, 1988

Triacylglycerol Lipase-Mediated Release of Arachidonic for Renal Medullary Prostaglandin Synthesis YOHKO Department

of

FUJIMOTO,l KIYOMI

KAZUYUKI NISHIOKA, YUKARI SADO, AND TADASHI FUJITA

Hygienic Chemistry, Osakn Received

May

University

22,1987,

of Pharma,ceutical

and in revised

form

Acid

HASE,

Sciences, Ma,tsubartr, Osaka 580, Japan October

7, 198’7

The effect of triacylglycerol lipase or triarachidonin on the synthesis of prostaglandin E2 in rabbit kidney medulla slices was examined. Prostaglandin Ez generation was enhanced by exogenous triacylglycerol lipase, indicating that arachidonic acid released from medullary triacylglycerols is readily available for prostaglandin biosynthesis. Triarachidonin stimulated prostaglandin Ez production in a dose-dependent manner. Moreover, the kidney medulla slices showed triacylglycerol lipase activity using trimargarin as substrate. The addition of mepacrine made the stimulatory effect of triarachidonin on prostaglandin Ez formation more pronounced. Studies utilizing EGTA and p-bromophenacyl bromide revealed that triacylglycerol lipase of kidney medulla is independent of Ca2+ and sensitive to a sulfhydryl inhibitor. These results suggest the presence of triacylglycerol lipase and triacylglycerol as a possible candidate for providing free arachidonic acid to cyclooxygenase in kidney medulla. (o 19x8 Academic PRESS, I,,~.

ries have shown that renal medullary lipid droplets and medullary tissue contain large amounts of triacylglycerols which are rich in arachidonic acid. These studies suggested the possibility that triacylglycerols rich in arachidonic acid, as well as phospholipids, could supply the substrate for the prostaglandin-generating system of the kidney; however, no evidence for a direct role of this lipid in furnishing arachidonic acid for prostaglandin synthesis has yet been provided. In an attempt to clarify this possibility, we investigated the effect of exogenous triacylglycerol lipase or triarachidonin on the synthesis of prostaglandin E2 in rabbit kidney medulla slices.

The release of arachidonic acid from membrane phospholipids, the major cellular depository of the fatty acid, is the initial step in the biosynthesis of prostaglandins and thromboxanes (l-3). Phospholipase Az and in some cells the combined activities of phospholipase C and other lipases catalyze the deacylation of arachidonic acid from phospholipids, a rate-limiting step in prostanoid biosynthesis as it determines the amount of free arachidonic acid available to cyclooxygenase (2-6). Recently, by using exogenously applied phospholipase C (7) and diarachidonin as an artificial substrate (8), we were able to report the presence of diacylglycerol lipase and monoacylglycerol lipase in rabbit kidney medulla and the importance of the phospholipase C-diacylglycerol lipase pathway in prostaglandin synthesis by the kidney medulla. On the other hand, previous studies from our (9) and other (10-12) laborato1 To whom 0003-9861/88 Copyright All rights

correspondence

should

$3.00

0 1988 by Academic Press, Inc. of reproduction in any form reserved.

MATERIALS

AND

METHODS

Materials. Triacylglycerol lipase (pancreatic, 300 units/mg of protein) was obtained from BoehringerMannheim (Mannheim FRG), and trimargarin (triheptadecanain) was obtained from Nu-Chek Prep (Elysian, MN). Triarachidonin, linolenic acid, and

