PROSTAGLANDINS

METABOLISM OF CYCLIC AMP IN ISOLATED RENAL TUBULES: EFFECTSOF PROSTAGLANDINS AND PARATHYROID HORMONE I M. G. Currle and D. M. Biddulph Department of Anatomy Bowman Gray School of Medicine Winston-Salem, North Carolina 27103 ABSTRACT Concentrations of cycllc AMP (cAMP) were in¢reased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E] (PGE]) with maximal effects of PGE1 belng 6 - 8 fold greater than those of PTH during a 10 min period. However, cAMP concentratlons in cells treated wlth ]-methyl-3-isobutylxanthlne (MIX) were increased with rnaxlrnal concentrations of elther hormone to the sarne degree. Sirnilar effects of both horrnones were observed on adenylate cy¢lase actlvity in renal homogenates. Sirnultaneous addition of hormones produced ¢hanges in both ¢AMP concentrations in intact tubules as weil as adenylate cyclase actlvlty of homogenates whi¢h were not cornpletely addltlve. Degradatlon of cAMP, estlmated in intact tubules as the dlfference in cAMP levels in the presence and absence of MIX, was increased by both horrnones, however, changes were 2 -3 fold greater in tubules exposed to PTH than to PGE]. Neither hormone dlrectly altered ¢AMP phosphodlesterase (PDE) actlvlty in either 30,000 x g supernatant or pellets frorn renal cortlcal hornogenates. The results suggest that both hormones increase the productlon of cAMP in renal cortlcal tubules and rnay share a cornrnon target cell type in this response. Degradation of cAMP, however, is differentlally effected by the two horrnones, probably reflectlng differences exerted on intracellular rnechanisrnsregulatlng the enzyrnatlc hydrolysls of cAMP.

1

This work was supported by a grant (to D.M.B.) from the National Science Foundatlon, PCM 75-07661.

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INTRODUCTION Adenylate cyclase, in a variety of cell types, has been reported to be stimulated by prostaglandlns (PG), partlcularly the E series, by interaction with speclfic membrane receptors coupled with the enzyme (1 -4). This effect on the adenylate cyclase--cAMP system is thought to mediate at least some of the physloIogical effects of these compounds (2), although in some tissues antagonistic effects of PGE1 on polypeptide-dependent cAMP accumulation have been reported (5, 6). In still other systems, PGE has been proposed as a required intermediate in polypeptide-mediated, cAMP formation (7, 8). Effects of PGE on cAMP metabolism in renal cortex and interactions with parathyroid hormone (PTH) on the cAMP system in this tissue have been controversial, with both antagonistic (9, 10) as weil as addltive (11) effects of the two hormones reported. The present study was designed to provlde additional information concerning the effects of these two hormones on the metabolism of cAMP utilizlng short term ~n vltro ~ncubations of isolated renal cortical tubules. MATERIALS AND METHODS Chernicals Bovlne parathyrold hormone (PTH) was purchased from W[Ison and Co., Chicago, III. (300 USP U/mg proteln). Dilutions of PTH were made wlth acldilied (pH 3.0) isotonic saline whlch also servêd as the control solutlon. Prostaglandin E1 (PGE1) was a gift from Dr. John Pike, Upjohn Co., and was dissolved in absolute ethanol. Desired concentratlons of PGE 1 were prepared by diluting the stock solution wffh incubation medla. Final concentrations of ethanol in the incubations did not exceed 0.4% and were duplicated in control incubations. All enzymes and nucleotides used in this study were purchased from Sigma Chemical Co. Anlon--exchange resin (AG1-X2)was purchased from BioRad Co. Reagents for assay of cAMP were purchased from Diagnostic Products Corp. Trltlated nucleotides and adenosine were purchased from New England Nuclear. Other chemlcals used were the best grades available and were obtained from standard suppllers. Isolated Renal Cortical Tubules Isolated renal cortical tubules were prepared by enzymatic dissociation of renal cortex as prevlously detailed (12, 13). Tubules, representing 1 - 1.5mg protein per flask were incubated in 5 ml sillconized, erlenmeyer flasks at 37°C in an oscillatory water bath in 1.5 ml Krebs-Ringer-Tris buffer, pH 7.4 containing 1 mM MgCI2, 1 mM CaCI2, 1.8 mg/tal glucose and 20 mM Tris-HCL. In some expeHments 1-methyl-3-isobutylxanthine (MIX), a potent inhibitor of cAMP phosphodiesterase (14)was added to obtaln final concentratlons deslgnated in the text. Parathyroid hormone or prostaglandins were added in .I ml volumes following a 5 min preincubatlon period. Control solutions conslsted of acld sallne and

