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Interactions between parathyroid hormone and prostaglandins on renal cortical cyclic AMP
PROSTAGLANDINS
INTERACTIONS BETWEEN PARATHYROID HORMONE AND PROSTAGLANDINS ON RENAL CORTICAL CYCLIC AMP' Mark G. CurTie
and David M. Biddulph
Department of Anatomy Bowman Gray School of Medicine Winston-Salem, North Carolina 27103 ABSTRACT Effects of parathyroid hormone (PTH) and several prostaglandins (PGs) on cyclic AMP (CAMP) metabolism were studied and compared in isolated renal cortical tubules from male hamsters. Both production and intracellular degradation of CAMP were increased by PTH and each of the PGs tested (PGE2, PGEI, PG12). Production of CAMP was increased to similar levels by maximal concentrations of PTH and each PG, however, degradation of CAMP was significantly higher in response to PTH than with any of the PGs. This difference in intracellular degradation of CAMP was responsible for the much higher concentrations of CAMP in renal cortical tubules exposed to PGs (PGE1, PGE2, PG12) than to PTH. Submaximal amounts of each PG produced additive increases in CAMP concentrations in the presence of maximal amounts of PTH. Additivity of the combined responses was lost, however, as the PGs concentrations reached their maximas. The results suggest that renal PGs (PGE2 and PGI1) may modulate the effects of PTH on CAMP concentrations in renal cortical tubules.
' This work was supported by a grant to D.M.B. from the National Science Foundation, PCM 75-07661.
2 This work is taken in part from a thesis submitted to the Department of Anatomy, Bowman Gray School of Medicine of Wake Forest University, in partial fulfillment of requirements for the degree of Doctor of Philosophy. Present address: Department of Pharmacology, Washington University School of Medicine, St. Louis, MO.
MAY
1981 VOL. 21 NO. 5
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PROSTAGLANDINS
INTRGWCTION Prostaglandins (PGs) have been reported to modulate the physiological actions of many peptide hormones (1). As hormonal modulators, PGs may either amplify or inhibit the effects of a hormone. There are many instances of PGs acting as hormonal modulators in the kidney (2). PGs are thought both to attenuate the renal actions of antidiuretic hormone and to amplify the vasodilatory effect of bradykinin (2). Although effects of PGs on PTH-dependent calcium and phosphate transport have not been reported, several studies have shown that prostaglandin El (PGE1) antagonizes effects of parathyroid hormone (PTH) on elevating cyclic AMP (CAMP) concentrations in rat renal cortical slices (2,3-6). Since CAMP is thought to mediate the actions of PTH (7), PGs have been proposed as an important local negative feedback mechanism for the renal effects of PTH (3-6). However, this inhibitory effect of PGEl on PTH-induced increases in CAMP was not observed in rabbit kidney slices (8). Using hamster renal cortical tubules, we also did not observe an inhibitory effect of PGE1, on the PTH response (9, 10). In our initial studies with PTH and PGE1, maximal concentrations of these two agonists, when added together, produced a nonadditive response; however, the combination of a submaximal concentration of PGEl with PTH resulted in an additive response (9). We proposed that the local release of PGs could amplify the effect of PTH on CAMP. In order to further test this hypothesis, effects of the naturally-occuring PGs (PGE, and PGI,) have been tested and compared with effects of PTH in the present study. MATERIALS AND METHODS Materials Male golden hamsters, Mesocricetus auratus were obtained from Engle Laboratories and were maintained on PurimLaboratory Chow and water ad libitum prior to experiments. Bovine parathyroid hormone (PTH) was purchased from Wilson and Co. (300 USP U/mg protein). Dilutions of PTH were made with acidified (pH 3.0) isotonic saline. Prostaglandin El (PGEI), prostaglandin E2 (PGE*), prostaglandin I2 (PGI,), and 6-ketoprostaglandin F,, (6-keto-PGF1,) were generously supplied by Dr. John Pike, Upjohn Co. PGEl and PGE, were dissolved in absolute ethanol and diluted to desired concentrations with incubation media such that the final ethanol concentration did not exceed 0.4%. PGI, and 6-keto PGF1, were dissolved and diluted to desired concentrations in a 20 mM Tris buffer, pH 9.0 just prior to use in experiments. Reagents for assay of CAMP were purchased from Diagnostic Products Corp. Anionexchange resin (AGl-X2) was purchased from BioRad Co. Tritiated nucleotides and adenosine were purchased from New England Nuclear. l-methyl-3isobutylxanthine (MIX) and all enzymes used in this study were obtained from Sigma Chemical Co. Other chemicals used were the best grades available and were obtained from standard suppliers.
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Preparation
of Isolated
Renal Tubules
Isolated renal cortical tubules were prepared as previously described (10). Kidneys were rapidly removed, hemisected and transferred to ice-cold Hanks solution containing 1.8 mg/ml glucose. Corticies were separated from medullary tissue, pooled and minced finely with surgical scissors in 10 ml of an enzyme solution consisting of 0.4 mg/ml collagenase, 1 mg/ml hyaluronidase and 1.8 mg/ml glucose dissolved in Hanks solution containing 1 mM calTissues were incubated at 37°C for 60 min with air as the gas phase cium. in an oscillatory water bath and filtered through a single layer of nylon with an additional 40 ml enzyme-free solution. Filtrates were centrifuged at 50 x 9 for 2 min at room temperature. The pellets were resuspended in Hanks-glucose solution and centrifuged again at 30 x CJ for 2 min. This washing procedure was repeated twice. The final pellet was the preparation used for tubule incubations and contained approximately 40% of the original cortical protein. Incubation,
Extraction
and Assay of Cyclic
Nucleotides
Tubules, representing 2-3 mg protein per flask were incubated in 5 ml siliconized, Erlenmeyer flasks at 37°C in an oscillatory water bath in 1.5 ml Krebs-Ringer-Tris buffer, pH 7.4 containing 1 mM MgC12, 1 lllMCaC12, 1.8 mg/ml glucose and 20 mM Tris-HCl. In some experiments, designated in the text, MIX was added to obtain a final concentration of 5 mM. Parathyroid hormone or prostaglandins were added in 0.1 ml volumes following a 5 min preincubation period. Control solutions consisted of acid saline and ethanol. Final concentrations of ethanol did not exceed 0.4%. In some experiments tubules were pretreated for 15 min in trypsin (2.0 mg/ml) at 37°C. Pellets were then washed twice prior to further treatment. Trypsin inhibitor (Sigma) was added in 3-fold excess during subsequent incubation of tubules. Incubations were terminated by addition of 1 volume cold 5% TCA, followed by homogenization. Homogenates were centrifuged at 1,000 x 9 and supernatants transferred to glass tubes, extracted 4 times with 10 volumes of ether, boiled to remove residual ether and lyophilized. Pellets were dried, resuspended in 1 N NaOH and assayed for protein by either the method of Lowry -: et al (11) or the biuret reaction. Lyophilized supernatants were dissolved in Tris buffer, pH 7.4 and assayed for CAMP by the method of Gilman (12) as modified by Tovey -et al. (13). Known amounts of CAMP added to either tissue extracts or TCA and carried through the entire extraction procedure, were recovered quantitatively. Incubation of sample aliquots with excess phosphodiesterase for 60 min at 37°C resulted in complete hydrolysis of CAMP. Serial dilution of samples assayed for CAMP were linear over a lo-fold range. All samples were assayed in either duplicate or triplicate. In some experiments tubule suspensions were rapidly centrifuged (1000 x CJ for 1 min) prior to addition of TCA. Cyclic AMP was assayed in both the tubule pellet and supernatant (medium) of each sample.
