Possible modulation renal prostaglandin
of rat production
by oxygen
TERRY V. ZENSER, MONTE J. LEVITT, AND BERNARD B. DAVIS Geriatric Research, Education and Clinical Center, Veterans Administration Hospital, and Department of Medicine and Biochemistry, St. Louis University, St. Louis, Missouri
ZENSER, TERRY V., MONTE J. LEVITT, AND BERNARD B. DAVIS. Possible modulation of rat renal prostaglandin production by oxygen. Am. J. Physiol. 233(6): F539-F543, 1977 or Am. J. Physiol.: Renal Fluid Electrolyte Physiol. 2(6): F539F543, 1977. -The effect of oxygen on in vitro prostaglandin production by slices of rat renal inner medulla was evaluated. Oxygen increased prostaglandin production in a dose-response fashion. Double reciprocal plots of the relationship revealed a half-maximal stimulation of prostaglandin (PG) E, production by 15.1% or 143 PM 0, and of PGFz, by 16.8% or 160 PM 0,. An increased prostaglandin production at 95% 0, was measurable within 2 min of incubation and was maximal at approximately 15 min. Oxygen also increased inner medullary cyclic AMP content with a similar time course. Lowering oxygen did not alter the response of the inner medulla to increase cyclic AMP content when exposed to vasopressin. PGE., increased cyclic AMP at 5% 0, but not at 95%, while PGF, was ineffective at either oxygen concentration. In the presence of structurally dissimilar prostaglandin synthetase inhibitors, indomethacin and sodium meclofenamate, there was a reduction of basal cyclic AMP content as well as prostaglandin production. PGE2 increased cyclic AMP at 95% Oe, in the presence of prostaglandin synthetase inhibitors while PGF& remained inactive. This suggests that the oxygen-mediated increases in cyclic AMP content may be due to endogenously produced PGE,. The data are consistent with oxygen modulating inner medullary production of prostaglandins, and with endogenously produced prostaglandins exerting a hormone-like action in the inner medulla. cyclic AMP; system
vasopressin;
renal
medulla;
renal
countercurrent
MEDULLA OF THE KIDNEY is the site ofrenal synthesis of prostaglandins (7, 9). That synthesis appears to proceed in the interstitial cells of the inner medulla (6), and results in the immediate release of prostaglandins rather than their accumulation in the cells (7). This localization of the renal prostaglandin synthetic mechanism to the inner medulla places the process in a very unique environment. The countercurrent arrangement of flow of tubule fluid through the loops of Henle and of blood through the vasa recta results in the generation of an increased solute concentration in the interstitial fluid of the inner medulla. This well-known mechanism provides the mammalian kidney with the capacity to elaborate a hypertonic urine, and during antidiuresis results in an inner medullary solute concentration which approximates that of THE INNER
63125
urine, approx. 2,800 mosmol/kg H,O in the rat (4). In addition to maintaining a high inner medullary solute concentration, the countercurrent system also functions to maintain a low ambient oxygen concentration in the inner medulla. Therefore, the inner medullary PO, during antidiuresis has been estimated to be in the range of 20-40 mmHg (2, 15). While inner medullary PO, is low, that fact has not generally been considered to be a major factor in either limiting or modulating inner medullary metabolic processes. However, we have recently observed a dependency of tissue cyclic AMP content in slices of inner medulla on ambient PO,. Increasing PO, increased the medullary cyclic AMP content, and the relationship between these two parameters was linear (11). Both indomethacin and meclofenamic acid inhibited oxygeninduced increases in cyclic AMP content. This suggested that the observed effect was dependent on an augmentation of prostaglandin production with increasing oxygen concentration. This paper is a report of the investigations of the effects of oxygen concentration on prostaglandin production, cyclic AMP generation, and the interrelationship between the two in the inner medulla of the rat kidney. MATERIALS
Experimental
AND
METHODS
Procedure
Unlabeled prostaglandin (PG) E, , EZ, F1, and F, were generously provided by The Upjohn Company, Kalamazoo, Mich. Sodium meclofenamate was kindly donated by Dr. James E. Gleichert, Parke, Davis & Co., Ann Arbor, Mich. Tritiated PGE, (117 Ci/mmol), PGF, (178 Ci/mmol), cyclic AMP (38 Ci/mmol) and Aquasol were purchased from New England Nuclear, Boston, Mass. Rabbit antisera to either PGE’s or PGF’s were obtained from Regis Chemical Co., Morton Grove, Ill. Antibodies, Inc., Davis, Calif. was the source of goat antisera to rabbit gamma globulins. Normal rabbit serum was obtained from Miles Laboratories, Elkhart, Ind. Indomethacin and arginine vasopressin (grade VI, synthetic) were purchased from Sigma Chemical ComMO. 1-Methyl-3-isobutylxanthine pany, St. Louis, (MIX) was purchased from Aldrich Chemical Co., Milwaukee, Wis. Cyclic AMP binding protein was prepared as described by Gilman (13). Gas mixtures were obtained from Liquid Carbonic, St. Louis, MO. All other
Downloaded from www.physiology.org/journal/ajprenal at American Univ of Beirut (193.188.128.021) on June 19, 2019.