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ethanol. Final concentrations of ethanol did not exceed 0.4%. Incubations were terminated by addition of 1 volume cold 5% TCA, followed by homogenization. Homogenates were centrlfuged at 1,000 x g and supernatants transferred to glass tubes, extracted 4 times wlth 10 volumes of ether, boiled to remove residual ether and lyophilized. Pellets were dried, resuspended in 1 N NaOH and assayed for proteln by either the method of Lowry et al. (15) or the bluret reactlon. /yophll ized supernatants were d issolved in Tr~'sbü'ffer, pH 7.4 and assayed for cAMP by the method of Gilman (16) as modlfled by Tovey et al. (17). Addltlons of known amounts ofcAMPadded to elther tissue extracts or TCA and carried through the entlre extraction procedure, were recovered quantitatlvely. Incubation of sample aliquots in excess phosphodiesterase for 60 min at 37°C resulted in complete hydrolysis of cAMP. Serial dilutlon of samples assayed for cAMP were linear over a 10 fold range. All samples were assayed in elther duplicate or trlplicate. In some experiments tubule suspenslons were rapidly centrifuged (1000 x g for 1 min) prior to addition of TCA. Cyclic AMP was assayed in both the tubule pellet and supernatant (medla) of ea¢h sample. Preparatio n and Assay 0.f Adeny_late Cyclase Washed partlcles from cortical homogenates were prepared for assay of adenylate cyclase activlty. Cortex was homogenized in 0.25 M sucrose, filtered through nylon and centrlfuged at 4,000 x g for 20 min at 2°C. The resulting pellet was washed with fresh sucrose and recentrifuged at the same temperature, speed and length of time as initially. Aliquots of the pellet (1.0 mg proteln/flask) were incubated at 37°C in 1.5 ml Krebs-Ringer-Tris buffer pH 7.4 containing ómM MgCI2, 1 mM ATP, 12mM theophylllne and an ATP-generatlng system (2.4mM phosphoenolpyruvate and 68 Mg,/ml pyruvate kinase). Other conditions were as described for tubule experlments. Preparation and Assay of cAMP Phosphodlesterase (PDE) PDE activity was measured in renal cortical homogenates by the two-step method of Thompson and Appleman (18). Renal cortlcal tissue was homogenized and sonicated in .25 M sucrose containing . 1M Tris and . 1 mM EDTA (pH 7.6). Homogenates were then centrifuged at 4°C at 30,000 x g for 30 min. Aliquots of both the supernatant and pellet were assayed in a buffer (0.4 ml total vol) containing 40mM Tris, (RH 8.0) 10mM MgCI2, 3.75mM mercaptoethanol, 0.1 mM or 4MM cyclic AMP, 3H- cyclic AMP ~60,000 cpm)and .2 ml of the enzyme preparation. Agents tested for effects on PDE actlvlty were added in 20 MI vol before the enzyme was added to initiate the reactlon. Incubations were for 5 min at 30°C wlth enzyme actlvity belng linear over this time period. Reactions were terminated by immersing reaction tubes into a dry ice-acetone bath for exactly 12 sec followed by boiling for 45 sec. Each tube was then incubated for 10 min at 30°C in the presence of excess 5' nucleotidase. Thls reactlon was terminated by addition of 1 ml of a 1: lslurry of BioRad anion--exchange resin (AGI-X2, 200-400 mesh)

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contalning 40% methanol as descrlbed by Thompson et al. (19). Recovery of adenoslne without methanol was 50-60% but increased to 90-95% after methanol addition. Bindlng of cAMP by the resin (95-98%) was not altered by the presence or absence of methanol. Contents of the tubes were centrifuged at 1,000 x g for 20 min and .4 ml aliquots of the supernatants were added to sclntillation vials and counted for radloactivity. Enzyme actlvities were determlned such that no more than 30% of the substrate was hydrolyzed durlng the reactlon. RESULTS Addition of elther PTH or PGE to incubations of isolated renal cortical tubules produced slgnificant (p< .01) increases in cAMP concentratlons (Fig. 1). Maximal amounts of elther hormone resulted in changes whlch were 6 fold greater with PGE 1 than with P T H . Concentrations of MIX between .01-SmM progresslvely increased ~he accumulation of cAMP in renal tubules exposed to maximal amounts of elther hormone (Fig. 1). Maximal effects of MIX on the PTH response (10 fold increase) was, however, considerably greater than w[th PGE 1 (2 fold increase). In experlments in whlch tubules were separated from media, 93-96% of the total cAMP was found to be associated wlth tubules dur[ng a 5 min incubation (data not shown). This percentage was not altered by either hormone elther in the presence or absence of 5mM MIX. Theophylline, however, a less potent inhlbitor of PDE in intact teils, significantly |ncreased the release of cAMP into medla with approxlmately 40% of the total cAMP, during a 5 min incubation, be|ng found in the media. Relative amounts of cAMP produced and degraded in |ntact renal tubules were estlmated for both hormonal responses by compar[ng cAMP concentratlons in the presence and absence of maximal concentrations of MIX (5 mM). Product|on of cAMP was equated wlth accumulatlon of thls nucleotide in the presence of MIX while degradatlon was estlmated as the dlfference in cAMP levels in the presence and absence of MIX. Estimated product|on of cAMP progressively |ncreased in respanse to both hormones durlng a 10 min perlod and did not dlffer quantitat|vely between hormones at any time interval examined (Fig. 2). Lossof cAMP, however/was at least 2 fold greater in renal tubules exposed to PTH than to PGE1 durlng the 10 min perlod (Fig. 2). Approx|mately 90-95% of the cAMP produced in response to PTH was degraded, as estimated by thls method, while only 30-40% of the cAMP produced by PGE 1 was degraded. Comparlson of productlon and degradat|on of cAMP in isolated tubules exposed to varylng concentratlons of PTH and PGE 1 are deplcted in Fig. 3. Both 1 and 5 min response intervals were examlned slnce« in the absence of MIX, PTH effects were essentially maximal by 1 min while 5 min was required to generate the maximal response to PGE 1 . At either tlme, maximal effects of these hormones on

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'/7

240

200

160

u)

120

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_

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c~ 80

40

w

ù

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0

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.01

I

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1.0

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Figure 1. Effect of 1-methyl-3-1sobutyl xanth[ne (MIX) on PTH (7U/ml; closed clrcles) and PGE1 (15 Isg/'ml; triangles) mediated increases in cAMP in isolated renal cortlcal tubules. Incubations were for 5 mln following addition of hormones. Open circles represent control tubules. Points are means of 6 - 9 samples wlth vertical bars representlng SE.

productlon of cAMP ( .7 U/ml, PTH; 15~g/'ml, PGE1) dld not differ signiflcantly from each other. Degradat|on of cAMP, however, was aga|n 2 -3 fold higher in tubules exposed to PTH than to PGE 1 . increase in the degradatlon of cAMP in response to PTH occurred over the same range of concentrafions (.007- .7 U/ml) whlch produced increases in productlon of cAMP and were about 90-95% of the

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PROSTAGLANDINS cAMP produced at elther time [nterval. In contrast to the effects of PTH, no signlficant change in degradation of cAMP in response to PGE1 was observed at 1 min desplte [ncreases in the productlon of cAMP by as rauch as 10 fold. At 5 min, degradation of cAMP was progressively increased by concentrations of PGE 1 which also increased cAMP production (.01-15 t~/ml) but remained signlficantly (p< .01) Iower, for a g[ven change in cAMP produced, than in PTH-treated tubules.

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200

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Figure 2. Effects of PTH (7U/ml) and PGE 1 (15Hg/ml) on accumulation and Ioss of cAMP in |solated renaJ cortical tubules. Accumulatlon of cAMP was measured in the presence of 5 mM MIX wh|le Ioss of cAMP was calculated as the dlfference in CAMP concentrations in the presence and absence of MIX. Open symbols represent changes in control incubat[ons. Concentrations of cAMP in the absence of MIX are the differences between accumulatlon and Joss at any part|cuJar intervaJ. Points are means of 3 - 6 samples + SE or SE of the dlfference

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Figure 3. Effects of PTH and PGE on accumulatlon and Ioss of cAMP in isolated renal cortical tubules. Incubations were for either 1 (upper panels) or 5 (Iower panels) min following addition of hormones at indicated concentrations. Accumulation and Ioss of cAMP were as described in Fig. 2. Points are means of 3 - ö samples + SE or SE of the difference Since these results suggested approximately equal maximal effects of PTH and PGE 1 on production of cAMP, hut accompanying effects on degradation of the nucleotlde which were markedly different, effects of both hormones on PDE and adenylate cyclase (AC) act[vltx were measured directly in renal homogenates. The hlghest PDE activ[ty as measured at both high (100WM) and Iow (4 I.g~A) cAMP ¢oncentrations was Iocated within the cxtosolic (30,000 x g supernatant) fraction of