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1981 VOL. 21 NO. 5
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PROSTAGLANDINS
,Preparation and Assay of cAMP Phosphodiesterase (PDE) PDE a c t i v i t y was measured in renal c o r t i c a l homogenates by the twostep method o f Thompson and Appleman (14). Renal c o r t i c a l tissue was homogenized and sonicated in 0.25 M sucrose containing 0.I M T r i s and 0.I mM EDTA (pH 7.6). Homogenates were then centrifuged at 4°C at 50,000 x f o r 30 min. Aliquots of the supernatant were assayed in a b u f f e r (0.4 ml t o t a l vol) containing 40 mM T r i s , (pH 8.0) I0 mM MgCl2, 3,75 mM mercaptoethanol, 0.I mM or 4 ~M c y c l i c AMP, 3H-cyclic AMP (60,000 cpm) and 0.2 ml of the enzyme preparation. Agents tested f o r effects on PDE a c t i v i t y were added in 20 ~I vol before the enzyme was added to i n i t i a t e the reaction. Incubations were f o r 5 min at 30°C with enzyme a c t i v i t y being l i n e a r over t h i s time period. Reactions were terminated by immersing reaction tubes i n t o a dry ice-acetone bath f o r exactly 12 sec followed by b o i l i n g f o r 45 sec. Each tube was then incubated f o r I0 min at 30°C in the presence of excess 5' nucleotidase. This reaction was terminated by addition of 1 ml of a I : I s l u r r y of BioRad anion-exchange resin (AGIX2, 200-400 mesh) containing 40% methanol as described by Thompson et a l . (15). Contents of the tubes were centrifuged at 1,000 x ~ f o r min and 0.4 ml a l i q u o t s of the supernatants were added to s c i n t i l l a t i o n v i a l s and counted f o r r a d i o a c t i v i t y . Enzyme a c t i v i t i e s were determined such that no more than 30% of the substrate was hydrolyzed during the reaction. S t a t i s t i c a l analysis of the data employed a t w o - t a i l e d t test and a m u l t i p l e comparison t e s t (16). RESULTS We have previously reported t h a t the PDE i n h i b i t o r , l - m e t h y l - 3 i s o b u t y l x a n t h i n e (MIX), at a concentration of 5 mM maximally potentiates the i n t r a c e l l u l a r accumulation of cAMP in isolated renal tubules in response to PTH and PGs ( I 0 ) . To determine r e l a t i v e rates of production and degradation of cAMP in response to PTH and the prostaglandins investigated in the present study, paired incubations of c o r t i c a l tubules were carried out in the presence and absence of 5 mM MIX. Production of cAMP was defined as the amount of cAMP accumulated in the presence of 5 mM MIX. Degrat i o n of cAMP was estimated as the d i f f e r e n c e in cAMP concentration in the presence and absence of MIX. In a series of time course experiments, maximally e f f e c t i v e concent r a t i o n s of PTH (7 U/ml) and the prostaglandins (15 ~g/ml) produced s i g n i f i cant (p < .01) increases in both cAMP production and degradation at each time i n t e r v a l examined (Fig. I ) . PTH and the E-series of prostaglandins produced s i m i l a r changes in both the k i n e t i c s and the magnitude of hormoneinduced increases in cAMP production. The k i n e t i c s of cAMP production due to s t i m u l a t i o n by PGI2 were s i m i l a r to that caused by PTH and the E-series of prostaglandins, however, the magnitude of cAMP production was s i g n i f i c a n t l y (p < .01) lower in tubules exposed to PGI2 when compared to the other hormones. The stable metabolite of PGI2, 6-keto-PGF~, did not
808
MAY 1981 VOL. 21 NO. 5
PROSTAGLANDINS
400
PGI,
350 300 250
i
400
-
350
-
300
-
250
-
200
-
150 100 50 O-
0246810 Figure 1. Time-course curves representing the effects of PTH (7 U/ml), PG12 (15 vg/ml), PGEl (15 pg/ml) and PGE2 (15 pg/ml) on production and degradation of cyclic AMP in isolated renal tubules. Production of cyclic AMP was measured in the presence of 5 mM MIX, while degradation of cyclic AMP was calculated as the difference in cyclic AMP concentrations in the presence and absence of MIX. Open symbols represent changes in control incubations. Points are means of three to nine samples ? SE or SE of the difference. significantly elevate CAMP levels at concentrations tested (1 and 15 ug/ml). In contrast to the similarities between PTH and PGs on CAMP production, degradation of CAMP was significantly (p < .Ol) lower in tubules exposed to PGs than to PTH. Degradation of CAMP, relative to amounts produced, ranged from 25-50% in response to the PGs tested and increased during the time intervals investigated in a way which was similar for all PGs. Degradation
MAY 1981VOL. 21 NO. 5
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PROSTAGLANDINS of CAMP in response to PTH ranged from 85-95% of the amounts produced and was responsible for the much lower net concentrations of CAMP in tubules treated with PTH than with any of the PGs. Concentration-response experiments with each hormone (Fig. 2) demonstrated that increases in degradation of CAMP were effected over the same
350 PG12
300 250 200 150
I
I
I
I
I
04.607 .07 0.7 70 70.0 U/ml
I PGE2
300
/++
250
9 c,
200 150
04’.01 0.1 1.0 10.0 100.0 I
I
1
I
1
p9 /ml Figure
810
2. Concentration-response curves representing the effects of varying concentrations of PTH, PG12, PGEI, and PGEP on production and degradation of cyclic AMP in isolated renal tubules. Incubations were for 5 min following the addition either of hormone or the appropriate control. Production of cyclic AMP was measured in the presence of 5 rrEil MIX, while degradation of cyclic AMP was calculated as the difference in cyclic AMP concentrations in the presence and absence of MIX. Points are means of three to nine samples + SE or SE of the differences.
MAY 1981VOL. 21 NO. 5
PROSTAGLANDINS
concentration range of hormone which increased production of CAMP. As was noted in time-course experiments, production of CAMP reached similar levels with maximal concentrations of PTH and PGs with PGIp being slightly Degradation of CAMP, in response to the less effective than the E-series. PGs, was however only increased to about half that produced by PTH. Statistically significant increases in production of CAMP were produced by all three PGs at concentrations as low as 0.01 pg/ml (28 nM). These differences in the amounts of CAMP degradation between tubules exposed to PTH and the prostaglandins were apparently not due to the direct effects of these hormones on the enzyme phosphodiesterase, since phosphodiesterase activity in renal cortical homogenates (either crude or 50,000 x 9 supernatants) was not directly affected by the addition of maximal concentrations of any of these agents. Experiments in which tubules were assayed for CAMP separately from their respective incubation media showed that the observed differences in the amounts of CAMP degraded were not due to differences in amounts of CAMP released from the cells, Neither PTH nor the PGs had a significant (p > .05) effect on the percentage of CAMP released from the tubule cells. Since the isolated cortical tubule preparation used in these experiments consisted of a mixed population of cells, experiments were designed to determine whether PTH and the PGs were affecting CAMP changes in a common target cell population. Essentially, these experiments searched for competition between maximal concentrations of PTH and PGs and investigated interactions between submaximal concentrations of PGs and a maximal concentration of PTH. The combination of maximally effective concentrations of PTH and PGEP, either in the presence or absence of MIX resulted in responses which suqqested interactions between these two hormones (Table 1). In the absence of-MIX, the combination of the maximal concentrations of PTH (7 U/ml) and PGE2 (30 (p < .Ol) less than uq/ml) resulted in a response which was significantly the single PGE2 response. In the presence of MIX, the combined response was significantly (p < .Ol) greater than that induced by either hormone The alone but considerably less than the sum of PTH and PGE2 responses. combination of a submaximal concentration (.05 ug/ml) of PGE, with a maximal concentration of PTH (7 U/ml) in the absence of MIX caused an increase in the CAMP response which was significantly (p < .Ol) greater than either agent alone; the response however was not completely additive (Table 1). Combination studies were also done with PTH and PG12 (Table 2). The combination of the maximal concentrations of PTH and PG12 boththe presence and absence of MIX resulted in responses which were less than completely additive. In the presence or absence of MIX with an incubation period of 1 min, the combination of the maximal concentrations of PTH (7 U/ml) and PG12 (30 ug/ml) resulted in responses which were completely nonadditive (Table 2). However, the combination of PTH and PG12 in the presence of MIX resulted in a response which was significantly (p < .Ol) greater than with either hormone alone but was much less than completely additive when the incubation time was 5 min. The combination of a submaximal concentration (.05 ng/ml) of PGIp with a maximal concentration of PTH resulted in a response which was significantly (p < .Ol) greater than with either response alone.
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811
PROSTAGLANDINS
TABLE Effects
of PTH and PGE2 on Cyclic in Isolated
Treatment
1 AMP Concentrations
Renal Cortical
Tubules
CAMP Concentration
(pmole/mg
1 min incubation - MIX Control
protein)
5 min incubation
+ MIX
+ MIX
1.6 + 0.3
6.4 f 0.5
11.7 -I 0.3
23.3 f 1.8
146.7 + 5.4
333.1 f 27.6
PGEz (30 pg/ml)
80.1 + 0.7
97.7 + 3.0
PGE2 (0.05 pg/ml)
29.4 + 2.0
PTH
PTH
(7 U/ml)
335.8 k
___
8.8
_-_
(7 U/ml) 57.6 + 3.6*
184.9 ?r 4.1*
557.1 f 10.1*
PiE2 (30 ng/ml) PTH
(7 U/ml) 38.0 f 0.9*
___
_--
PiEz (0.05 ug/ml)
Tubules were incubated in the presence or absence of 5 mM MIX for 1 or 5 min after additions of the above agents. Values are the means of three to six incubations f SE. *
812
Significantly (p < 0.01) different in the combination.
from both single
MAY
responses
involved
1981 VOL. 21 NO. 5
PROSTAGLANDINS
TABLE 2 Effects
of PTH and
PG12on
in Isolated
Cyclic AMP Concentrations
Renal Cortical
Tubules
CAMP Concentration
Treatment
(pmole/mg
1 min incubation Control
MIX
5 min
7.6 f 0.6
23.1 * 1.4
149.3 + 6.7
PGIz (30 ug/ml)
64.5 k 2.2
74.9 + 2.1
PGIp(0.05ng/ml)
38.8 * 2.3
_--
63.9 f 1.8
157.5 f 5.7
PTH
(7 U/ml)
incubation
+ MIX
+ MIX
1.1 + 0.2
PTH
protein)
12.2 k
0.5
317.4 -I 22.4 238.8 f
2.4
--_
(7 U/ml) 459.8
+ 15.9*
P+G12 (30 pg/ml) PTH
(7 U/ml) 53.4 f 1.5*
_-_
_-_
PkIp (0.05 ug/ml)
Tubules were incubated in the presence or absence of 5 mM MIX for 1 or 5 min after additions of the above agents. Values are the means of three to six incubations + SE. *
Significantly (p < .Ol) different in the combination.
MAY 1981VOL. 21 NO. 5
from both single
responses
involved
813
PROSTAGLANDINS
Since the combination experiments revealed competition between the maximal concentrations of PTH and the PGs, experiments were designed to determine if the observed competition involved the PTH receptor. We have previously shown that mild trypsin treatment of these tubules selectively inhibits the PTH response without affecting the PGEl response (9). This effect of trypsin on the PTH response is thought to be due to a direct action of trypsin on the PTH receptor (17). The present study utilized trypsin treatment of the tubules to determine whether the PTH response The trypsin could be differentiated from the PGE2 and PGIp responses. treatment inhibited the PTH induced response by 70%; however, the responses due to stimulation by either PGE;! or PG12 were not significantly (p > .05) affected (Table 3).
TABLE Effects
of Pretreatment
with Trypsin
PGEl and PG12-induced Isolated
Cyclic
on PTH, PGE1,
AMP Responses
Renal Cortical
Treatment
in
Tubules
CAMP Concentration
- Trypsin Control
3
(pmoles/mg
+ Trypsin
protein)
% Inhibition
16.0 +
0.5
14.9 f
0.5
9
303.7 +
8.0
105.9 f
6.1
69
PGEl (30 ug/ml)
312.7 f 10.5
302.9 +
5.4
3
PGE2 (30 ug/ml)
332.6 f
9.1
311.8 it:10.1
7
PGI2 (30 us/ml)
269.3 f 11.4
238.6 k 10.4
12
PTH
(7 U/ml)
Tubules were pretreated for 15 min at 37" with trypsin (2 mg/ml) before washing and adding a 3-fold excess of trypsin inhibitor. Tubules were then incubated for 5 min in the presence of 5 mM MIX after the addition of the above agents. Values are the means of three to six incubations * SE.
814
MAY 1981 VOL. 21 NO. 5
PROSTAGLANDINS
study
Interactions (Fig. 3).
between
PGE2 and PGI;, were
PGE,
F
also examined
in the present
(30pg/ml)
. .. ..... ...........................,................. . .
Basal
,f.. ..,.... ,:.:. ~:,~:.:.~;.:.:.:.;..:.:.:‘:‘:‘:‘: ..,..._. i,.........~~.~.,~.~~~~~,~.~.~.~ . ..._._._. ...._._. ,._._ ... ._.,..._._. ,..._., L...... :,.,.. ,..,._._.,..._._........
40
.Ol
.I
PGI, Figure
MAY
1.0 IO 100
~9 /ml
3. Effects of simultaneous addition of PGEl (0.05 pg/ml or 30 pg/ml) and varying concentrations of PGI2 on cyclic AMP concentrations in isolated renal tubules. Incubations were for 5 min after addition of prostaglandins and included 5 mM MIX (PGI,, closed circles; PGE%, horizontal bars; PGE2 + PG12 at indicated concentrations, open circles). Each point is the mean of three to six samples +_ SE, Bars represent means of three to six values +_ SE.
1981 VOL. 21 NO. 5
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PROSTAGLANDINS
The simultaneous addition of a maximal concentration of PGI, (30.0 ug/ml) with an equivalent concentration of PGE2 produced a combined response which was significantly (p < .Ol) lower than the PGE;! response but signiCombination of a ficantly (p < .Ol) greater than the PG12 response. submaximal concentration (.05 pg/ml) of PGE, with varying concentrations of PG12 gave results, suggesting additivity of the two responses at low concentrations of PGI, (Fig. 3). Similar results were obtained using PGEl in place of PGE2 (data not shown).
DISCUSSION The present study demonstrates that PTH, PGI,, PGE, and PGEl increase CAMP concentrations in hamster renal cortical tubules, confirming Previously reported observations in renal cortical tissue from other Species The lowest effective concentration for PGI,, PGE, and (4, 5, 6, 8, 18). PGEl to cause an increase in CAMP production was 10 ng/ml (28 nM), a concentration similar to that reported in studies utilizing rat renal cortical In the hamster renal cortex, PTH and the PGs appear to tissue (4, 5, 18). Degradation of CAMP, affect the production of CAMP in a similar manner. however, seems to be differentially affected by the two different types In tubules exposed to PTH, CAMP breakdown was much greater of hormones. Neither PTH nor the PGs had a direct than in tubules exposed to PGs. effect on PDE activity in renal cortical homogenates, suggesting that the differences observed in intact tubule cells were due to some indirect Lack of direct effects of both PTH as well as PGEl on POE activeffect. ity of renal cortical homogenates have been reported in other species (5, 19) and suggests that some other intracellular factor, which is presumably differentially affected by PTH and PGs is regulating PDE activity. Intracellular calcium, which we have previously shown to be altered in this system by PTH (20) and which is known to regulate PDE activity in some tissues (21) may be involved. The present study also suggests that a common cell type within renal cortical tubules may be responsive to both PTH and the prostaThe results appear to be consistent with effects of glandins tested. PTH and PGs on overlapping but not conqruent populations of renal Production of CAMP, as measured in intact cells in the cortical cells. presence of MIX did not demonstrate summated responses to simultaneous addition of hormones at maximal concentrations. In renal cells without PDE inhibition, the combined response to PTH and PGE, was clearly less than with PGE2 alone. This diminished response to PGE2 in the presence of PTH may be reflecting the differential effects of these hormones on degradation of CAMP. The observed competition between PTH and the prostaglandins at maximal concentrations apparently does not involve a common receptor site. Pretreatment of the tubules with trypsin selectively inhibited the PTH response without affecting the prostaglandin-induced cyclic AMP response. The effect of try sin treatment is thought to be at the receptor level since the binding of P251 _ labeled PTH is greatly reduced in membranes previously treated
816
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PROSTAGLANDINS
We have previously reported that trypsin treatment rewith trypsin (17). duced PTH, but not PGEl-induced increases in Cyclic AMP (5-j).The current study verifies the previous work and extends it by comparing the effect of of trypsin treatment on the responses caused by PTH, PGEz and PGI,. Since the competition between PTH and the prostaglandins does not appear to be at the receptor level, the likely site of occurrence for the observed ComPetition appears to reside at the adenylate cyclase level. If this interpretation is correct, the presence of a multireceptor-coupled adenylate cyclase system is suggested in renal tubule cells in which the combined number of hormonespecific receptors is in excess of available adenylate cyclase. Such a relationship between hormone-specific receptors and adenylate cyclase has been implicated in the fat cell (22) and is in general agreement with the "spare receptor hypothesis" (23). We cannot rule out the possibility however, that PGs might inhibit the reaction of PTH Further work is with its receptor by a non-competitive mechanism. required to determine the exact nature of PTH-PGs interactions in this tissue. In the present study, the combination of a submaximal concentration of either PGEz or PG12 with PTH resulted in a partially additive response in the absence of MIX. The results suggest that the local release of these prostaglandins from renal cellular elements might act to amplify the PTH-induced accumulation of cyclic AMP. This proposal conflicts with the hypothesis proposed by Beck et al. (3), who showed that PGEl c-ireduced the PTH-induced increase in cyclic AMP in the rat and suggested that the response is attenuated by the local release of prostaglandins. Several recent studies concerning the effect of PGE, on the PTH-stimulated increases in cyclic AMP in rat kidney slices, generally support this hypothesis (4, 5, 6). In a study which utilized renal cortical slices from rabbits, however, no inhibitory action of PGEl on the PTH-induced accumulation of cyclic AMP was reported (8). Thus, the observations noted in the present study with hamsters and in the study with rabbits differ considerably from reported observations utilizing the rat kidney as a model. These differences possibly reflect sensitivity to PTH and prostaglandins. In the rat, PTH is a more potent agonist than PGs in causing increases in cyclic AMP (3, 5, 6). Thus, the combination of the weak agonist with the strong agonist would be expected to produce a response which is less than the strong agonist's response alone, if competition is occurring at some common point. However, PGE1, PGE, and PG12 are more potent agonists than PTH in causing net increases in cyclic AMP in both the hamster and the rabbit (8). Thus, the combination of PTH and PGs, in these two species, would be expected to produce a response which is greater than the PTH-response alone. Interactions between PTH and the more physiological prostaglandins used in the present study (PGE2 and PGI,) upon the tissue accumulation of CAMP to our knowledge have not been investigated previously. The results from experiments investigating interactions between the E-series of prostaglandins and PG12 suggested that these agonists stimulated CYClic AMP synthesis in a common population of cells. This conclusion iS based on the lack of additive responses to the combination of
MAY 1981VOL. 21 NO. 5
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PROSTAGLANDINS
maximal concentrations of the E-series of prostaglandins and PGIp. The combination of submaximal amounts of these agonists produced additive Nonadditive responses for maximal concentrations of the E-series responses. and PGI, have also been reported in rat renal cortex (24) and rat liver (25). The basis for this interaction between the E-series and PGI2, while not delineated by the present study, most probably involves competition at receptor sites, as has been reported in platelets (26), or competition between separate receptors for the two types of prostaglandins for a comnon adenylate cyclase, as has recently been suggested in liver Providing support for the occurrence of the latter possibility cells (25). in this system is the report based on binding studies of a receptor in the kidney which specifically binds PGE with high affinity (27).
In conclusion, the present study revealed that PTH, PGI,, PGEl and PGE, produced increases in cyclic AMP concentrations in isolated renal cortical tubules from hamsters. PTH and the prostaglandins appeared to affect the production of cyclic AMP in a similar manner; however, these two different types of hormones seemed to affect the degradation of cyclic AMP differentiThe combination studies suggested that PTH and these prostaglandins ally. might share a common target cell type in affecting a change in cyclic AMP concentrations. The results also suggested that the local release of prostaglandins from renal cellular elements might act as physiological amplifiers of the PTH-induced accumulation of cyclic AMP in the hamster. The implication of this hypothesis is that prostaglandins might augment the renal actions of PTH by amplifying the influence of the hormone on cyclic AMP, although we are unaware of any studies in which effects of PGs on tubular transport of calcium or phosphorous have been reported. Finally, the study points to the need for a thorough investigation of any effect PTH might have on prostaglandin metabolism in the kidney. ACKNOWLEDGEMENT The authors are indebted to the secretarial for typing of this manuscript.
aid of Daphne
B. Styers
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Dunn, M. J. and V. L. Hood. Prostaglandins and the kidney. Am. J. Physiol. 233: F169, 1977. Baer, P. cand J. C. McGiff. Hormonal systems and renal hemodyAnn. Rev. Physiol. 42: 589, 1980. namics. F. Michelis, R. 0. Fusco., J. B. Beck, N. P., F. R. DeRubertixM. Field and 8. B. Davis. Effect of prostaglandin El on certain renal actions of parathyroid hormone. J. Clin. Invest. 51: 2352, 1972. Morrison, A. R., J. Yates and S. Klahr. Effect of-@-ostaglandin El on the adenyl cyclase-cyclic AMP system and gluconeogenesis in rat renal cortical slices. Biochim. Biophys. Acta 421: 203, 1976. Araki, N., N. Nagata and N. Kimura. Effects ofTrathyroid hormone and prostaglandin El z vitro on release of cyclic AMP from kidney cortical tissue. Endocrinol. Jap. -24: 581, 1978.
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6)
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16) 17)
18) 19) 20) 21) 22)
Interaction between parathyroid Nagata, N., Y. Ono and N. Kimura. hormone and prostaglandin E, on cyclic AMP metabolism in rat kidney. Acta Endocrinologica 93: 339, 1980. Renal adenyl cyclase: anatomically Chase, L. R. and G. DTAurbach. Science separate sites for parathyroid hormone and vasopressin. 159: 545, 1968. Chase, L. R. Selective proteolysis of the receptor for parathyroid Endocrinology 96: 70, 1975. hormone in renal cortex. Metabolism of cyclic AMP in Currie, M. C., and il. M. Biddulph. Effects of prostaglandins and parathyroid isolated renal tubules: Prostaglandins 17: 211, 1979 hormone. and R. W. Wrenn. Effects and interBiddulph, Il. M., M. G. CuEie actions of parathyroid hormone and prostaglandins on adenosine 3', 5'-monophosphate concentrations in isolated renal tubules. Endocrinology 104: 1164, 1979. Rosebrough, A. L. Farr and R. J. Randall. Lowry, 0. H., KJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265, 1951. Gilman,. G. A protein binding assay for adenosine 3'5'-cyclic phosphate. Proc. Natl. Acad. Sci. 67: 305, 1970. A simple direct Tovey, K. C., K. G. Oldham and J. TiT M. Whelan. assay for cyclic AMP in plasma and other biological samples using an improved competitive binding technique. Clin. Chim Acta 56: 221, 1974. Thompson, W. J. and M. M. Appleman. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry -10: 311. 1971. Thompson, W. J., C. P. Ross, W. J. Pledger and S. J. Strada. Cyclic adenosine 3':5'-monophosphate phosphodiesterase: distinct forms in human lymphocytes and monocytes. J. Biol. Chem. _'251: 4922, 1976. Duncan, 0. B. Multiple range and multiple F-tests. Biometrics 11: 1, 1955. McIntosh, C. H. and R. D. Hesch. Characterization of the parathyrin receptor in renal plasma membranes by labelled hormone and labelled antibody binding techniques. Biochim. Biophys. Acta 26: 535, 1976. Zense
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23) 24)
25)
26) 27)
Stephenson, R. P. A modification of the receptor theory. Br. J. Pharmacol. 11: 379, 1976. Herman, C. A.,7. V. Zenser and B. B. Davis. Comparison of the effect of prostaglandin I2 and prostaglandin E2 stimulation of the rat kidney adenylate cyclase-cyclic AMP systems. Biochim. Biophys. Acta 582: 496, 1979. Robertson, R. p,, D. R. Storm, N. H. Anderson and K. R. Westcott. Prostaglandin E and prostacyclin stimulate a common adenylate cyclase but bind to separate receptors in liver plasma membrane. Clinical Research 27: 446A, 1979 (Abstract). Siegl, A. M., J. BrSmith and M. J. Silver, K. C. Nicolaou and D. Ahern. Selective binding site for C3H] prostacyclin on platelets. J. Clin. Invest. 63: 215, 1979. Oien, H. G., E. MyBabiarz, D. D. Soderman, E. A. Ham and F. A. Kuehl, Jr. Evidence for a PGE-receptor in the rat kidney. Prostaglandins -17: 525, 1979.
Editor: Donald E. Wilson
820
Reed: 7-80
Acptd: 3-81
MAY
1981 VOL. 21 NO. 5
INTERACTIONS BETWEEN PARATHYROID HORMONE AND PROSTAGLANDINS ON RENAL CORTICAL CYCLIC AMP' Mark G. CurTie
and David M. Biddulph
Department of Anatomy Bowman Gray School of Medicine Winston-Salem, North Carolina 27103 ABSTRACT Effects of parathyroid hormone (PTH) and several prostaglandins (PGs) on cyclic AMP (CAMP) metabolism were studied and compared in isolated renal cortical tubules from male hamsters. Both production and intracellular degradation of CAMP were increased by PTH and each of the PGs tested (PGE2, PGEI, PG12). Production of CAMP was increased to similar levels by maximal concentrations of PTH and each PG, however, degradation of CAMP was significantly higher in response to PTH than with any of the PGs. This difference in intracellular degradation of CAMP was responsible for the much higher concentrations of CAMP in renal cortical tubules exposed to PGs (PGE1, PGE2, PG12) than to PTH. Submaximal amounts of each PG produced additive increases in CAMP concentrations in the presence of maximal amounts of PTH. Additivity of the combined responses was lost, however, as the PGs concentrations reached their maximas. The results suggest that renal PGs (PGE2 and PGI1) may modulate the effects of PTH on CAMP concentrations in renal cortical tubules.
' This work was supported by a grant to D.M.B. from the National Science Foundation, PCM 75-07661.
2 This work is taken in part from a thesis submitted to the Department of Anatomy, Bowman Gray School of Medicine of Wake Forest University, in partial fulfillment of requirements for the degree of Doctor of Philosophy. Present address: Department of Pharmacology, Washington University School of Medicine, St. Louis, MO.
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1981 VOL. 21 NO. 5
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PROSTAGLANDINS
INTRGWCTION Prostaglandins (PGs) have been reported to modulate the physiological actions of many peptide hormones (1). As hormonal modulators, PGs may either amplify or inhibit the effects of a hormone. There are many instances of PGs acting as hormonal modulators in the kidney (2). PGs are thought both to attenuate the renal actions of antidiuretic hormone and to amplify the vasodilatory effect of bradykinin (2). Although effects of PGs on PTH-dependent calcium and phosphate transport have not been reported, several studies have shown that prostaglandin El (PGE1) antagonizes effects of parathyroid hormone (PTH) on elevating cyclic AMP (CAMP) concentrations in rat renal cortical slices (2,3-6). Since CAMP is thought to mediate the actions of PTH (7), PGs have been proposed as an important local negative feedback mechanism for the renal effects of PTH (3-6). However, this inhibitory effect of PGEl on PTH-induced increases in CAMP was not observed in rabbit kidney slices (8). Using hamster renal cortical tubules, we also did not observe an inhibitory effect of PGE1, on the PTH response (9, 10). In our initial studies with PTH and PGE1, maximal concentrations of these two agonists, when added together, produced a nonadditive response; however, the combination of a submaximal concentration of PGEl with PTH resulted in an additive response (9). We proposed that the local release of PGs could amplify the effect of PTH on CAMP. In order to further test this hypothesis, effects of the naturally-occuring PGs (PGE, and PGI,) have been tested and compared with effects of PTH in the present study. MATERIALS AND METHODS Materials Male golden hamsters, Mesocricetus auratus were obtained from Engle Laboratories and were maintained on PurimLaboratory Chow and water ad libitum prior to experiments. Bovine parathyroid hormone (PTH) was purchased from Wilson and Co. (300 USP U/mg protein). Dilutions of PTH were made with acidified (pH 3.0) isotonic saline. Prostaglandin El (PGEI), prostaglandin E2 (PGE*), prostaglandin I2 (PGI,), and 6-ketoprostaglandin F,, (6-keto-PGF1,) were generously supplied by Dr. John Pike, Upjohn Co. PGEl and PGE, were dissolved in absolute ethanol and diluted to desired concentrations with incubation media such that the final ethanol concentration did not exceed 0.4%. PGI, and 6-keto PGF1, were dissolved and diluted to desired concentrations in a 20 mM Tris buffer, pH 9.0 just prior to use in experiments. Reagents for assay of CAMP were purchased from Diagnostic Products Corp. Anionexchange resin (AGl-X2) was purchased from BioRad Co. Tritiated nucleotides and adenosine were purchased from New England Nuclear. l-methyl-3isobutylxanthine (MIX) and all enzymes used in this study were obtained from Sigma Chemical Co. Other chemicals used were the best grades available and were obtained from standard suppliers.
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MAY 1981 VOL. 21 NO. 5
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Preparation
of Isolated
Renal Tubules
Isolated renal cortical tubules were prepared as previously described (10). Kidneys were rapidly removed, hemisected and transferred to ice-cold Hanks solution containing 1.8 mg/ml glucose. Corticies were separated from medullary tissue, pooled and minced finely with surgical scissors in 10 ml of an enzyme solution consisting of 0.4 mg/ml collagenase, 1 mg/ml hyaluronidase and 1.8 mg/ml glucose dissolved in Hanks solution containing 1 mM calTissues were incubated at 37°C for 60 min with air as the gas phase cium. in an oscillatory water bath and filtered through a single layer of nylon with an additional 40 ml enzyme-free solution. Filtrates were centrifuged at 50 x 9 for 2 min at room temperature. The pellets were resuspended in Hanks-glucose solution and centrifuged again at 30 x CJ for 2 min. This washing procedure was repeated twice. The final pellet was the preparation used for tubule incubations and contained approximately 40% of the original cortical protein. Incubation,
Extraction
and Assay of Cyclic
Nucleotides
Tubules, representing 2-3 mg protein per flask were incubated in 5 ml siliconized, Erlenmeyer flasks at 37°C in an oscillatory water bath in 1.5 ml Krebs-Ringer-Tris buffer, pH 7.4 containing 1 mM MgC12, 1 lllMCaC12, 1.8 mg/ml glucose and 20 mM Tris-HCl. In some experiments, designated in the text, MIX was added to obtain a final concentration of 5 mM. Parathyroid hormone or prostaglandins were added in 0.1 ml volumes following a 5 min preincubation period. Control solutions consisted of acid saline and ethanol. Final concentrations of ethanol did not exceed 0.4%. In some experiments tubules were pretreated for 15 min in trypsin (2.0 mg/ml) at 37°C. Pellets were then washed twice prior to further treatment. Trypsin inhibitor (Sigma) was added in 3-fold excess during subsequent incubation of tubules. Incubations were terminated by addition of 1 volume cold 5% TCA, followed by homogenization. Homogenates were centrifuged at 1,000 x 9 and supernatants transferred to glass tubes, extracted 4 times with 10 volumes of ether, boiled to remove residual ether and lyophilized. Pellets were dried, resuspended in 1 N NaOH and assayed for protein by either the method of Lowry -: et al (11) or the biuret reaction. Lyophilized supernatants were dissolved in Tris buffer, pH 7.4 and assayed for CAMP by the method of Gilman (12) as modified by Tovey -et al. (13). Known amounts of CAMP added to either tissue extracts or TCA and carried through the entire extraction procedure, were recovered quantitatively. Incubation of sample aliquots with excess phosphodiesterase for 60 min at 37°C resulted in complete hydrolysis of CAMP. Serial dilution of samples assayed for CAMP were linear over a lo-fold range. All samples were assayed in either duplicate or triplicate. In some experiments tubule suspensions were rapidly centrifuged (1000 x CJ for 1 min) prior to addition of TCA. Cyclic AMP was assayed in both the tubule pellet and supernatant (medium) of each sample.
MAY
1981 VOL. 21 NO. 5
807
PROSTAGLANDINS
,Preparation and Assay of cAMP Phosphodiesterase (PDE) PDE a c t i v i t y was measured in renal c o r t i c a l homogenates by the twostep method o f Thompson and Appleman (14). Renal c o r t i c a l tissue was homogenized and sonicated in 0.25 M sucrose containing 0.I M T r i s and 0.I mM EDTA (pH 7.6). Homogenates were then centrifuged at 4°C at 50,000 x f o r 30 min. Aliquots of the supernatant were assayed in a b u f f e r (0.4 ml t o t a l vol) containing 40 mM T r i s , (pH 8.0) I0 mM MgCl2, 3,75 mM mercaptoethanol, 0.I mM or 4 ~M c y c l i c AMP, 3H-cyclic AMP (60,000 cpm) and 0.2 ml of the enzyme preparation. Agents tested f o r effects on PDE a c t i v i t y were added in 20 ~I vol before the enzyme was added to i n i t i a t e the reaction. Incubations were f o r 5 min at 30°C with enzyme a c t i v i t y being l i n e a r over t h i s time period. Reactions were terminated by immersing reaction tubes i n t o a dry ice-acetone bath f o r exactly 12 sec followed by b o i l i n g f o r 45 sec. Each tube was then incubated f o r I0 min at 30°C in the presence of excess 5' nucleotidase. This reaction was terminated by addition of 1 ml of a I : I s l u r r y of BioRad anion-exchange resin (AGIX2, 200-400 mesh) containing 40% methanol as described by Thompson et a l . (15). Contents of the tubes were centrifuged at 1,000 x ~ f o r min and 0.4 ml a l i q u o t s of the supernatants were added to s c i n t i l l a t i o n v i a l s and counted f o r r a d i o a c t i v i t y . Enzyme a c t i v i t i e s were determined such that no more than 30% of the substrate was hydrolyzed during the reaction. S t a t i s t i c a l analysis of the data employed a t w o - t a i l e d t test and a m u l t i p l e comparison t e s t (16). RESULTS We have previously reported t h a t the PDE i n h i b i t o r , l - m e t h y l - 3 i s o b u t y l x a n t h i n e (MIX), at a concentration of 5 mM maximally potentiates the i n t r a c e l l u l a r accumulation of cAMP in isolated renal tubules in response to PTH and PGs ( I 0 ) . To determine r e l a t i v e rates of production and degradation of cAMP in response to PTH and the prostaglandins investigated in the present study, paired incubations of c o r t i c a l tubules were carried out in the presence and absence of 5 mM MIX. Production of cAMP was defined as the amount of cAMP accumulated in the presence of 5 mM MIX. Degrat i o n of cAMP was estimated as the d i f f e r e n c e in cAMP concentration in the presence and absence of MIX. In a series of time course experiments, maximally e f f e c t i v e concent r a t i o n s of PTH (7 U/ml) and the prostaglandins (15 ~g/ml) produced s i g n i f i cant (p < .01) increases in both cAMP production and degradation at each time i n t e r v a l examined (Fig. I ) . PTH and the E-series of prostaglandins produced s i m i l a r changes in both the k i n e t i c s and the magnitude of hormoneinduced increases in cAMP production. The k i n e t i c s of cAMP production due to s t i m u l a t i o n by PGI2 were s i m i l a r to that caused by PTH and the E-series of prostaglandins, however, the magnitude of cAMP production was s i g n i f i c a n t l y (p < .01) lower in tubules exposed to PGI2 when compared to the other hormones. The stable metabolite of PGI2, 6-keto-PGF~, did not
808
MAY 1981 VOL. 21 NO. 5
PROSTAGLANDINS
400
PGI,
350 300 250
i
400
-
350
-
300
-
250
-
200
-
150 100 50 O-
0246810 Figure 1. Time-course curves representing the effects of PTH (7 U/ml), PG12 (15 vg/ml), PGEl (15 pg/ml) and PGE2 (15 pg/ml) on production and degradation of cyclic AMP in isolated renal tubules. Production of cyclic AMP was measured in the presence of 5 mM MIX, while degradation of cyclic AMP was calculated as the difference in cyclic AMP concentrations in the presence and absence of MIX. Open symbols represent changes in control incubations. Points are means of three to nine samples ? SE or SE of the difference. significantly elevate CAMP levels at concentrations tested (1 and 15 ug/ml). In contrast to the similarities between PTH and PGs on CAMP production, degradation of CAMP was significantly (p < .Ol) lower in tubules exposed to PGs than to PTH. Degradation of CAMP, relative to amounts produced, ranged from 25-50% in response to the PGs tested and increased during the time intervals investigated in a way which was similar for all PGs. Degradation
MAY 1981VOL. 21 NO. 5
809
PROSTAGLANDINS of CAMP in response to PTH ranged from 85-95% of the amounts produced and was responsible for the much lower net concentrations of CAMP in tubules treated with PTH than with any of the PGs. Concentration-response experiments with each hormone (Fig. 2) demonstrated that increases in degradation of CAMP were effected over the same
350 PG12
300 250 200 150
I
I
I
I
I
04.607 .07 0.7 70 70.0 U/ml
I PGE2
300
/++
250
9 c,
200 150
04’.01 0.1 1.0 10.0 100.0 I
I
1
I
1
p9 /ml Figure
810
2. Concentration-response curves representing the effects of varying concentrations of PTH, PG12, PGEI, and PGEP on production and degradation of cyclic AMP in isolated renal tubules. Incubations were for 5 min following the addition either of hormone or the appropriate control. Production of cyclic AMP was measured in the presence of 5 rrEil MIX, while degradation of cyclic AMP was calculated as the difference in cyclic AMP concentrations in the presence and absence of MIX. Points are means of three to nine samples + SE or SE of the differences.
MAY 1981VOL. 21 NO. 5
PROSTAGLANDINS
concentration range of hormone which increased production of CAMP. As was noted in time-course experiments, production of CAMP reached similar levels with maximal concentrations of PTH and PGs with PGIp being slightly Degradation of CAMP, in response to the less effective than the E-series. PGs, was however only increased to about half that produced by PTH. Statistically significant increases in production of CAMP were produced by all three PGs at concentrations as low as 0.01 pg/ml (28 nM). These differences in the amounts of CAMP degradation between tubules exposed to PTH and the prostaglandins were apparently not due to the direct effects of these hormones on the enzyme phosphodiesterase, since phosphodiesterase activity in renal cortical homogenates (either crude or 50,000 x 9 supernatants) was not directly affected by the addition of maximal concentrations of any of these agents. Experiments in which tubules were assayed for CAMP separately from their respective incubation media showed that the observed differences in the amounts of CAMP degraded were not due to differences in amounts of CAMP released from the cells, Neither PTH nor the PGs had a significant (p > .05) effect on the percentage of CAMP released from the tubule cells. Since the isolated cortical tubule preparation used in these experiments consisted of a mixed population of cells, experiments were designed to determine whether PTH and the PGs were affecting CAMP changes in a common target cell population. Essentially, these experiments searched for competition between maximal concentrations of PTH and PGs and investigated interactions between submaximal concentrations of PGs and a maximal concentration of PTH. The combination of maximally effective concentrations of PTH and PGEP, either in the presence or absence of MIX resulted in responses which suqqested interactions between these two hormones (Table 1). In the absence of-MIX, the combination of the maximal concentrations of PTH (7 U/ml) and PGE2 (30 (p < .Ol) less than uq/ml) resulted in a response which was significantly the single PGE2 response. In the presence of MIX, the combined response was significantly (p < .Ol) greater than that induced by either hormone The alone but considerably less than the sum of PTH and PGE2 responses. combination of a submaximal concentration (.05 ug/ml) of PGE, with a maximal concentration of PTH (7 U/ml) in the absence of MIX caused an increase in the CAMP response which was significantly (p < .Ol) greater than either agent alone; the response however was not completely additive (Table 1). Combination studies were also done with PTH and PG12 (Table 2). The combination of the maximal concentrations of PTH and PG12 boththe presence and absence of MIX resulted in responses which were less than completely additive. In the presence or absence of MIX with an incubation period of 1 min, the combination of the maximal concentrations of PTH (7 U/ml) and PG12 (30 ug/ml) resulted in responses which were completely nonadditive (Table 2). However, the combination of PTH and PG12 in the presence of MIX resulted in a response which was significantly (p < .Ol) greater than with either hormone alone but was much less than completely additive when the incubation time was 5 min. The combination of a submaximal concentration (.05 ng/ml) of PGIp with a maximal concentration of PTH resulted in a response which was significantly (p < .Ol) greater than with either response alone.
MAY 1981VOL. 21 NO. 5
811
PROSTAGLANDINS
TABLE Effects
of PTH and PGE2 on Cyclic in Isolated
Treatment
1 AMP Concentrations
Renal Cortical
Tubules
CAMP Concentration
(pmole/mg
1 min incubation - MIX Control
protein)
5 min incubation
+ MIX
+ MIX
1.6 + 0.3
6.4 f 0.5
11.7 -I 0.3
23.3 f 1.8
146.7 + 5.4
333.1 f 27.6
PGEz (30 pg/ml)
80.1 + 0.7
97.7 + 3.0
PGE2 (0.05 pg/ml)
29.4 + 2.0
PTH
PTH
(7 U/ml)
335.8 k
___
8.8
_-_
(7 U/ml) 57.6 + 3.6*
184.9 ?r 4.1*
557.1 f 10.1*
PiE2 (30 ng/ml) PTH
(7 U/ml) 38.0 f 0.9*
___
_--
PiEz (0.05 ug/ml)
Tubules were incubated in the presence or absence of 5 mM MIX for 1 or 5 min after additions of the above agents. Values are the means of three to six incubations f SE. *
812
Significantly (p < 0.01) different in the combination.
from both single
MAY
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involved
1981 VOL. 21 NO. 5
PROSTAGLANDINS
TABLE 2 Effects
of PTH and
PG12on
in Isolated
Cyclic AMP Concentrations
Renal Cortical
Tubules
CAMP Concentration
Treatment
(pmole/mg
1 min incubation Control
MIX
5 min
7.6 f 0.6
23.1 * 1.4
149.3 + 6.7
PGIz (30 ug/ml)
64.5 k 2.2
74.9 + 2.1
PGIp(0.05ng/ml)
38.8 * 2.3
_--
63.9 f 1.8
157.5 f 5.7
PTH
(7 U/ml)
incubation
+ MIX
+ MIX
1.1 + 0.2
PTH
protein)
12.2 k
0.5
317.4 -I 22.4 238.8 f
2.4
--_
(7 U/ml) 459.8
+ 15.9*
P+G12 (30 pg/ml) PTH
(7 U/ml) 53.4 f 1.5*
_-_
_-_
PkIp (0.05 ug/ml)
Tubules were incubated in the presence or absence of 5 mM MIX for 1 or 5 min after additions of the above agents. Values are the means of three to six incubations + SE. *
Significantly (p < .Ol) different in the combination.
MAY 1981VOL. 21 NO. 5
from both single
responses
involved
813
PROSTAGLANDINS
Since the combination experiments revealed competition between the maximal concentrations of PTH and the PGs, experiments were designed to determine if the observed competition involved the PTH receptor. We have previously shown that mild trypsin treatment of these tubules selectively inhibits the PTH response without affecting the PGEl response (9). This effect of trypsin on the PTH response is thought to be due to a direct action of trypsin on the PTH receptor (17). The present study utilized trypsin treatment of the tubules to determine whether the PTH response The trypsin could be differentiated from the PGE2 and PGIp responses. treatment inhibited the PTH induced response by 70%; however, the responses due to stimulation by either PGE;! or PG12 were not significantly (p > .05) affected (Table 3).
TABLE Effects
of Pretreatment
with Trypsin
PGEl and PG12-induced Isolated
Cyclic
on PTH, PGE1,
AMP Responses
Renal Cortical
Treatment
in
Tubules
CAMP Concentration
- Trypsin Control
3
(pmoles/mg
+ Trypsin
protein)
% Inhibition
16.0 +
0.5
14.9 f
0.5
9
303.7 +
8.0
105.9 f
6.1
69
PGEl (30 ug/ml)
312.7 f 10.5
302.9 +
5.4
3
PGE2 (30 ug/ml)
332.6 f
9.1
311.8 it:10.1
7
PGI2 (30 us/ml)
269.3 f 11.4
238.6 k 10.4
12
PTH
(7 U/ml)
Tubules were pretreated for 15 min at 37" with trypsin (2 mg/ml) before washing and adding a 3-fold excess of trypsin inhibitor. Tubules were then incubated for 5 min in the presence of 5 mM MIX after the addition of the above agents. Values are the means of three to six incubations * SE.
814
MAY 1981 VOL. 21 NO. 5
PROSTAGLANDINS
study
Interactions (Fig. 3).
between
PGE2 and PGI;, were
PGE,
F
also examined
in the present
(30pg/ml)
. .. ..... ...........................,................. . .
Basal
,f.. ..,.... ,:.:. ~:,~:.:.~;.:.:.:.;..:.:.:‘:‘:‘:‘: ..,..._. i,.........~~.~.,~.~~~~~,~.~.~.~ . ..._._._. ...._._. ,._._ ... ._.,..._._. ,..._., L...... :,.,.. ,..,._._.,..._._........
40
.Ol
.I
PGI, Figure
MAY
1.0 IO 100
~9 /ml
3. Effects of simultaneous addition of PGEl (0.05 pg/ml or 30 pg/ml) and varying concentrations of PGI2 on cyclic AMP concentrations in isolated renal tubules. Incubations were for 5 min after addition of prostaglandins and included 5 mM MIX (PGI,, closed circles; PGE%, horizontal bars; PGE2 + PG12 at indicated concentrations, open circles). Each point is the mean of three to six samples +_ SE, Bars represent means of three to six values +_ SE.
1981 VOL. 21 NO. 5
815
PROSTAGLANDINS
The simultaneous addition of a maximal concentration of PGI, (30.0 ug/ml) with an equivalent concentration of PGE2 produced a combined response which was significantly (p < .Ol) lower than the PGE;! response but signiCombination of a ficantly (p < .Ol) greater than the PG12 response. submaximal concentration (.05 pg/ml) of PGE, with varying concentrations of PG12 gave results, suggesting additivity of the two responses at low concentrations of PGI, (Fig. 3). Similar results were obtained using PGEl in place of PGE2 (data not shown).
DISCUSSION The present study demonstrates that PTH, PGI,, PGE, and PGEl increase CAMP concentrations in hamster renal cortical tubules, confirming Previously reported observations in renal cortical tissue from other Species The lowest effective concentration for PGI,, PGE, and (4, 5, 6, 8, 18). PGEl to cause an increase in CAMP production was 10 ng/ml (28 nM), a concentration similar to that reported in studies utilizing rat renal cortical In the hamster renal cortex, PTH and the PGs appear to tissue (4, 5, 18). Degradation of CAMP, affect the production of CAMP in a similar manner. however, seems to be differentially affected by the two different types In tubules exposed to PTH, CAMP breakdown was much greater of hormones. Neither PTH nor the PGs had a direct than in tubules exposed to PGs. effect on PDE activity in renal cortical homogenates, suggesting that the differences observed in intact tubule cells were due to some indirect Lack of direct effects of both PTH as well as PGEl on POE activeffect. ity of renal cortical homogenates have been reported in other species (5, 19) and suggests that some other intracellular factor, which is presumably differentially affected by PTH and PGs is regulating PDE activity. Intracellular calcium, which we have previously shown to be altered in this system by PTH (20) and which is known to regulate PDE activity in some tissues (21) may be involved. The present study also suggests that a common cell type within renal cortical tubules may be responsive to both PTH and the prostaThe results appear to be consistent with effects of glandins tested. PTH and PGs on overlapping but not conqruent populations of renal Production of CAMP, as measured in intact cells in the cortical cells. presence of MIX did not demonstrate summated responses to simultaneous addition of hormones at maximal concentrations. In renal cells without PDE inhibition, the combined response to PTH and PGE, was clearly less than with PGE2 alone. This diminished response to PGE2 in the presence of PTH may be reflecting the differential effects of these hormones on degradation of CAMP. The observed competition between PTH and the prostaglandins at maximal concentrations apparently does not involve a common receptor site. Pretreatment of the tubules with trypsin selectively inhibited the PTH response without affecting the prostaglandin-induced cyclic AMP response. The effect of try sin treatment is thought to be at the receptor level since the binding of P251 _ labeled PTH is greatly reduced in membranes previously treated
816
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We have previously reported that trypsin treatment rewith trypsin (17). duced PTH, but not PGEl-induced increases in Cyclic AMP (5-j).The current study verifies the previous work and extends it by comparing the effect of of trypsin treatment on the responses caused by PTH, PGEz and PGI,. Since the competition between PTH and the prostaglandins does not appear to be at the receptor level, the likely site of occurrence for the observed ComPetition appears to reside at the adenylate cyclase level. If this interpretation is correct, the presence of a multireceptor-coupled adenylate cyclase system is suggested in renal tubule cells in which the combined number of hormonespecific receptors is in excess of available adenylate cyclase. Such a relationship between hormone-specific receptors and adenylate cyclase has been implicated in the fat cell (22) and is in general agreement with the "spare receptor hypothesis" (23). We cannot rule out the possibility however, that PGs might inhibit the reaction of PTH Further work is with its receptor by a non-competitive mechanism. required to determine the exact nature of PTH-PGs interactions in this tissue. In the present study, the combination of a submaximal concentration of either PGEz or PG12 with PTH resulted in a partially additive response in the absence of MIX. The results suggest that the local release of these prostaglandins from renal cellular elements might act to amplify the PTH-induced accumulation of cyclic AMP. This proposal conflicts with the hypothesis proposed by Beck et al. (3), who showed that PGEl c-ireduced the PTH-induced increase in cyclic AMP in the rat and suggested that the response is attenuated by the local release of prostaglandins. Several recent studies concerning the effect of PGE, on the PTH-stimulated increases in cyclic AMP in rat kidney slices, generally support this hypothesis (4, 5, 6). In a study which utilized renal cortical slices from rabbits, however, no inhibitory action of PGEl on the PTH-induced accumulation of cyclic AMP was reported (8). Thus, the observations noted in the present study with hamsters and in the study with rabbits differ considerably from reported observations utilizing the rat kidney as a model. These differences possibly reflect sensitivity to PTH and prostaglandins. In the rat, PTH is a more potent agonist than PGs in causing increases in cyclic AMP (3, 5, 6). Thus, the combination of the weak agonist with the strong agonist would be expected to produce a response which is less than the strong agonist's response alone, if competition is occurring at some common point. However, PGE1, PGE, and PG12 are more potent agonists than PTH in causing net increases in cyclic AMP in both the hamster and the rabbit (8). Thus, the combination of PTH and PGs, in these two species, would be expected to produce a response which is greater than the PTH-response alone. Interactions between PTH and the more physiological prostaglandins used in the present study (PGE2 and PGI,) upon the tissue accumulation of CAMP to our knowledge have not been investigated previously. The results from experiments investigating interactions between the E-series of prostaglandins and PG12 suggested that these agonists stimulated CYClic AMP synthesis in a common population of cells. This conclusion iS based on the lack of additive responses to the combination of
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maximal concentrations of the E-series of prostaglandins and PGIp. The combination of submaximal amounts of these agonists produced additive Nonadditive responses for maximal concentrations of the E-series responses. and PGI, have also been reported in rat renal cortex (24) and rat liver (25). The basis for this interaction between the E-series and PGI2, while not delineated by the present study, most probably involves competition at receptor sites, as has been reported in platelets (26), or competition between separate receptors for the two types of prostaglandins for a comnon adenylate cyclase, as has recently been suggested in liver Providing support for the occurrence of the latter possibility cells (25). in this system is the report based on binding studies of a receptor in the kidney which specifically binds PGE with high affinity (27).
In conclusion, the present study revealed that PTH, PGI,, PGEl and PGE, produced increases in cyclic AMP concentrations in isolated renal cortical tubules from hamsters. PTH and the prostaglandins appeared to affect the production of cyclic AMP in a similar manner; however, these two different types of hormones seemed to affect the degradation of cyclic AMP differentiThe combination studies suggested that PTH and these prostaglandins ally. might share a common target cell type in affecting a change in cyclic AMP concentrations. The results also suggested that the local release of prostaglandins from renal cellular elements might act as physiological amplifiers of the PTH-induced accumulation of cyclic AMP in the hamster. The implication of this hypothesis is that prostaglandins might augment the renal actions of PTH by amplifying the influence of the hormone on cyclic AMP, although we are unaware of any studies in which effects of PGs on tubular transport of calcium or phosphorous have been reported. Finally, the study points to the need for a thorough investigation of any effect PTH might have on prostaglandin metabolism in the kidney. ACKNOWLEDGEMENT The authors are indebted to the secretarial for typing of this manuscript.
aid of Daphne
B. Styers
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