ZENSER,
F540 chemicals were purchased in the highest available grade from standard sources. Male Sprague-Dawley rats weighing 250-300 g were from Eldridge Laboratory Animals, Barnhart, MO. Tissue Incubation The rats were anesthetized with ethyl ether, and the kidneys were removed quickly and placed in cold 0.85% NaCl. The kidneys were bisected in the coronal plane and slices obtained with a Stadie-Riggs microtome were placed on filter paper moistened with cold 0.85% NaCl. Each slice was then carefully dissected to isolate the inner medulla. The moist tissue was weighed to the nearest milligram on an analytical balance. Slices weighing 9-14 mg were placed in a 25ml flask containing 2 ml of incubation medium. The medium consisted of Krebs-Ringer bicarbonate buffer at pH 7.4 containing 1 mg/ml each glucose and bovine serum albumin. The potent inhibitor of cyclic nucleotide phosphodiesterase MIX (2 mM) was also present in the incubation medium. Prior to incubation each flask was purged with gas and the oxygen concentration of the medium was verified with an oxygen monitor (Yellow Springs Instrument Co. model 53). The slices were first incubated for 20 min with 0% 02, 5% C02, and 95% N, and then transferred for 20 min to flasks previously equilibrated with 0, 5, 10, 20, or 95% 02, with 5% CO, and the balance N,. In a separate series of experiments the first 20.min incubation was in 5% 02, 5% CO*, and 90% N,. The second incubation was in either 5% 02, 5% CO*, 90% N, or 95% 02, 5% CO,. As above, 2 mM MIX was in the incubation medium. PGE, 2.9 x 10m4 M, PGF2, 2.9 x lob4 M, or vasopressin 3.5 x lo-* M were added to appropriate flasks as indicated in the results. Such additions were made 5 min prior to the termination of the second incubation. When indomethacin (100 pg/ml) or sodium meclofenamate (50 pg/ml) were used they were present throughout the initial and the second incubation. For the experiments examining the time course of Os-mediated increases in inner medullary PGE, production and cyclic AMP content, slices were preincubated at 0% 02, 5% COt2, 95% N, for 20 min. They were then transferred to flasks gassed with 95% 02, 5% CO,. At the times indicated in text (2, 5, 10, 15, and 20 min), the reaction was terminated and the medium analyzed for PGE, and the slice for cyclic AMP. Care was taken to gas the flasks during transfer with the appropriate gas mixture. All incubations were performed at 37°C with 100 cycle/min agitation in a Dubnoff metabolic incubator. At the end of this incubation period, the tissue was removed, homogenized, and extracted for cyclic AMP. The incubation medium was immediately frozen and stored at -35OC for later analys1s. Prostaglandin
Analysis
The incubation mediums were analyzed generally within 1 wk, although it was found that storage at -35°C for more than 1 mo did not alter the PG content. A trace amount of tritiated PGF2, or PGE, (approx.
LEVITT,
AND
DAVIS
1,000 counts/min or 3 pg) was added to each 0.5.ml aliquot of thawed medium. After adjustment to pH 3.03.5 with 1.0 N HCl, each aliquot was extracted twice with 1.5 ml of ethyl acetate. Combined extracts were evaporated to dryness at 40°C under N,. Dried extracts were dissolved in 0.5 ml of assay buffer at pH 7.0 consisting of 0.01 M sodium phosphate, 0.15 M sodium chloride, and 1% normal rabbit serum. An aliquot of 0.050 ml was removed to determine the recovery of PG during extraction (85-95%). Reported values were corrected for recovery. Additional aliquots were analyzed in duplicate to determine the PG content. Radioimmunoassay for PGE and PGF were performed as described previously (16). Antibody plus [“H]PG (2,000 counts/min, 5.0 pg) and PG standards or unknowns (0.05 ml) were incubated in 1 .O ml of pH 7.0 buffer at 4°C. After 4 h, goat antiserum was added to rabbit gamma globulins and incubation continued for 20 h at 4°C. The precipitate was collected by centrifugation at 3,000 rpm for 30 min, dissolved in 1.0 ml of 0.1 N NaOH, and counted. Standards and unknowns were routinely run in duplicate with the reproducibility between duplicate samples averaging 3.4%. Unknowns exhibited linearity with respect to sample dilutions and combinations of unknowns and standards were additive. Cross-reactivity of PGE antibody with PGFzu or PGD, and PGF antibody with PGE, and PGD, was approx. 1% at the 50% displacement level. Thus, the assay technique would distinguish among the three major classes of prostaglandins known to be produced by the kidney (5). The PGE antibody would not, however, distinguish PGE, from PGE, , nor would the PGF antibody distinguish PGF1, from PGF2,. However, it has been previously reported that only the 2 series prostaglandins are produced by the kidney (8, 12). Preliminary experiments utilizing radiolabeled PGE, indicated no detectable PGE, metabolism by the inner medulla, a finding consistent with previous reports (1). Therefore, it was assumed that under the conditions of these experiments the PGE and PGF antibodies were measuring PGE, and PGF2,, respectively. Logit transformations of the standard curves resulted in linear functions between 0.1 and 10 ng PG per tube, equivalent to 2.0200 ng PG/ml of incubation medium. Prostaglandin wet content is expressed as nanograms per milligram weight of tissue. Cyclic AMP Analysis Cyclic AMP was extracted
by homogenizing
slices in
0.5 ml of 50 mM sodium acetate buffer (pH 4.0) at 95°C (17). The homogenate was heated for 5 min at that temperature. Cooled homogenates were spun at 3,000 rpm for 10 min at 4°C. Cyclic AMP was determined in the supernatant by the protein-binding method of Gilman (13). Cyclic AMP recovery was monitored by addition of 2,000 counts/min of cyclic [ 3H]AMP to the extraction buffer. Each reported result was corrected for recovery and represents the mean t SE of three incubation samples, each analyzed in duplicate. Statistical differences were evaluated by Student t test for unpaired values.
Downloaded from www.physiology.org/journal/ajprenal at American Univ of Beirut (193.188.128.021) on June 19, 2019.
OXYGEN
MODULATION
OF
RENAL
PROSTAGLANDIN
F541
PRODUCTION
RESULTS
There was an increase in the inner medullary production of both PGE, and PGFza with increasing oxygen concentration. Values for PGE, at 0, 5, 10, 20, and 95% were 1.2 t 0.2, 4.8 t 1.0, 9.9 t 1.6, 14.5 t 2.2, and 27.0 t 3.5 ng/mg wet tissue wt per 20 min, respectively. The values for PGF% were 0.2 t 0.01, 0.6 t 0.02, 1.2 t 0.2, and 2.5 t 0.3 ng/mg wet tissue wt per 20 min, respectively. While the values for PGF, were considerably less than those for PGE, the 0, concentrations which gave the half-maximal production of PGF2,, 16.8% or 160 PM, was approximately that of PGE2, 15.1% or 143 PM. The Vmax for PGE, and PGF2, was 25.6 and 3.0 ng/mg wet wt per 20 min, respectively (Fig. 1). The time course of the oxygen response at 95% O3 is illustrated in Fig. 2. A response was measurable within 2 min (the earliest time examined), and appeared to be maximal within 15 min. The time curve for PGFa was similar to that of PGE,. In the experiments in which 5% 0, was used in the preincubation phase, PGE, production was 6.8 t 0.5 ng/mg wet wt per 20 min when the gas phase of the second incubation was 5% 0’) and 21.6 t 2.0 when it was 95% 0,. There Was an effect of oxygen to increase cyclic AMP content in slices of inner medulla (Table 1). At 5% 0, the cyclic AMP content was 3.6 t 0.4 pmol cyclic AMP/ mg wet wt and this increased to 21 t 2 at 95% 0,. Figure 2 also contains the time curve for oxygen-induced increases in cyclic AMP content. The curve can
be seen to exhibit characteristics similar to the one for PGE,. As shown in Table 1, the responsiveness to vasopressin was maintained at the lower oxygen concentration. Cyclic AMP content at 95% 0, with vasopressin 3.5 x lo+ M (58 _+ 6 pmol cyclic AMP/mg wet wt) was not measurably different from the value at 5% O2 (45 t 6). Since the basal cyclic AMP content was lower at 5%, the relative cyclic AMP increase was actually greater at 5% than at 95% Oz. PGEz, 2.9 x 10m4 M, caused an increase in cyclic AMP content at 5% 0, but not at 95% 0, (Table 1). PGE*, however, increased cyclic AMP content at 95% O2 when sodium meclofenamate (50 pg/ml) 14 t 0.9 pmol cyclic AMP/ i
u
15
Of
2
5,
101 Time [minutes]
154 xi-
FIG. 2. Time course of O,-mediated increases in inner medullary PGE production and cyclic AMP content at 95% 0,. Slices were preincubated at 0% 0, for 20 min and then transferred to flasks with 95% 0, for indicated times. Values for prostaglandins and cyclic AMP are cumulative from 0 time to indicated time.
I
5
I
r
I
15
10
l/O
2
20
25
TABLE 1. Effects of prostaglandins, vasopressin, meclofenamate on inner medullary cyclic AMP content at high and low oxygen tension
Control AVP PGE, PGF, pm01 cyclic AMP/mg wet tissue wt
ImMl
95% 0, x: SE + Meclofenamate, i SE
l/O
2 WI
FIG. 1. Double reciprocal plots of effects of medium substrate (0,) on inner medullary PGE, (A) and PGF,, (B) production. Following preincubation at 0% 0, for 20 min, gas phase oxygen concentration was varied from 5 to 95% for 20 min.
and
5% 0, x SE + Meclofenamate, 2 SE
22 2
58 6
24 3
23 4
3.8 0.4
54 6
14 0.9
4.2 0.3
4.5 0.4
45 6
11 0.9
4.3 0.5
3.5 0.4
46 5
12 1.1
4.0 0.3
50 pg/ml
50 pglml
Slices were incubated at 5% 0, for 20 min as indicated to flasks containing 5% or 95% 0, vasopressin (AVP) (3.5 x 10m8 M), PGE, (2.9 (2.9 x 1O-4 M) were present during the last 5
and then transferred for 15 min. Arginine x 10e4 M), and PGF, min of incubation.
Downloaded from www.physiology.org/journal/ajprenal at American Univ of Beirut (193.188.128.021) on June 19, 2019.
F542
ZENSER,
mg wet wt was added to the incubation medium. PGF2,, 2.9 x 10m4 M, had no effect on cyclic AMP content at either 5% 0, or 95% 0, in the presence of either indomethacin or meclofenamate. At the concentrations used both indomethacin and meclofenamate blocked the effect of 95% 0, to increase the production of PGE, (cl.0 ng/mg wet tissue), PGF, (~0.2 ng/mg wet tissue), and cyclic AMP (2.8 t 0.4 pmol/mg wet tissue). However, neither indomethacin nor meclofenamate blocked the increase in cyclic AMP content mediated by vasopressin (54 t 6 pmol/mg wet tissue). DBCXJSSION
The data indicate that in slices of rat inner medulla there is a relationship between ambient oxygen and production of both PGE, and PGF&. Analysis of that relationship estimates the half-maximal value for prostaglandin production as 143 PM 0, for PGE, and 160 PM for PGF2,. These oxygen concentrations correspond to P%‘s of 115 mmHg and 128 mmHg, respectively, and are just above the upper limits of the Po,‘s of the arterial blood in normal conditions. They are considerably higher than the PO& of 20-40 mmHg or approximately 3-5% O2 which are estimated to occur in the inner renal medulla during antidiuresis (2, 15). It has been proposed that intramedullary PO, is low because of the countercurrent exchange function of the blood flow to that area of the kidney. Of interest is the fact that the efficiency of the countercurrent exchange process is very sensitive to the rate of blood flow. Increases in flow rate decrease efficiency of exchange (16). A range of inner medullary PO& has been reported (3). The inner medulla contains a system, therefore, which could possibly meter the amount of oxygen available at the site of the prostaglandin synthetic system. Our data suggest that the effect of oxygen to stimulate PGE, production at 25 mmHg is approximately 20% of the half-maximal value. The theoretical maximal value for inner medullary PO, under physiological circumstances would be the oxygen concentration of the renal artery, approx. 100 mmHg. That value is approx. 86% of the half-maximal effect of oxygen on PGE, synthesis. In addition, increases in prostaglandin production produced by oxygen concentrations within the physiological range are associated with increases in medullary cyclic AMP content (Fig. 2). These in vitro findings are consistent with the concept that 0, plays a role in the physiological regulation of prostaglandin production by the inner medulla of the kidney. These experiments were designed to test the effect of oxygen on prostaglandin production. It has been postulated by Flower and Blackwell (12) that phospholipase A*, which provides arachidonic acid as substrate for the
LEVITT,
AND
DAVIS
cycle-oxygenase reaction, could exert a regulatory action in prostaglandin synthesis. It has been shown that hormones such as bradykinin and angiotensin II which increase prostaglandin production do so at least in part by increasing availability of arachidonic acid (10, 19). The experiments of Flower and Blackwell (12), and Danon et al. (10) were performed at 95% 02, a concentration of oxygen which would not be expected to seriously limit prostaglandin production. In our experiments an attempt was made to isolate oxygen as a variable. The data indicate that oxygen, in concentrations ambient to the inner medulla, can affect the rate of prostaglandin synthesis. These results are in no way incompatible with the concept or the results of Flower and Blackwell (12). Both substrates would be expected to be important in the determination of the rate of bimolecular reaction. The data do not distinguish among the various mechanisms by which oxygen might exert this effect. In general, it is possible that oxygen either increases the availability of the fatty acid precursor, arachidonic acid, of the prostaglandins or that it increases the catalytic efficiency of prostaglandin synthetase, a reaction which utilizes oxygen as a substrate. Oxygen has an effect of increasing cyclic AMP content in slices of inner medulla, but vasopressin-induced increases in cyclic AMP content were similar at low and high oxygen. Sodium meclofenamate decreased cyclic AMP content at 95% but not at 5% oxygen. That agent had no effect on vasopressin-induced increases in cyclic AMP content. Those observations combined with the fact that PGE, increased inner medullary cyclic AMP content at 5% but not at 95% oxygen and that PGF2, had no effect on cyclic AMP content at either oxygen concentration, suggests that the oxygen-induced increase in cyclic AMP content is mediated by endogethe ability of nously produced PGE,. Furthermore, PGE, to increase cyclic AMP content at 95% oxygen in the presence but not in the absence of inhibitors of prostaglandin syn .thetase suggests that it is possible to generate enough endogenous PGE, to exert a maximal effect on cyclic AMP generation. Whether or not there is a physiological process mediated by this series of reactions remains to be determined. The data, however, are all consistent with endogenously produced prostaglandins exerting a hormone-like action in the inner medulla. We thank Mrs. Maryanne B. Sandusky, Ms. Liz Newkirk, and Ms. Peggy Egan for their skilled technical assistance, and Mrs. Dorothy Nabors for secretarial assistance. Appreciation is expressed to Dr. John E. Pike for providing the prostaglandins, and to Dr. Charles M. Coleman for providing the prostaglandin antisera. This work was supported by the Veterans Administration. Received
for publication
25 March
1977.
REFERENCES 1. ANGGARD,
E., S. 0. BOHMAN, J. E. GRIFFIN III, C. LARSSON, AND A. B. MAUNSBACH. Subcellular localization of the prostaglandin system in the rabbit renal papilla. Acta Physiol. Scud. 84: 231-246, 1972. 2. APERIA, A. C., AND A. A. LIEBOW. Implications of urine p0, for renal medullary blood flow. Am. J. Physiol. 206: 499-504, 1964. 3. AUKLAND, K., AND J. KROG. Influence of various factors on
urine oxygen tension in the dog. Actu Physiol. Stand. 52: 350365, 1961. 4. BERLINER, R. W., AND C. M. BENNETT. Concentration of urine in the mammalian kidney. Am. J. Med. 42: 777-789, 1967. 5. BLACKWELL, G. J., R. J. FLOWER, AND J. R. VANE. Some characteristics of prostaglandin synthesizing system in rabbit kidney microsomes. Biochim. Biophys. Acta 398: 178-190, 1975.
Downloaded from www.physiology.org/journal/ajprenal at American Univ of Beirut (193.188.128.021) on June 19, 2019.
OXYGEN
MODULATION
OF
RENAL
PROSTAGLANDIN
F543
PRODUCTION
6. CAVALU), T. Fine structural localization of endogenous peroxidase activity in inner medullary interstitial cells of the rat kidney. Lab. Invest. 31: 458-464, 1974. 7. CROWSHAW, K. The incorporation of [ 1-14C]arachidonic acid into lipids of rabbit renal slices and conversion to prostaglandins E, and F,. Prostaglandins 3: 607-620, 1973. 8. CROWSHAW, K., J. C. MCGIFF, J. C. STRAND, A. J. LONIGRO, AND N. A. TERRAGNO. Prostaglandins in dog renal medulla. J. Pharm. Pharmacol. 22: 302-304, 1970. 9. CROWSHAW, K., AND J. Z. SZLYK. Distribution of prostaglandins in rabbit kidney. Biochem. J. 116: 421-424, 1970. 10. DANON, A., L. C T. CHANG, B. J. SWEETMAN, A. S. NIES, AND J. A. OATES. Synthesis of prostaglandins by the rat renal papilla in vitro. Biochim. Biophys Acta 388: 71-83, 1975. 11. DERUBERTIS, F. R., T. V. ZENSER, P. A. CRAVEN, AND B. B. DAVIS. Modulation of the cyclic AMP content of rat renal inner medulla by oxygen: possible role of local prostaglandins. J. Clin.. Invest. 58: 1370-1378, 1976. 12. FLAIWER, R. J., AND G. J. BLACKWELL. The importance of phospholipase A, in prostaglandin biosynthesis. Biochem. Pharmacol. 25: 285-291, 1976. 13. GILMAN, A. G. A protein binding assay for adenosine 3’:5’-cyclic
14.
15.
16.
17.
18.
19.
monophosphate. Proc. NatZ. Acad. Sci. US 67: 305-312, 1970. LEE, J. B., K. CROWSHAW, B. H. TAKMAN, K. A. ATTREP, AND J. 2. G~UGOUTAS. The identification of prostaglandins Ez, F, and A, from rabbit kidney medulla. Biochem. J. 105: 1251-1260, 1967. RENNIE, D. W., R. B. REEVES, AND J. R. PAPPENHEIMER. Oxygen pressure in the urine and its relation to intrarenal blood flow. Am. J. Physiol. 195: 120-132, 1958. WIRZ, H., AND R. DIRIX. Urinary concentration and dilution. In: Handbook of PhysioZogy. Renal PhysioZogy, edited by J. Orloff and R. W. Berliner. Washington, D.C.: Am. Physiol. Sot. 1973, p. 415-430. ZENSER, T. V., AND B. B. DAVIS. Mechanism of inhibition of organic acid transport in rabbit renal cortex by cyclic AMP. MetaboZism 25: 1137-1142, 1976. ZENSER, T. V., M. J. LEVITT, AND B. B. DAVIS. Effects of 0, and solute concentration on PGE and PGF production by rat kidney. Prostaglandins 13: 143-53, 1977. ZUSMAN, R. M., AND H. R. KEISER. Prostaglandin E, biosynthesis by rabbit renomedullary interstitial cells in tissue culture. J. BioZ. Chem. 252: 2069-2071, 1977.
Downloaded from www.physiology.org/journal/ajprenal at American Univ of Beirut (193.188.128.021) on June 19, 2019.
of rat production
by oxygen
TERRY V. ZENSER, MONTE J. LEVITT, AND BERNARD B. DAVIS Geriatric Research, Education and Clinical Center, Veterans Administration Hospital, and Department of Medicine and Biochemistry, St. Louis University, St. Louis, Missouri
ZENSER, TERRY V., MONTE J. LEVITT, AND BERNARD B. DAVIS. Possible modulation of rat renal prostaglandin production by oxygen. Am. J. Physiol. 233(6): F539-F543, 1977 or Am. J. Physiol.: Renal Fluid Electrolyte Physiol. 2(6): F539F543, 1977. -The effect of oxygen on in vitro prostaglandin production by slices of rat renal inner medulla was evaluated. Oxygen increased prostaglandin production in a dose-response fashion. Double reciprocal plots of the relationship revealed a half-maximal stimulation of prostaglandin (PG) E, production by 15.1% or 143 PM 0, and of PGFz, by 16.8% or 160 PM 0,. An increased prostaglandin production at 95% 0, was measurable within 2 min of incubation and was maximal at approximately 15 min. Oxygen also increased inner medullary cyclic AMP content with a similar time course. Lowering oxygen did not alter the response of the inner medulla to increase cyclic AMP content when exposed to vasopressin. PGE., increased cyclic AMP at 5% 0, but not at 95%, while PGF, was ineffective at either oxygen concentration. In the presence of structurally dissimilar prostaglandin synthetase inhibitors, indomethacin and sodium meclofenamate, there was a reduction of basal cyclic AMP content as well as prostaglandin production. PGE2 increased cyclic AMP at 95% Oe, in the presence of prostaglandin synthetase inhibitors while PGF& remained inactive. This suggests that the oxygen-mediated increases in cyclic AMP content may be due to endogenously produced PGE,. The data are consistent with oxygen modulating inner medullary production of prostaglandins, and with endogenously produced prostaglandins exerting a hormone-like action in the inner medulla. cyclic AMP; system
vasopressin;
renal
medulla;
renal
countercurrent
MEDULLA OF THE KIDNEY is the site ofrenal synthesis of prostaglandins (7, 9). That synthesis appears to proceed in the interstitial cells of the inner medulla (6), and results in the immediate release of prostaglandins rather than their accumulation in the cells (7). This localization of the renal prostaglandin synthetic mechanism to the inner medulla places the process in a very unique environment. The countercurrent arrangement of flow of tubule fluid through the loops of Henle and of blood through the vasa recta results in the generation of an increased solute concentration in the interstitial fluid of the inner medulla. This well-known mechanism provides the mammalian kidney with the capacity to elaborate a hypertonic urine, and during antidiuresis results in an inner medullary solute concentration which approximates that of THE INNER
63125
urine, approx. 2,800 mosmol/kg H,O in the rat (4). In addition to maintaining a high inner medullary solute concentration, the countercurrent system also functions to maintain a low ambient oxygen concentration in the inner medulla. Therefore, the inner medullary PO, during antidiuresis has been estimated to be in the range of 20-40 mmHg (2, 15). While inner medullary PO, is low, that fact has not generally been considered to be a major factor in either limiting or modulating inner medullary metabolic processes. However, we have recently observed a dependency of tissue cyclic AMP content in slices of inner medulla on ambient PO,. Increasing PO, increased the medullary cyclic AMP content, and the relationship between these two parameters was linear (11). Both indomethacin and meclofenamic acid inhibited oxygeninduced increases in cyclic AMP content. This suggested that the observed effect was dependent on an augmentation of prostaglandin production with increasing oxygen concentration. This paper is a report of the investigations of the effects of oxygen concentration on prostaglandin production, cyclic AMP generation, and the interrelationship between the two in the inner medulla of the rat kidney. MATERIALS
Experimental
AND
METHODS
Procedure
Unlabeled prostaglandin (PG) E, , EZ, F1, and F, were generously provided by The Upjohn Company, Kalamazoo, Mich. Sodium meclofenamate was kindly donated by Dr. James E. Gleichert, Parke, Davis & Co., Ann Arbor, Mich. Tritiated PGE, (117 Ci/mmol), PGF, (178 Ci/mmol), cyclic AMP (38 Ci/mmol) and Aquasol were purchased from New England Nuclear, Boston, Mass. Rabbit antisera to either PGE’s or PGF’s were obtained from Regis Chemical Co., Morton Grove, Ill. Antibodies, Inc., Davis, Calif. was the source of goat antisera to rabbit gamma globulins. Normal rabbit serum was obtained from Miles Laboratories, Elkhart, Ind. Indomethacin and arginine vasopressin (grade VI, synthetic) were purchased from Sigma Chemical ComMO. 1-Methyl-3-isobutylxanthine pany, St. Louis, (MIX) was purchased from Aldrich Chemical Co., Milwaukee, Wis. Cyclic AMP binding protein was prepared as described by Gilman (13). Gas mixtures were obtained from Liquid Carbonic, St. Louis, MO. All other
Downloaded from www.physiology.org/journal/ajprenal at American Univ of Beirut (193.188.128.021) on June 19, 2019.
ZENSER,
F540 chemicals were purchased in the highest available grade from standard sources. Male Sprague-Dawley rats weighing 250-300 g were from Eldridge Laboratory Animals, Barnhart, MO. Tissue Incubation The rats were anesthetized with ethyl ether, and the kidneys were removed quickly and placed in cold 0.85% NaCl. The kidneys were bisected in the coronal plane and slices obtained with a Stadie-Riggs microtome were placed on filter paper moistened with cold 0.85% NaCl. Each slice was then carefully dissected to isolate the inner medulla. The moist tissue was weighed to the nearest milligram on an analytical balance. Slices weighing 9-14 mg were placed in a 25ml flask containing 2 ml of incubation medium. The medium consisted of Krebs-Ringer bicarbonate buffer at pH 7.4 containing 1 mg/ml each glucose and bovine serum albumin. The potent inhibitor of cyclic nucleotide phosphodiesterase MIX (2 mM) was also present in the incubation medium. Prior to incubation each flask was purged with gas and the oxygen concentration of the medium was verified with an oxygen monitor (Yellow Springs Instrument Co. model 53). The slices were first incubated for 20 min with 0% 02, 5% C02, and 95% N, and then transferred for 20 min to flasks previously equilibrated with 0, 5, 10, 20, or 95% 02, with 5% CO, and the balance N,. In a separate series of experiments the first 20.min incubation was in 5% 02, 5% CO*, and 90% N,. The second incubation was in either 5% 02, 5% CO*, 90% N, or 95% 02, 5% CO,. As above, 2 mM MIX was in the incubation medium. PGE, 2.9 x 10m4 M, PGF2, 2.9 x lob4 M, or vasopressin 3.5 x lo-* M were added to appropriate flasks as indicated in the results. