203
Biochimica et Biophysica Acta, 421 (1976) 203.--..209
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27822
E F F E C T OF P R O S T A G L A N D I N El ON THE ADENYL CYCLASE-CYCLIC AMP SYSTEM AND GLUCONEOGENESIS IN RAT R E N A L CORTICAL SLICES
AUBREY R. MORRISON, JESSE YATES and SAULO KLAHR Renal Division, Department of Internal Medicine, Washington University School of Medicine, 4550 Scott Avenue, St. Louis, Mo. 63110 (U.S.A.)
(Received July 21st, 1975)
Summary Prostaglandin E, was found to increase the formation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) by renal cortical slices. This increased release of cyclic AMP was not influenced by the absence of Ca 2÷ in the incubating media. The enhanced production of cyclic AMP was probably mediated by stimulation of membrane-bound adenylate cyclase activity. An increase in adenyl cyclase activity was observed with increasing concentrations of prostaglandin El. Furthermore, prostaglandin E, augmented glucose production from a-ketoglutarate. This effect on gluconeogenesis was abolished by the removal of Ca 2÷ from the incubating medium. These effects are similar to those described for parathyroid hormone and suggest that the renal cortex is a prostaglandindependent system. Prostagiandin E 1 decreased cyclic AMP production and glucose production (from a-ketoglutarate) in response to submaximal doses of parathyroid hormone, suggesting that prostagiandin may be important in modulating the intracellular action of parathyroid hormone in the kidney cortex.
Introduction The prostaglandins are long-chain polyunsaturated fatty acids that affect, at very low concentrations, several aspects of mammalian cell function. The presence within the renal medulla of a prostaglandin-synthesizing enzyme system (prostaglandin synthetase) was demonstrated by Hamberg [1 ]. Compared with renal medulla the renal cortex has much less biosynthetic activity [2]. However, the activity to synthesize prostaglandins in the cortex cannot be assumed to be negligible since the activity in the medulla is very high, surpassed only by that of seminal vesicles. The finding that the prostaglandins affect adenyl cy-
204
clase activity in a number of different mammalian tissues [3] and are synthesized in situ and rapidly degraded in the circulation, has led many authors to suggest that they may act as intracellular modulators of the "classical" hormones [4]. Lipson and Sharp [5] have shown that prostaglandin E, can increase Na ÷ transport and osmotic water flow in the toad bladder. Grantham and Orloff [6] demonstrated using the isolated perfused renal tubule that prostaglandin E, increased net water movement along an osmotic gradient but it blunted the response to submaximal concentrations of antidiuretic hormone. Beck et al. [7] have examined the effect of prostaglandin E, in renal cortex. They were unable to demonstrate an increase in adenylate cyclase activity by prostaglandin E~, however, at concentrations of 1 0 - 4 M prostaglandin E, blunted the cyclic AMP response to 1 unit/ml of parathyroid hormone. The present experiments were designed to reexamine the effect of prostaglandin E, on the adenyl cyclase-cyclic AMP system of renal cortex. The effect of prostaglandin E, on gluconeogenesis, using a-ketoglutarate, fructose and glycerol as substrates, was also examined. Materials and Methods
Measurements of cyclic AMP in cortical slices Adult Holtzman rats weighing 200--300 g were killed by a blow on the head and the kidneys rapidly removed and placed in ice-cold Krebs-Ringer phosphate buffer, pH 7.4. Food was not withheld prior to the experiment. Cortical slices were obtained using a Stadie-Riggs microtome. They were then incubated for 1 h at 37°C in Krebs-Ringer buffer of the following composition in mmol/l; Na*, 130; K ÷, 4; Ca :+, 1; Mg 2÷, 1.4; CI-, 136; PO~-, 2.4. Similar experiments were also run with 0 mM Ca :+ in the incubating medium. The buffer also contained glucose, 10 mM; theophylline, 10 -2 M, and lyophilized bovine serum albumin, 0.25%. After this pre-incubation period, slices weighing 15--30 mg were added to 1 ml of Krebs-Ringer phosphate buffer containing 10 mM glucose, 10 -2 M theophylline and 0.25% bovine serum albumin. In the experimental flasks three concentrations of prostaglandin E, were used, 10 -9, 10 -~ and 10 -s M. In addition, another flask containing parathyroid hormone, 1 unit/ml, was used. The slices were incubated for 20 min at 37°C in a D u b n o f f metabolic shaker using a gas mixture of CO2/O: (5 : 95, v/v). The reaction was terminated by removing the slices and quickly freezing them in freon. They were then placed into boiling 50 mM sodium acetate/acetic acid buffer, pH 4, for 10 min. They were homogenized by hand using a glass homogenizer; an aliquot was removed for protein determination and the remainder was centrifuged at 5000 × g and the supernatant assayed for cyclic AMP. The incubation media were also assayed for cyclic AMP and the total cyclic AMP produced per mg of tissue protein was calculated. Cyclic AMP was measured by a modification of the method of Gilman [8]. All measurements were done in duplicate and the mean of each pair of values used. Protein determinations were done by the method of Lowry et al. [9].
205
Adenyl cyclase assay For the adenyl cyclase assay the membranes were prepared by the method of Marcus and Aurbach [10] and stored in small aliquots at --70°C. Adenyl cyclase activity was measured by the method of Salomon et al. [ 1 ]. Both the Dowex columns (Dowex 50AG W-X8) and the alumina columns (Aluminum Oxide, Woelm neutral) were reused five times. Recycling of Dowex columns. At the completion of each experiment 2 ml of I M HC1 were added to each Dowex column and the column stored with no further treatment. Prior to reusing, the columns were washed with 10 ml of distilled water. Reuse of Alumina columns. Prior to reuse, the alumina columns were washed with 8 ml 0.1 M imidazole • HCI 7.5.
Measurements of gluconeogenesis Kidney slices weighing 10--25 mg were obtained after killing 2 0 0 - 3 0 0 g Holtzman rats by a blow on the head. These slices were pre-incubated in substrate-free Krebs-Ringer phosphate buffer, pH 7.4, for 1 h prior to the experiment. At the end of the hour, slices were transferred to 1 ml of KrebsRinger buffer, pH 7.4, containing either 10 mM a-ketoglutarate, 10 mM fructose or 10 mM glycerol, or no substrate. Experimental flasks contained prostaglandin E~ at a concentration of 10 -6 M. Control flasks contained alcohol at the same concentration as that present in the flasks with prostaglandin E1 since the prostaglandin El stock solution was made up in absolute alcohol at a concentration of 10 mg/ml. Slices were, therefore, incubated without substrate, with substrate, with substrate and prostaglandin Et, and with substrate and alcohol. After 1 h of incubation an aliquot of the incubation medium was obtained and assayed for glucose. The slice was then removed, blotted gently and weighed. Glucose was assayed by a fluorimetric technique using the hexokinase method [12]. All measurements were done in duplicate. Reagents. 3H-labeled cyclic 3',5'-AMP was purchased form New England Nuclear (Boston, Mass.) and [a-32P]ATP from International Chemical and Nuclear Corp. Parathyroid hormone was from Beckman, Bovine Parathyroid Hormone Synthetic 1-34, Lot No. 26013. Prostaglandin was a gift from Dr. John Pike, Upjohn Co., Kalamazoo, Mich. (PGEI, V-10136, Lot No. 10315-VDV-115). Results are expressed as mean -+S.E. Statistical analyses were performed using Student's t-test for paired data. Results Table I summarizes the data on cyclic AMP production by renal cortical slices in the presence of 1 mM Ca 2+ and in the absence of external Ca 2+. It can be seen that prostaglandin El increased cyclic AMP production in a dosedependent fashion at prostaglandin E1 concentrations of 10 -9 to 10 -s M which was the highest concentration tested. Control values for cyclic AMP were 15.2 + 2.0 pmol/mg protein per 20 min; 25.3 -+ 1.8 pmol/mg protein with 10 -9 prostaglandin El (P < 0.001), with increasing concentration of prostaglandin E~, cyclic AMP production increased to 33.0 _+ 2.8 and 48.0 _+ 4.9, P < 0.02.
206 By comparison, with 1 unit/ml of parathyroid hormone, cyclic AMP values were 67.8 + 5.9 pmol/mg protein per 20 min. Since the prostaglandin E, was dissolved in absolute alcohol at a concentration of 10 mg/ml, and there is evidence suggesting that alcohol can increase adenylate cyclase activity in rat liver and kidney plasma membranes [13], we incubated kidney slices with 0.35% alcohol, an amount equivalent to the alcohol content present in the flasks when 10 -s M prostaglandin EI was used. This a m o u n t of alcohol did not increase cyclic AMP production significantly. The production of cyclic AMP by cortical slices was unaffected by omission of Ca 2÷ from the external medium. The increase in cyclic AMP production by prostaglandin E, was similar to the effects seen in the presence of Ca 2÷. In an attempt to establish that indeed cyclic AMP was being measured, additional experiments were done in which the aliquots to be assayed were exposed to phosphodiesterase prior to assay. Secondly, a known quantity of cold cyclic AMP was added to the medium prior to assay and then assayed for cyclic AMP by a modification of Gilman's assay. The results indicated that indeed cyclic AMP was the substance being measured. Table II shows the effect of prostaglandin El on renal cortical adenylate cyclase activity. Control values for renal cortical cyclase averaged 215 _+ 12 pmol/mg protein per 10 min. Addition of 10 -9 M prostaglandin E, increased adenylate cyclase activity to 309 +- 15 pmol (P < 0.01). Prostaglandin EI at concentrations of 10 -s M produced a further increase in adenylate cyclase activity to 381 -+ 14 pmol/mg protein per 10 min. These results demonstrate increasing adenylate cyclase activity with increasing concentrations of prostaglandin El. The corresponding values for parathyroid hormone are shown for comparison. The values with parathyroid hormone averaged 716 +- 34 pmol/mg protein per 10 min. Thus, 5 units/ml parathyroid hormone increased the activity of renal cortical adenylate cyclase more than 3-fold. Parathyroid hormone has been shown to increase renal gluconeogenesis [14,15] presumably by increasing cyclic AMP levels. Since prostaglandin E, had similar effects on renal cortical cyclic AMP production and adenylate cyclase activity, we examined the effect of 10 -6 M prostaglandin E~ on glucose production by renal cortical slices using 10 mM a-ketoglutarate as substrate. These results are summarized in Table III. In the absence of substrate, glucose TABLE I EFFECTS OF PROSTAGLANDIN E l AND PARATHYROID HORMONE ON CYCLIC AMP PRODUCTION IN THE PRESENCE AND ABSENCE OF Ca2+IN THE INCUBATING MEDIUM VMues
are the
mean
-+ S . E .
of six experiments
in the
case
of
1 mM
external
Ca 2+and
four
with n o external C a 2+.
Cyclic A M P I mM
produced/rag protein per 2 0 m i n
external C a 2+
N o external C a 2+
Control Prostaglandin
E l , 10 -9 M
;.5.2 ± 2.0 2,5.3 +- 1.8
14.0 -' 1.7 23.0 *- 1.0
Prostaglandin
E l , 10 -7 M
33.0
± 2.8
30.7
-+
4.4
Prostaglandin
E l , I0 -S M
48.0
± 4.9
45.7
-~
4.1
6 7 . 8 ~: 5 . 9 ..................................................
82.0
~ 15.0
Parathyroid
hormone,
I unit/ml
experiments
207 TABLE
II
EFFECTS OF PROSTAGLANDIN TIVITY OF RENAL CORTICAL
E l AND PARATHYROID MEMBRANES
HORMONE
ON ADENYLCYCLASE
AC-
V a l u e s are t h e m e a n +- S . E . o f f o u r e x p e r i m e n t s pmol of cyclic AMP formed/mg protein per 10 min Control Prostaglandin El, 10 -9 M Prostaglandin El, 10 -2 M Parathyroid hormone, 5 units/ml
215 309 381 716
± -+ ± ±
12 15 14 34
production was 1.87 ± 0.48 pmol/g wet weight per h. Addition of 10 mM a-ketoglutarate increased glucose production to 11.9 + 0.77 pmol/g wet weight per h. Prostaglandin El at a concentration of 10 -6 M increased glucose production further to 13.5 ± 0.59, a value significantly different (P < 0.05) from that obtained with a-ketoglutarate alone. Using alcohol at the same concentration as that present in 10 -6 M prostaglandin E~ the value averaged 11.50 + 0.69 which was not significantly different from control. These experiments were done at a concentration of 0.25 mM Ca 2÷ in the incubation medium. Omission of Ca 2÷ from the incubation medium decreased baseline glucose production to 9.47 ± 0.82 pmol/g wet weight per h and addition of 10 -6 M prostaglandin El caused no increase in glucose production, 9.42 -+ 0.88 pmol/g wet weight per h. The parathyroid hormone-induced increase in gluconeogenesis, produced by an increase in cyclic AMP, is presumably mediated by stimulating the activity of the key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase. Pagliara and Goodman [16] have shown that there is no increase in glucose production induced by cyclic AMP when fructose and glycerol are used as substrates. When we tested the effects of prostaglandin El on glucose production from fructose and glycerol, we found that the values for glucose production in the absence and presence of 10-6M prostaglandin El were not significantly different. Glucose production from glycerol and fructose averaged 6.0 -+ 0.9 and 71 ± 8.1 pmol/g wet weight per h, respectively. In the presence of 10 -6 M prostaglandin the values were not significantly different and averaged 6.3 ± 0.4 and 78.9 -+ 6.7 ~mol/g wet weight per h, respectively.
TABLE
Ill
EFFECTS OF PROSTAGLANDIN THE PRESENCE AND ABSENCE
E l ON GLUCOSE PRODUCTION O F E X T E R N A L C a 2+
FROMa-KETOGLUTARATE
IN
V a l u e s are t h e m e a n ± S . E . o f e i g h t e x p e r i m e n t s . Glucose production (~mol/g wet wt per h) 0 . 2 5 m M e x t e r n a l C a 2+ No substrate
N o e x t e r n a l C a 2+
1 . 8 7 -+ 0 . 4 8
1 . 6 9 -+ 0 . 1 5
10 mM c,-ketoglutarate
11.9
± 0.77
9.47 ± 0.82
~ - K e t o g l u t a r a t e + 1 0 -6 M p r o s t a g l a n d i n E I
13.5
*- 0 . 5 9
9.42 ± 0.88
~-Ketoglutarate + alcohol diluent
11.5
~ 0.69
8.98 ± 0.85 .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
208 Additional experiments demonstrated a decreased stimulation of glucose production by submaximal doses of parathyroid hormone when 10 -6 M prostaglandin E~ was added. Parathyroid hormone, 1 unit/ml, increased glucose production from 9.4 -+ 0.5 pmol glucose/mg protein per h to 11.2 + 0.5 pmol glucose/mg protein per h. 10 -6 M prostaglandin E~ increased glulucose production to 12.0 + 0.4. Addition of parathyroid hormone, 1 unit/ml, and prostaglandin E~, 10 -6 M, increased glucose production to 10.3 -+ 0.4, a value which was not statistically different from control but statistically different from parathyroid hormone alone (P < 0.05). Discussion
These results demonstrate that the renal cortex responds to prostaglandin E 1 with an increase in cyclic AMP production. An increase in cyclic nucleotide production by prostaglandins has been demonstrated in whole fat pads, lung, spleen [3], thyroid [17], ovary [18], and renal medulla [19]. Beck et al. [7] did not find an increase in cyclic AMP production in renal cortical slices exposed to 10 .4 M prostaglandin El. However, they showed that prostaglandin E~ significantly reduced the cyclic AMP generation produced by submaximal concentrations of parathyroid hormone. Our results demonstrate an increase in cyclic AMP production with increasing concentrations of prostaglandin E~, and we have confirmed the decrease in cyclic AMP production in response to a submaximal concentration of parathyroid hormone when prostaglandin E~ is added. The finding that the increase in cyclic AMP generation by prostaglandin E was not influenced by removal of Ca 2÷ from the external medium in the present experiments is similar to the increase in cyclic AMP by prostaglandin E~ observed by Yamasita et al. [17] in thyroid slices and similar to the effects of parathyroid hormone on cyclic AMP generation in renal cortical tubules [15]. This increase in cyclic AMP is probably a result of membrane-receptor interaction with stimulation of adenylate cyclase activity as demonstrated by increased activity of this enzyme with increasing concentrations of prostaglandin E~. We were unable to show a significant effect of prostaglandin El on the stimulation of adenyl cyclase produced by 5 units/ml of parathyroid hormone. This is in contrast to the findings of Beck et al. [7] who showed that prostaglandin E~ significantly decreased the activation of adenylate cyclase activity by submaximal doses of parathyroid hormone. However, since we are dealing with a broken cell preparation and the adenylate cyclase activity may be modified by a number of factors, including Mg/ATP ratios [ 1 0 ] , concentration of Mg 2÷ in the external medium [20] and the levels of ATP and other nucleotides, viz. GTP [20], GMP, the conditions of our experiments may not have been optimal for the demonstration of an effect of prostaglandin E~ on the parathyroid hormone activation of adenylate cyclase. Prostaglandin E~ produced the same metabolic changes as those demonstrated for parathyroid hormone by Nagata and Rasmussen [15] in renal cortical tubules and by Kurokawa et al. [14] in isolated renal tubules. Prostaglandin E~ increased glucose production from a-ketoglutarate. Omission of Ca 2÷ from external media lowered baseline glucose production and completely blocked
209 any further stimulation in response to prostaglandin El. Furthermore, glucose production from glycerol and fructose was not increased by prostaglandin E1 suggesting that the effect of prostaglandin E, was at the level of the key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase. Cyclic AMP has been shown by Pagliara and Goodman [16] to increase renal gluconeogenesis from a-ketoglutarate and glutamine but not from fructose and glycerol. This would then suggest that prostaglandin El, like parathyroid hormone, increases gluconeogenesis by increasing the intracellular content of cyclic AMP. Finally, the increase in gluconeogenesis produced by submaximal concentrations of parathyroid hormone was decreased in the presence of 10-6M prostaglandin El. In view of these observations, it is suggested that prostaglandin E, may play a physiological role in the renal cortex by modulating the response to parathyroid hormone.
Acknowledgments This work was supported by U.S.P.H.S. NIAMD Grants AM-05248 and AM09976. During the course of this study, Dr. Morrison was the recipient of fellowships from the National Kidney Foundation and the Kidney Foundation of Eastern Missouri and Metro-East. The authors wish to express their appreciation to Mr. Orlando Moncada for his technical assistance and to Mrs. Patricia Verplancke for her secretarial assistance.
References 1 2 3 4 5 6 7 8 9 I0 11 12 13 14 15 16 17 18 19 20
H a m b e r g , M. ( 1 9 6 9 ) F E B S L e t t . 5, 1 2 7 L a r s s o n , C. a n d A n g g a r d , E. ( 1 9 7 3 ) E u r . J. P h a r m a c o l . 2 1 , 3 0 B u t c h e r , R.W. a n d R a n d , C.C. ( 1 9 6 8 ) J . Biol. C h e m . 2 4 3 , 1 7 1 3 K u e h l , Jr., F . A . ( 1 9 7 3 ) P r o s t a g l a n d i n s a n d C y c l i c A M P ( K a h n , R . H . a n d L a n d s , W.E.M., eds.), p. 2 2 3 , A c a d e m i c Press, New Y o r k L i p s o n , L.C. a n d S h a r p , G . W . G . ( 1 9 7 1 ) A m . J. P h y s i o l . 2 2 0 , 1 0 4 6 G r a n t h a m , J . J . a n d O r l o f f , J. ( 1 9 6 8 ) J. Clin. Invest. 4 7 , 1 1 5 4 B e c k , N., D e R u b e r t i s , F . R . , Michelis, M . F . , F u s c o , R . D . , Field, J . B . a n d Davis, B. ( 1 9 7 2 ) J. Clin. Invest. 51, 2 3 5 2 G i l m a n , A . G . ( 1 9 7 0 ) P r o c . Natl. A c a d . Sci. U.S. 6 7 , 3 0 5 L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 M a r c u s , R. a n d A u r b a c h , G . D . ( 1 9 6 9 ) E n d o c r i n o l o g y 8 5 , 801 S a l o m o n , Y., L o n d o n , C. a n d R o d b e l l , R. ( 1 9 7 4 ) A n a l . B i o c h e m . 58, 5 4 1 L o w r y , O . H . a n d P a s s o n n e a u , J . V . ( 1 9 7 2 ) A F l e x i b l e M e t h o d o f E n z y m a t i c A n a l y s i s , p. 1 7 4 , A c a d e m ic Press, N e w Y o r k M a s h i t e r , K. a n d Field, J . G . ( 1 9 7 4 ) Fed. P r o c . 3 3 , 7 8 K u r o k a w a , Y., O h n o , T. a n d R a s m u s s e n , H. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 3 , 32 N a g a t a , N. a n d R a s m u s s e n , H. ( 1 9 7 0 ) B i o c h i m . B i o p h y s . A c t a 2 1 5 , 17 Pagliara, A.S. a n d G o o d m a n , D . A . ( 1 9 6 9 ) J. Clin. Invest. 4 8 , 1 4 0 8 Y a m a s i t a , K., B l o o m , G. a n d Field, J . B . ( 1 9 7 1 ) E n d o c r i n o l o g y 2 0 , 9 4 3 K u e h l , J r . , P.A., H u m e s , J . L . , T a r n o , H . J . , Cirino, V.J. a n d H a m , E . A . ( 1 9 7 0 ) S c i e n c e 1 6 9 , 8 8 3 B e c k , N.P., K a n e k o , T., Z o r , U., Field, J . B . a n d Davis, B.B. ( 1 9 7 1 ) J. Clin. Invest. 50, 2 4 6 1 B i r n b a u m e r , L. ( 1 9 7 3 ) B i o c h l m . B i o p h y s . A c t a 3 0 0 , 1 2 9
Biochimica et Biophysica Acta, 421 (1976) 203.--..209
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27822
E F F E C T OF P R O S T A G L A N D I N El ON THE ADENYL CYCLASE-CYCLIC AMP SYSTEM AND GLUCONEOGENESIS IN RAT R E N A L CORTICAL SLICES
AUBREY R. MORRISON, JESSE YATES and SAULO KLAHR Renal Division, Department of Internal Medicine, Washington University School of Medicine, 4550 Scott Avenue, St. Louis, Mo. 63110 (U.S.A.)
(Received July 21st, 1975)
Summary Prostaglandin E, was found to increase the formation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) by renal cortical slices. This increased release of cyclic AMP was not influenced by the absence of Ca 2÷ in the incubating media. The enhanced production of cyclic AMP was probably mediated by stimulation of membrane-bound adenylate cyclase activity. An increase in adenyl cyclase activity was observed with increasing concentrations of prostaglandin El. Furthermore, prostaglandin E, augmented glucose production from a-ketoglutarate. This effect on gluconeogenesis was abolished by the removal of Ca 2÷ from the incubating medium. These effects are similar to those described for parathyroid hormone and suggest that the renal cortex is a prostaglandindependent system. Prostagiandin E 1 decreased cyclic AMP production and glucose production (from a-ketoglutarate) in response to submaximal doses of parathyroid hormone, suggesting that prostagiandin may be important in modulating the intracellular action of parathyroid hormone in the kidney cortex.
Introduction The prostaglandins are long-chain polyunsaturated fatty acids that affect, at very low concentrations, several aspects of mammalian cell function. The presence within the renal medulla of a prostaglandin-synthesizing enzyme system (prostaglandin synthetase) was demonstrated by Hamberg [1 ]. Compared with renal medulla the renal cortex has much less biosynthetic activity [2]. However, the activity to synthesize prostaglandins in the cortex cannot be assumed to be negligible since the activity in the medulla is very high, surpassed only by that of seminal vesicles. The finding that the prostaglandins affect adenyl cy-
204
clase activity in a number of different mammalian tissues [3] and are synthesized in situ and rapidly degraded in the circulation, has led many authors to suggest that they may act as intracellular modulators of the "classical" hormones [4]. Lipson and Sharp [5] have shown that prostaglandin E, can increase Na ÷ transport and osmotic water flow in the toad bladder. Grantham and Orloff [6] demonstrated using the isolated perfused renal tubule that prostaglandin E, increased net water movement along an osmotic gradient but it blunted the response to submaximal concentrations of antidiuretic hormone. Beck et al. [7] have examined the effect of prostaglandin E, in renal cortex. They were unable to demonstrate an increase in adenylate cyclase activity by prostaglandin E~, however, at concentrations of 1 0 - 4 M prostaglandin E, blunted the cyclic AMP response to 1 unit/ml of parathyroid hormone. The present experiments were designed to reexamine the effect of prostaglandin E, on the adenyl cyclase-cyclic AMP system of renal cortex. The effect of prostaglandin E, on gluconeogenesis, using a-ketoglutarate, fructose and glycerol as substrates, was also examined. Materials and Methods
Measurements of cyclic AMP in cortical slices Adult Holtzman rats weighing 200--300 g were killed by a blow on the head and the kidneys rapidly removed and placed in ice-cold Krebs-Ringer phosphate buffer, pH 7.4. Food was not withheld prior to the experiment. Cortical slices were obtained using a Stadie-Riggs microtome. They were then incubated for 1 h at 37°C in Krebs-Ringer buffer of the following composition in mmol/l; Na*, 130; K ÷, 4; Ca :+, 1; Mg 2÷, 1.4; CI-, 136; PO~-, 2.4. Similar experiments were also run with 0 mM Ca :+ in the incubating medium. The buffer also contained glucose, 10 mM; theophylline, 10 -2 M, and lyophilized bovine serum albumin, 0.25%. After this pre-incubation period, slices weighing 15--30 mg were added to 1 ml of Krebs-Ringer phosphate buffer containing 10 mM glucose, 10 -2 M theophylline and 0.25% bovine serum albumin. In the experimental flasks three concentrations of prostaglandin E, were used, 10 -9, 10 -~ and 10 -s M. In addition, another flask containing parathyroid hormone, 1 unit/ml, was used. The slices were incubated for 20 min at 37°C in a D u b n o f f metabolic shaker using a gas mixture of CO2/O: (5 : 95, v/v). The reaction was terminated by removing the slices and quickly freezing them in freon. They were then placed into boiling 50 mM sodium acetate/acetic acid buffer, pH 4, for 10 min. They were homogenized by hand using a glass homogenizer; an aliquot was removed for protein determination and the remainder was centrifuged at 5000 × g and the supernatant assayed for cyclic AMP. The incubation media were also assayed for cyclic AMP and the total cyclic AMP produced per mg of tissue protein was calculated. Cyclic AMP was measured by a modification of the method of Gilman [8]. All measurements were done in duplicate and the mean of each pair of values used. Protein determinations were done by the method of Lowry et al. [9].
205
Adenyl cyclase assay For the adenyl cyclase assay the membranes were prepared by the method of Marcus and Aurbach [10] and stored in small aliquots at --70°C. Adenyl cyclase activity was measured by the method of Salomon et al. [ 1 ]. Both the Dowex columns (Dowex 50AG W-X8) and the alumina columns (Aluminum Oxide, Woelm neutral) were reused five times. Recycling of Dowex columns. At the completion of each experiment 2 ml of I M HC1 were added to each Dowex column and the column stored with no further treatment. Prior to reusing, the columns were washed with 10 ml of distilled water. Reuse of Alumina columns. Prior to reuse, the alumina columns were washed with 8 ml 0.1 M imidazole • HCI 7.5.
Measurements of gluconeogenesis Kidney slices weighing 10--25 mg were obtained after killing 2 0 0 - 3 0 0 g Holtzman rats by a blow on the head. These slices were pre-incubated in substrate-free Krebs-Ringer phosphate buffer, pH 7.4, for 1 h prior to the experiment. At the end of the hour, slices were transferred to 1 ml of KrebsRinger buffer, pH 7.4, containing either 10 mM a-ketoglutarate, 10 mM fructose or 10 mM glycerol, or no substrate. Experimental flasks contained prostaglandin E~ at a concentration of 10 -6 M. Control flasks contained alcohol at the same concentration as that present in the flasks with prostaglandin E1 since the prostaglandin El stock solution was made up in absolute alcohol at a concentration of 10 mg/ml. Slices were, therefore, incubated without substrate, with substrate, with substrate and prostaglandin Et, and with substrate and alcohol. After 1 h of incubation an aliquot of the incubation medium was obtained and assayed for glucose. The slice was then removed, blotted gently and weighed. Glucose was assayed by a fluorimetric technique using the hexokinase method [12]. All measurements were done in duplicate. Reagents. 3H-labeled cyclic 3',5'-AMP was purchased form New England Nuclear (Boston, Mass.) and [a-32P]ATP from International Chemical and Nuclear Corp. Parathyroid hormone was from Beckman, Bovine Parathyroid Hormone Synthetic 1-34, Lot No. 26013. Prostaglandin was a gift from Dr. John Pike, Upjohn Co., Kalamazoo, Mich. (PGEI, V-10136, Lot No. 10315-VDV-115). Results are expressed as mean -+S.E. Statistical analyses were performed using Student's t-test for paired data. Results Table I summarizes the data on cyclic AMP production by renal cortical slices in the presence of 1 mM Ca 2+ and in the absence of external Ca 2+. It can be seen that prostaglandin El increased cyclic AMP production in a dosedependent fashion at prostaglandin E1 concentrations of 10 -9 to 10 -s M which was the highest concentration tested. Control values for cyclic AMP were 15.2 + 2.0 pmol/mg protein per 20 min; 25.3 -+ 1.8 pmol/mg protein with 10 -9 prostaglandin El (P < 0.001), with increasing concentration of prostaglandin E~, cyclic AMP production increased to 33.0 _+ 2.8 and 48.0 _+ 4.9, P < 0.02.
206 By comparison, with 1 unit/ml of parathyroid hormone, cyclic AMP values were 67.8 + 5.9 pmol/mg protein per 20 min. Since the prostaglandin E, was dissolved in absolute alcohol at a concentration of 10 mg/ml, and there is evidence suggesting that alcohol can increase adenylate cyclase activity in rat liver and kidney plasma membranes [13], we incubated kidney slices with 0.35% alcohol, an amount equivalent to the alcohol content present in the flasks when 10 -s M prostaglandin EI was used. This a m o u n t of alcohol did not increase cyclic AMP production significantly. The production of cyclic AMP by cortical slices was unaffected by omission of Ca 2÷ from the external medium. The increase in cyclic AMP production by prostaglandin E, was similar to the effects seen in the presence of Ca 2÷. In an attempt to establish that indeed cyclic AMP was being measured, additional experiments were done in which the aliquots to be assayed were exposed to phosphodiesterase prior to assay. Secondly, a known quantity of cold cyclic AMP was added to the medium prior to assay and then assayed for cyclic AMP by a modification of Gilman's assay. The results indicated that indeed cyclic AMP was the substance being measured. Table II shows the effect of prostaglandin El on renal cortical adenylate cyclase activity. Control values for renal cortical cyclase averaged 215 _+ 12 pmol/mg protein per 10 min. Addition of 10 -9 M prostaglandin E, increased adenylate cyclase activity to 309 +- 15 pmol (P < 0.01). Prostaglandin EI at concentrations of 10 -s M produced a further increase in adenylate cyclase activity to 381 -+ 14 pmol/mg protein per 10 min. These results demonstrate increasing adenylate cyclase activity with increasing concentrations of prostaglandin El. The corresponding values for parathyroid hormone are shown for comparison. The values with parathyroid hormone averaged 716 +- 34 pmol/mg protein per 10 min. Thus, 5 units/ml parathyroid hormone increased the activity of renal cortical adenylate cyclase more than 3-fold. Parathyroid hormone has been shown to increase renal gluconeogenesis [14,15] presumably by increasing cyclic AMP levels. Since prostaglandin E, had similar effects on renal cortical cyclic AMP production and adenylate cyclase activity, we examined the effect of 10 -6 M prostaglandin E~ on glucose production by renal cortical slices using 10 mM a-ketoglutarate as substrate. These results are summarized in Table III. In the absence of substrate, glucose TABLE I EFFECTS OF PROSTAGLANDIN E l AND PARATHYROID HORMONE ON CYCLIC AMP PRODUCTION IN THE PRESENCE AND ABSENCE OF Ca2+IN THE INCUBATING MEDIUM VMues
are the
mean
-+ S . E .
of six experiments
in the
case
of
1 mM
external
Ca 2+and
four
with n o external C a 2+.
Cyclic A M P I mM
produced/rag protein per 2 0 m i n
external C a 2+
N o external C a 2+
Control Prostaglandin
E l , 10 -9 M
;.5.2 ± 2.0 2,5.3 +- 1.8
14.0 -' 1.7 23.0 *- 1.0
Prostaglandin
E l , 10 -7 M
33.0
± 2.8
30.7
-+
4.4
Prostaglandin
E l , I0 -S M
48.0
± 4.9
45.7
-~
4.1
6 7 . 8 ~: 5 . 9 ..................................................
82.0
~ 15.0
Parathyroid
hormone,
I unit/ml
experiments
207 TABLE
II
EFFECTS OF PROSTAGLANDIN TIVITY OF RENAL CORTICAL
E l AND PARATHYROID MEMBRANES
HORMONE
ON ADENYLCYCLASE
AC-
V a l u e s are t h e m e a n +- S . E . o f f o u r e x p e r i m e n t s pmol of cyclic AMP formed/mg protein per 10 min Control Prostaglandin El, 10 -9 M Prostaglandin El, 10 -2 M Parathyroid hormone, 5 units/ml
215 309 381 716
± -+ ± ±
12 15 14 34
production was 1.87 ± 0.48 pmol/g wet weight per h. Addition of 10 mM a-ketoglutarate increased glucose production to 11.9 + 0.77 pmol/g wet weight per h. Prostaglandin El at a concentration of 10 -6 M increased glucose production further to 13.5 ± 0.59, a value significantly different (P < 0.05) from that obtained with a-ketoglutarate alone. Using alcohol at the same concentration as that present in 10 -6 M prostaglandin E~ the value averaged 11.50 + 0.69 which was not significantly different from control. These experiments were done at a concentration of 0.25 mM Ca 2÷ in the incubation medium. Omission of Ca 2÷ from the incubation medium decreased baseline glucose production to 9.47 ± 0.82 pmol/g wet weight per h and addition of 10 -6 M prostaglandin El caused no increase in glucose production, 9.42 -+ 0.88 pmol/g wet weight per h. The parathyroid hormone-induced increase in gluconeogenesis, produced by an increase in cyclic AMP, is presumably mediated by stimulating the activity of the key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase. Pagliara and Goodman [16] have shown that there is no increase in glucose production induced by cyclic AMP when fructose and glycerol are used as substrates. When we tested the effects of prostaglandin El on glucose production from fructose and glycerol, we found that the values for glucose production in the absence and presence of 10-6M prostaglandin El were not significantly different. Glucose production from glycerol and fructose averaged 6.0 -+ 0.9 and 71 ± 8.1 pmol/g wet weight per h, respectively. In the presence of 10 -6 M prostaglandin the values were not significantly different and averaged 6.3 ± 0.4 and 78.9 -+ 6.7 ~mol/g wet weight per h, respectively.
TABLE
Ill
EFFECTS OF PROSTAGLANDIN THE PRESENCE AND ABSENCE
E l ON GLUCOSE PRODUCTION O F E X T E R N A L C a 2+
FROMa-KETOGLUTARATE
IN
V a l u e s are t h e m e a n ± S . E . o f e i g h t e x p e r i m e n t s . Glucose production (~mol/g wet wt per h) 0 . 2 5 m M e x t e r n a l C a 2+ No substrate
N o e x t e r n a l C a 2+
1 . 8 7 -+ 0 . 4 8
1 . 6 9 -+ 0 . 1 5
10 mM c,-ketoglutarate
11.9
± 0.77
9.47 ± 0.82
~ - K e t o g l u t a r a t e + 1 0 -6 M p r o s t a g l a n d i n E I
13.5
*- 0 . 5 9
9.42 ± 0.88
~-Ketoglutarate + alcohol diluent
11.5
~ 0.69
8.98 ± 0.85 .
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208 Additional experiments demonstrated a decreased stimulation of glucose production by submaximal doses of parathyroid hormone when 10 -6 M prostaglandin E~ was added. Parathyroid hormone, 1 unit/ml, increased glucose production from 9.4 -+ 0.5 pmol glucose/mg protein per h to 11.2 + 0.5 pmol glucose/mg protein per h. 10 -6 M prostaglandin E~ increased glulucose production to 12.0 + 0.4. Addition of parathyroid hormone, 1 unit/ml, and prostaglandin E~, 10 -6 M, increased glucose production to 10.3 -+ 0.4, a value which was not statistically different from control but statistically different from parathyroid hormone alone (P < 0.05). Discussion
These results demonstrate that the renal cortex responds to prostaglandin E 1 with an increase in cyclic AMP production. An increase in cyclic nucleotide production by prostaglandins has been demonstrated in whole fat pads, lung, spleen [3], thyroid [17], ovary [18], and renal medulla [19]. Beck et al. [7] did not find an increase in cyclic AMP production in renal cortical slices exposed to 10 .4 M prostaglandin El. However, they showed that prostaglandin E~ significantly reduced the cyclic AMP generation produced by submaximal concentrations of parathyroid hormone. Our results demonstrate an increase in cyclic AMP production with increasing concentrations of prostaglandin E~, and we have confirmed the decrease in cyclic AMP production in response to a submaximal concentration of parathyroid hormone when prostaglandin E~ is added. The finding that the increase in cyclic AMP generation by prostaglandin E was not influenced by removal of Ca 2÷ from the external medium in the present experiments is similar to the increase in cyclic AMP by prostaglandin E~ observed by Yamasita et al. [17] in thyroid slices and similar to the effects of parathyroid hormone on cyclic AMP generation in renal cortical tubules [15]. This increase in cyclic AMP is probably a result of membrane-receptor interaction with stimulation of adenylate cyclase activity as demonstrated by increased activity of this enzyme with increasing concentrations of prostaglandin E~. We were unable to show a significant effect of prostaglandin El on the stimulation of adenyl cyclase produced by 5 units/ml of parathyroid hormone. This is in contrast to the findings of Beck et al. [7] who showed that prostaglandin E~ significantly decreased the activation of adenylate cyclase activity by submaximal doses of parathyroid hormone. However, since we are dealing with a broken cell preparation and the adenylate cyclase activity may be modified by a number of factors, including Mg/ATP ratios [ 1 0 ] , concentration of Mg 2÷ in the external medium [20] and the levels of ATP and other nucleotides, viz. GTP [20], GMP, the conditions of our experiments may not have been optimal for the demonstration of an effect of prostaglandin E~ on the parathyroid hormone activation of adenylate cyclase. Prostaglandin E~ produced the same metabolic changes as those demonstrated for parathyroid hormone by Nagata and Rasmussen [15] in renal cortical tubules and by Kurokawa et al. [14] in isolated renal tubules. Prostaglandin E~ increased glucose production from a-ketoglutarate. Omission of Ca 2÷ from external media lowered baseline glucose production and completely blocked
209 any further stimulation in response to prostaglandin El. Furthermore, glucose production from glycerol and fructose was not increased by prostaglandin E1 suggesting that the effect of prostaglandin E, was at the level of the key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase. Cyclic AMP has been shown by Pagliara and Goodman [16] to increase renal gluconeogenesis from a-ketoglutarate and glutamine but not from fructose and glycerol. This would then suggest that prostaglandin El, like parathyroid hormone, increases gluconeogenesis by increasing the intracellular content of cyclic AMP. Finally, the increase in gluconeogenesis produced by submaximal concentrations of parathyroid hormone was decreased in the presence of 10-6M prostaglandin El. In view of these observations, it is suggested that prostaglandin E, may play a physiological role in the renal cortex by modulating the response to parathyroid hormone.
Acknowledgments This work was supported by U.S.P.H.S. NIAMD Grants AM-05248 and AM09976. During the course of this study, Dr. Morrison was the recipient of fellowships from the National Kidney Foundation and the Kidney Foundation of Eastern Missouri and Metro-East. The authors wish to express their appreciation to Mr. Orlando Moncada for his technical assistance and to Mrs. Patricia Verplancke for her secretarial assistance.
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