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renal homogenates being 4 - 9 fold greater than in the accompanying (30,000 x g) pellet (Table 1). The cytosolic enzyme was stimulated by both imldazole and c GMP while both partlculate and cytosollc enzymes were rnaximally inhiblted (90- 100%) by 0.5mM MIX (Table 1). Neither PTH (7U/tal) or PGE 1 (15Mg/tal) produced significant changes in e[ther the cytosolic or partlculate enzyme actlvity at either high (100BM) or Iow (4MM) substrate concentrations of cAMP (Table 1). Preincubation of intact tubules for 5 min with maximal concentrations of either hormone also had no effect on PDE activity assayed in either particulate or cytosollc fractlons (data not shown). Table 1. Effects of parathyrold hormone (PTH) and prostaglandin E1 (PGE 1) on cAMP phosphodlesterase (PDE) activity in renal cortical homogenates.

Treatment

Control PTH (7M/ml) PGE 1(15Mg/ml )

PDE Activity (nmoles/min/mg proteln) 4 FM cAMP 100 pM cAMP Cytosol Pellet Cytosol Pellet 0.50 _ .01 0.46 -I- .02 0.54+_ .03

0.07 4- .01 0.06 + .01 0.05_+ .01

3.6 -I- 0.2 3.5 + 0.3 3.5_+ 0.1

1.0 + 0.1 1.0 -I- 0.I 1.1_+ 0.2

MIX (0.5 mM) Imidazole (40mM)

0.4 -I- 0. I* 4 . 8 + 0.1"

undetected 1.2-1- 0.1

cGMP (5HM)

4.5_. 0.1"

1.1_+ 0.2

Incubations were for 5 min following addition of enzymes and substances at concentratlons deplcted. Enzyme was prepared from 30,000 xg supernatants (cytosol) or pellets and assayed at substrate (cAMP) concentratlon of either 4 or 100 MM. Values are means of tripllcate determinations __. SE. *Values differlng significantly (p< .01) from controls. In contrast, both hormones slgnlflcantly (p< .01) stimulated AC activity by about 4 fold (Table 2). Simultaneous addition of both hormones at maxlmal concentratlons resulted in an increase beyond either hormone alone but only about 20% of the expected increase was obtained assuming completely additive responses. Simultaneous addition of both hormones to intact tubules resulted in cAMP levels which were significantly less than with PGE 1 alone in the absence of MIX (Table 2). In the presence of 5mM MIX, the combined response was signiflcantly greater than with elther hormone alone, but only 40-50% of the expected increase was obtained assumlng completely addltive responses. These results resembled those from comparable experiments usmg adenylate cyclase.

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PROSTAGLANDINS Table 2. Effects of parathyroid hormone (PTH) and prostaglandin EI(PGE 1) on adenylate cyclase (AC) activity Tn renal homogenates and cAMP accumulatlon in intact renal tubules. Treatment

Control

AC Act|vlty pmoles/min/mg protein

28"1" 1

cAMP Accumulation pmoles/5mln/mg protein + MIX - MIX 11_+ 0.3

3 ~ 0.2

NaF (10mM)

223 4- 2*

PTH (7U/ml)

107± 3*

300_.+ 11.1"

204- 0.8*

PGE1(15Hg/ml )

103_.+ 2*

309-1- 9.2*

1604- 2.1"

PTH 7U/ml + PGE 1 15Hg/ml

127_ 3*

453 4- 13.1"

142___ 1.8"

Incubatlons were for 5 min followlng add|tion of enzyme or renal tubules. Test substances were at concentmtlons depicted. AC act|vlty was assayed in 4,000 xg pellets. Concentrations of cAMP were measured in intact renal tubules in the presence (+) or absence 4-) of 5 mM 1-methyl-3-isobutylxanthine (MIX). Values are means of 3 -6 determinat|ons + SE. Values dlfferlng slgnificantly (p < .01)from control value.

DISCUSSlON

The present study demonstrates that both PTH and PGE 1 increase concentrations of cAMP in isolated renal cortical tubules from hamsters, confirm|ng observat|ons of others in renal tissue from other spec[es 410, 11). In the hamster PGE 1 induced increases in cAMP were observed to be 6 - 8 fold greater than with PTH. These quantltative differences between PTH and PGE 1 appear to be just opposite to that reported in rat renal t|ssue 410) in which effects of PGE 1 on cAMP levels in renal sllces, at concentrat|ons comparable to those of the present study, produced changes which were considerably less than with maximal amounts of PTH. The larger effe¢ts of PGE 1 than PTH on cAMP levels in hamster renal tubules, appear to be due to relative differences in the amounts of cAMP degraded wlthln the tubule teils which accompany effe¢ts of both hormones on cAMP synthesis. Maximal effects of PTH and PGE 1 on both adenylate cyclase, as weil as accumulat[on of cAMP in intact cells in the presence of maximal concentratlons of the phosphod|esterase inhibitor MIX, were quantltat[vely similar. However, degradation of cAMP, as evaluated in inta¢t cells by the dlfference in cAMP levels in PDE-inhib|ted and noninhlblted tubules clearly differed between tubules exposed to PTH and PGE 1 . Degradatlon of cAMP |n tubules exposed to PTH increased in d|rect relationshlp

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PROSTAGLANDINS

with increases in the productlon of cAMP, as e,Adenced fr0m both time--course and dose-response experiments wlth about 9 0 - 9 5 % of the cAMP produced in response to PTH being degraded before its accumulation could be detected. In tubules exposed to PGE 1, degradatlon of cAMP was conslstently less than with PTH uncler conclitions where no dlfferences existed in effects of the two hormones on procluctlon of cAMP. Thls was clearly demonstrated in kinetic experirnents using maximal concentrations of each hormone, as weil as dose-response experiments in whlch cAMP clegradatlon could be compared under conclltlons where product[on of cAMP, in response to a glven amount of either hormone, was equal. These apparent differences in the degradatlon of cAMP observed in intact renal tubule cells shoulcl reflect comparable clifferences in cAMP-PDE activity, although it is clear from the present study that dlrect effects of either hormone on elther the soluable or partlculate form of the enzyme do not occur. Distributlon of cAMPPDE actlvlty in renal cortex from hamsters resemblecl that reportecl in the rat (14) wlth about 8 0 - 9 0 % of the total activlty detected in the soluable (cytosolic) fractlon. The enzyme, as has been prevlously reportecl (14, 20, 21) was stlmulated by both imldazole and cGMP and was inhlbltecl up to 90% by .SmM MIX. Lack of dlrect effects of elther hormone on PDE activity has also been observed by others (10, 22, 23) and suggests that some other intracellular factor, whlch is presumably differentially affected by PTH and PGE I is regulating PDE act[vity. Intracellular calcium, whlch we have previously shown to be altered in thls system by PTH (12) and which is known to regulate PDE activlty in some tissues (24, 25) may be involvecl. Studles to test this hypothesis are currently in progress. The present work also suggests that a common cel l type withln renal cortical tubules may be responsive to both PTH and PGE 1 . Production of cAMP, as measured in either homogenates (AC actlvlty) or in intact cells in the presence of MIX d[d not clernonstrate summatecl responses to slmultaneous aclclitlon of both hormones at maximal concentrations. In cel ls without PDE inhlbltion, the combined response was clearly less than that with PGE 1 alone. Thls dimlnlshed response to PGE 1 in the presence of PTH may be refle¢ting the differential effects of these hormones on degradation of cAMP. Further work is in progress to more completely deflne the nature of PTH-PGE I interactlons on the adenylate cyclase system. ACKNOWLEDGEMENT The authors are indebted to Nancy Wood for expert technical asslstance and to Mrs. Mary Frances Stubbs for secretarial assistance.

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REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17)

Samuelsson, B., E. Granstrom, K. Green, M. Hamberg, and S. Hammarstr~m. Prostaglandins. Ann. Rev. Biochem. 44: 898, 1975. Kuehl, F. A,, Jr. Prostaglandlns, cyc-'c'Ticnucteotides and cell functlon. Prostaglandins 5: 325, 1974. Brunton, L. L.-~-R. W. Wiklund, P. M. VanArsdale, and A. G. Gilman. Binding of 3H-prostaglondin E1 to putotive receptors I inked to adenylate cyclase of cultured cell clones. J. Biol. Chem. 251: 3037, 1976. Lin, M. T. and C. V. Rao. 3H-prostaglandin binding to dispersed bovine luteal cells: evidence for discrete prostaglandin receptors. Biochem. Biophys. Res. Comm. 78: 510, 1977. Dunn, M. J. and V. L-'~-Hood. Prostaglandins and the kidney. Am. J. Physiol. 233: F169, 1977. Butcher, R. W. and C. E. Baird. Effects of prostaglandins on adenoslne 3',5'-monophosphate levels in fat and other tissues. J. Biol. Chem. 243: 1713, 1968. Kuehl, F. A., Jr., J. L. Humes, J. Tarnoff, V. J. Cirillo and E. A. Harn. Prostaglandin receptor site: evidence for an essential role in the actlon of luteinizing hormone. Science 169: 88, 1970. Sato, S., M. Szabo, K. Kowals"l