be addressed. 368

TRIACYLGLYCEROL

LIPASE

HYDROLYSIS

mepacrine (quinacrine) were obtained from Sigma Chemical Co. (St. Louis, MO). p-Bromophenacyl bromide and EGTA’ were purchased from Wako Pure Chemical Industries (Osaka, Japan). All other reagents were analytical grade. Tissue. Male rabbits (Z-2.5 kg) were used in the present study. The kidneys were removed from anesthetized (sodium pentobarbital, 30 mg/kg) rabbits and rapidly chilled in ice-cold 0.9% NaCl. Kidney medulla slices were prepared as described elsewhere (13). Incubation of medulla slices. In all experiments, rabbit kidney medulla slices (0.4 g) were preincubated in 4.0 ml of 0.15 M KCV0.02 M Tris/HCl buffer, pH 7.4, at 4°C for 5 min. After preincubation, the medium was changed to a new solution containing the indicated concentrations of triacylglycerol lipase or triarachidonin. Triarachidonin was first 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 E2 production in medulla slices. Incubation was continued for another 30 min to measure prostaglandin Ez. In experiments utilizing EGTA, slices were preincubated for 30 min at 37°C in the Tris/HCl buffer with or without 2 mM EGTA. At t = 30 min, slices were washed briefly and transferred to fresh buffer with or without 2 mM EGTA, and the incubation was continued to t = 60 min to measure prostaglandin Ez. Triarachidonin was added at t = 30 min to the incubations. Determination of prostaglandin formation. After incubation the medium was assayed for prostaglandin Ez content by a high-pressure liquid chromatographic method as described in our previous paper (9). Briefly, prostaglandin Ez extracted with ethyl acetate (approx pH 3) was measured after its basecatalyzed conversion to prostaglandin Bz (14). Peak heights were measured for the quantification of the extracted prostaglandin Bz relative to a prostaglandin Bz standard prepared from authentic prostaglandin E,. Assay of triacylglycerol lipase activity. To measure triacylglycerol lipase activity of medulla slices, trimargarin (50 pM) was employed as substrate. Triacylglycerol lipase activity is expressed as micrograms margaric acid released from the substrate per gram of tissue into the medium. Medullary slices (0.4 g slices in 4.0 ml of buffer) were incubated in the Tris/ HCl buffer containing 2 mg/ml fatty acid-free bovine serum albumin at 37°C for 30 min. After incubation, the medium was assayed for margaric acid content as described previously (15). To separate the lipid fractions (free fatty acids, triacylglycerols etc.), the total lipids extracted with chloroform/methanol (2/l, v/v)

*Abbreviation used: EGTA, aminoethyl ether)N,hP-tetraacetic

ethylene acid.

glycol

bis(fi-

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MEDULLA

369

were applied on the thin-layer plate (silica gel H; 0.5 mm thick, Merck) under a stream of Nz and then developed with light petroleum (bp 30-70”C)/diethyl ether/acetic acid (80/30/l, by vol). The free fatty acid zone was extracted, and methyl esters were prepared and quantitatively determined by gas-liquid chromatography. The concentration of margaric acid in the medium was evaluated from the peak area calculated by the triangulation, in comparison with the known amounts of linolenic acid (10 qg), which appeared as a new peak. Statistics. Results are means + SE. Statistical significance was determined by Student’s f test. RESULTS

AND

DISCUSSION

Previous studies have shown that renal medullary lipid droplets and medullary tissue contain large amounts of triacylglycerols which are rich in arachidonic acid (9-12). Therefore, we first examined the influence of triacylglycerol lipase on prostaglandin E2 synthesis in rabbit kidney medulla slices. As shown in Fig. 1, exogenous triacylglycerol lipase stimulated medullary generation of prostaglandin Ez at concentrations ranging from 50 to 250 units/ml. Maximal effect on prostaglandin E2 production was observed at 150 units triacylglycerol lipase/ml (approximately 1.2-fold compared with the control). No further stimulation was noted when the concentration of triacylglycerol lipase was raised to 250 units/ml. Trlacylglycerol lipase did not have a toxic effect on prostaglandin Ez synthesis, even at the highest concentration. These results indicate that arachidonic acid released from medullary triacylglycerols is readily available for prostaglandin biosynthesis. Figure 2 illustrates the effects of various concentrations of triarachidonin on the release of prostaglandin Ez from rabbit kidney medulla slices. Triarachidonin showed a dose-dependent stimulation of prostaglandin Ez production. At three concentrations (25, 50, and 100 PM), triarachidonin increased prostaglandin Ez release l.l- to 1.3-fold compared with the control. Recently we have reported that exogenous phospholipase C or diarachidonin has a powerful stimulatory effect on prostaglandin E2 synthesis of rabbit kidney

370

FUJIMOTO

50

150 Concentration

250

(units/ml)

ET

AL

comparison might be questionable, the findings of this study and the above considerations strongly suggest that diacylglycerol and monoacylglycerol lipase activities are higher than triacylglycerol lipase activity in kidney medulla and that the reaction catalyzed by triacylglycerol lipase becomes the rate-limiting step in furnishing arachidonic acid from renal medulla triacylglycerols for prostaglandin synthesis. To verify further the presence of triacylglycerol lipase in kidney medulla, we measured this enzyme activity using trimargarin as substrate (Fig. 3). Medullary slices were incubated with bovine serum albumin (2 mg/ml) to facilitate “trapping” released margaric acid. The release of margaric acid was quantified by gas-

FIG. 1. Influence of triacylglycerol lipase on prostaglandin Ea release from rabbit kidney medulla slices. Slices were incubated for 30 min at 37°C in 0.15 M KCV0.02 M Tris/HCl buffer in the presence of different concentrations of triacylglycerol lipase. Each point represents the mean for five experiments (SE values were less than 5%). The mean value for prostaglandin Ea release at zero triacylglycerol lipase (5.40 fig/g wet wt of tissue) was subtracted from the corresponding values at the different triacylglycerol lipase concentrations and the obtained differences, which represent net triacylglycerol lipase-stimulated prostaglandin E, release, were plotted.

medulla slices and that the enhancement of prostaglandin EZ formation elicited by phospholipase C or diarachidonin can be ascribed to increased availability of free arachidonic acid by diacylglycerol lipase and monoacylglycerol lipase (7, 8). In these studies, we observed that phospholipase C (2 units/ml) and diarachidonin (100 /*M) resulted in an increase in the prostaglandin Ez release of about 3.9- and 2.1fold, respectively, compared with the control, and diarachidonin produced a twofold greater increase in prostaglandin Ez release than that seen in response to exogenous arachidonic acid at the same concentration. It seems likely that phospholipase C or diarachidonin has a more pronounced stimulatory effect on prostaglandin E2 formation than triacylglycerol lipase or triarachidonin. Although a direct

c‘ 0

25

50 Concentration

100 (url)

FIG. 2. Effect of triarachidonin on prostaglandin Ez release from rabbit kidney medulla slices. Slices were incubated for 30 min at 37°C in 0.15 M KCV0.02 M Tris/HCl buffer in the presence of different concentrations of triarachidonin. Each point represents the mean for seven experiments (SE values were less than 5%). The mean value for prostaglandin Ea release at zero triarachidonin (5.48 pg/g wet wt of tissue) was subtracted from the corresponding values at the different triarachidonin concentrations and the obtained differences, which represent net triarachidonin-stimulated prostaglandin Ea release, were plotted.

TRIACYLGLYCEROL

Trimargarln Epinephrine

-

LIPASE

HYDROLYSIS

+

+

-

+

FIG. 3. Triacylglycerol lipase activity in control and epinephrine-stimulated rabbit kidney medulla slices by using trimargarin as the substrate. Slices were incubated for 30 min at 37°C in 0.15 M KW0.02 M Tris/HCl buffer with bovine serum albumin (2 mg/ml). Trimargarin (50 pM) was used as the substrate for triacylglycerol lipase. Values are the means + SE (n = 4). Epinephrine concentration = 1 mM. N.D. = not detectable.

IN

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KIDNEY

been shown that phospholipase C activation does not depend on cytoplasmic increases of calcium concentration, at variance with phospholipase AZ (20, 21). The inhibition by EGTA of basal prostaglandin Ez formation appears to be mediated via inhibition of a Ca2+-dependent phospholipase A2 (22-24) that cleaves arachidonic acid from phospholipids, primarily phosphatidylcholine. Triarachidonin significantly increased prostaglandin E2 release at 50 pM compared with the control. Even in the presence of EGTA triarachidonin significantly stimulated prostaglandin E2 production compared to the value observed with EGTA alone. This result reveals that Ca2+ dependence does not seem to be a feature of triacylglycerol lipase activity. Our present results, together with the recent demonstration that activation of diacylglycerol or monoacylglycerol lipase does not depend on Ca2+ (8,25,26), suggest that this whole pathway might be independent of Ca2+, as well as the phospholipase C-diacylglycerol lipase pathway (8, 25). This contrasts with the release of arTABLE

liquid chromatography. In the control experiment, incubated in the absence of trimargarin, margaric acid did not appear in the medium. When the slices were incubated in the presence of trimargarin (50 pM) the liberation of margaric acid was observed, indicating the presence of triacylglycerol lipase in kidney medulla. In addition epinephrine (1 mM) enhanced triacylglycerol lipase activity 5.2-fold. This result suggests that kidney medulla is sensitive to the adipokinetic action of epinephrine, in agreement with our data in kidney cortex (16) and with the reports of other laboratories in adipose tissue (17-19). Furthermore, we determined the effect of EGTA on triarachidonin-induced prostaglandin E, release from rabbit kidney medulla slices (Table I). EGTA reduced the production of basal prostaglandin Ee by 59% at a concentration of 2 mM. It has

371

MEDULLA

I

OF EGTA ONTRIARACHIDONIN-INDUCED PROSTAGLANDIN E2 RELEASEFROMRABBIT KIDNEYMEDULLASLICES

EFFECT

Prostaglandin E:: (pg/g wet wt of tissue) No triarachidonin

Treatment None EGTA

4.05 t 0.19 (2

mM)

1.67 t 0.10"

+50 /.LM triarachidonin 5.13 f 0.15” 2.30 + O.lOb

Note. Slices were preincubated for 30 min at 37°C in 0.15 M KCl/0.02 M Tris/HCl buffer with or without 2 mM EGTA. At t = 30 min, slices were washed briefly and transferred to fresh buffer with or without 2 mM EGTA, and the incubation was continued to t = 60 min to measure prostaglandin E2. Triarachidonin, 50 pM, was added at t = 30 min to the incubations. Values are means f SE (n = 7). a Significantly different (P < 0.01) from control. *Significantly different (P < 0.01) from EGTAtreated value.

372

FUJIMOTO

achidonic acid from phosphatidylcholine via phospholipase AZ which is a well-established calcium-dependent pathway. Mepacrine and p-bromophenacyl bromide have been shown to inhibit the release of arachidonic acid and the generation of arachidonate-oxygenated products from blood platelets (27). We reported that mepacrine at a concentration of 1.6 mM, and p-bromophenacyl bromide at 0.1 mM, inhibited exogenous phospholipase AZ-stimulated prostaglandin Ez production by 48 and 58% respectively (7). In the same study, we suggested that p-bromophenacyl bromide was a direct inhibitor of phospholipase AZ and that mepacrine might work in an indirect way. Mepacrine has also been reported to inhibit phosphatidylinositol-specific phospholipase C from platelets by binding to phospholipids (28). As shown in Table II, mepacrine (1.6 mM) failed to inhibit triarachidonin-stimulated prostaglandin E2 formation, indicating that triacylglycerol lipase acts exclusively on triacylglycerols. Triarachidonin in the presence of mepacrine stimulated the release of prostaglandin Ez at a high level as compared with triarachidonin alone (triarachidonin-induced prostaglandin Ez release of 1.07 pg/g of tissue was enhanced by treatment with

TABLE EFFECTS

OF MEPACRINE

ET

mepacrine to 1.43 ,ug/g of tissue). Dise et a,l. (29) reported that mepacrine interacted directly with membrane phospholipids, primarily phosphatidylethanolamine, to form less polar derivatives and could alter membrane architecture. Thus, it can be conceived that the breakdown of triacylglycerols by triacylglycerol lipase occurs rapidly in the presence of mepacrine. By contrast, the stimulation of prostaglandin Ez release induced by triarachidonin was blocked by p-bromophenacyl bromide (0.1 mM). It has been shown that p-bromophenacyl bromide can inhibit phosphatidylinositol-specific phospholipase C from platelets, possibly by interaction with thiol groups (28, 30). Our data were interpreted as indicating that sulfhydryl groups were essential for the activation of triacylglycerol lipase in kidney medulla. A similar tendency was observed after treatment with p-chloromercuribenzoic acid, which reacts preferentially with thiol groups; the release of fatty acids from rabbit kidney cortical slices enhanced by epinephrine was blocked by pchloromercuribenzoic acid (16). The results of this work suggest the presence of triacylglycerol lipase and triacylglycerol as a possible candidate for providing free arachidonic acid to cy-

II

AND p-BROMOPHENACYL BROMIDE ON TRIARACHIDONIN-INDUCED RELEASE FROM RABBIT KIDNEY MEDULLA SLICES Prostaglandin

Treatment None Mepacrine (1.6 mM) p-Bromophenacyl bromide (0.1 mM)

AL.

No triarachidonin

Ea (pg/g

PROSTAGLANDIN

Ez

wet wt of tissue)

+50 WM triarachidonin

Difference

5.60 f 0.07 1.88 k 0.08”

6.67 + 0.12” 3.31 k 0.26“

1.07 1.43

4.26 i 0.28’

4.59 f 0.14

0.33

Note. Slices were incubated for 30 min at 3’7°C in 0.15 M KCl/0.02 M Tris/HCl buffer. Values are means f SE (n = 4). Effects of mepacrine and p-bromophenacyl bromide on triarachidonin-induced prostaglandin Ea release were determined as the difference in the amounts of prostaglandin Ea released in the presence and absence of triarachidonin. a Significantly different (P < 0.01) from control. b Significantly different (P < 0.02) from control. c Significantly different (P < 0.01) from mepacrine-treated value.

TRIACYLGLYCEROL

LIPASE

HYDROLYSIS

clooxygenase in kidney medulla. Also, a study of some properties of triacylglycerol lipase was performed. However, it does not necessarily support that the triacylglycerol lipase pathway plays a major role in arachidonic acid release for prostaglandin synthesis in kidney medulla. Under normal physiological conditions phospholipids seem to be a major source of substrate for prostaglandin biosynthesis. Direct deacylation of phospholipids by phospholipase A2 has been recognized as a favorable pathway, since this represents the simplest mode of release of arachidonic acid (23, 31). It is generally accepted that glucocorticoids, which are widely used for the treatment of nephrotic syndrome, induce the synthesis of phospholipase inhibitory proteins and cause reduction of prostaglandin synthesis in renomedullary interstitial cells and other cell types (32-34). It is against this background that recent studies have reported that the urinary excretion of prostaglandin E2 and prostaglandin Fz, increases in rats receiving glucocorticoids (35, 36). If glucocorticoids prevent the hydrolysis of phospholipids then there must be an alternative pathway for the synthesis of prostaglandins. With regard to this Erman et al. (37) have shown that chronic treatment with dexamethasone increases the level of unesterilied 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 the “phospholipase pathway” is blocked by glucocorticoids in certain pathological states, prostaglandin synthesis is diverted to the triacylglycerol lipase pathway. REFERENCES 1. VOGT, W. (1978) Adv. ProstaglwLdin Thrumboxane Res. 3, X9-95. 2. FLOWER, R. J., AND BLACKWELL, G. J. (19763 Bitr them. Pharmacol. Z&285-291. 3. ISAKSON, P. C., RAZ, A., DENNY, S. E., WYCHE, A.,

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4.

5. 6. 7. 8. 9. 10. II. 12. 13. 14.

15.

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

28.

29.

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KIDNEY

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AND NEEDLEMAN, P. (1977) Prostaglandins 14, 853-871. BELL, R. L., KENNERLY, D. A., STRANDFORD, N., AND MAJERUS, P. (1979) Proc. N&l. Acad. Sci. USA 76,3228-3241. BILLAH, M. M., LAPETINA, E. G., AND CUATRECASAS, P. (1981) J. Biol. Chem. 256,5399-5403. CRAVEN, P. A., AND DERUBERTIS, F. R. (1983) J. Biol. Chem. 258,4814-4823. FUJIMOTO, Y., AKAMATSU, N., HATTORI, A., AND FUJITA, T. (1984) Biochem. J. 218, 69-74. FUJIMOTO, Y., UNO, H., KAGEN, C., UENO, T., AND F~JITA, T. (1985) Biochem. J. 232, 625-628. FUJIMOTO, Y., TANIOKA, II., KESHI, I., AND FuJITA, T. (1983) Biochem. J. 212,167-171. BOJESEN, I. (1974) Lipids 9.835-843. COMAI, K., FARBER, S. J., AND PAIJLSRUD, J. R. (1975) Lipids 10,551-561. DANON, A., HEIMBERG, M., AND OATES, J. A. (1975) Biochim. Biophys. Acta 388, 318-330. FUJIMOTO, Y., AND FUJITA, T. (1982) Biochim. Biophys. Acfn 710, 82-86. JOUVENAZ, G. H., NUGTEREN, D. H., BEERTHUIS, R. K., AND VAN DORP, D. A. (1970) Biochim. Biophys. Acta 202, 231-234. YASUDA, M., FUJITA, T., HIGASHIO, T., OKAHARA, T., ABE, Y., ASD YAMAMOTO, K. (1980) Pflusers Arch. 385,111-116. FUJIMOTO, Y., SHIMIZU, K., ENMI, K., AND FUJITA, T. (1982) Biochim. Biophys. Actn 713,688-691. BIITCHER, R. W., AND SUTHERLAND, E. W. (1967) Ann. 1V. y Acad. Sci. 139, 849-859. JUNGAS, R. L., AND BALL, E. G. (1963) Biochemistry 2.383-388. SCHIMMEL, R. J., AND GOODMANN, H. M. (1972) Endocrinologq 90, 1391-1395. RITTENHOUSE, S. E. (1984) Biochem. J. 222, 103-110. SIMON, M. F., CHAP, H., AND DOUSTE-BLAZY, L. (1984) FEBS Left. 170,43-48. BROEKMAN, M. J., WARD, J. W.. AND MAR~IJS, A. J. (1980) J. C!in. Invest. 66, 275-283. MCKEAN, M. L., SMITH, J. B., AND SILVER, M. J. (1981) J. Biol. Chem. 256, 1522-1524. BILLAH, M. M., AND LAPETINA, E. G. (1982) J. Biol. Chem. 257, 5196-5200. AtJTHI, K. S., LAGARDE, M., AND CRAWFORD, N. (1985) FEBS Left. 180,95-101. CHAU, L. Y., AND TAI, H. H. (1981) Biochem. Biophys. Res. Commun. 100, 1688-1695. VALLEE, E., GOIJ~AT, J., NAVARRO, J., AND DELAHAYES, J. F. (1979) J. Pharm. Pharmacol. 31, 588-592. HOFMANN, S. L., PRESCOTT, S. M., AND MAJERUS, P. W. (1982) Arch. Biocham. Biophys. 215, 237-244. DISE, C. A., BURCH, J. W., AND GOODMAN, D. B. P. (1982) J. Rio/. Chem. 257, 4701-4704.

374

FUJIMOTO

30. KYGER, E. M., AND FRANSON, R. C. (1984) Bie chim

Biophys.

Acta

794,96-103.

31. RIITENHOUSE-SIMMONS, S., RUSSELL, F. A., AND DEYKIN, D. (1976) Biochem. Biophys. Res. Cornmun. 70,295-301. 32. CLOIX, J. F., COLARD, O., ROTHHUT, B., AND RUSSO-MARIE, F. (1983) Brit. J Ph,armacoL 79, 313-321. 33. HIRATA, F., SCHIFFMANN, E., VENKATASUBRAMANIAN, K., SALOMON, D., AND AXELROD, J. (1980) Proc. Na,tl. Acad. Sci. USA 77, 2533-2536.

ET

AL.

34. BLACKWELL, G. J., CARNUCCIO, R., DIROSA, M., FLOWER,R.J.,LANGHAM,C.S.J.,PARENTE,L., PERSICO, P., RUSSELL-SMITH, N. C., AND STONE, D. (1982) Brit. J. Pharmacol. 76, 185-194. 35. NASJLETTI, A., ERMAN, A., CAGEN, L. M., AND BAER, P. G. (1984) Endocrinology 114, 1033-1040. 36. RAMOS-FRENDO, B., ELOY, L., AND GRUNFELD, J. P. (1985) J. Hypertens. 3,461-467. 37. ERMAN,A.,BAER,P.G.,ANDNASJLETTI,A.(~~~~) .I BioL

Chem.

260.46’79-4683.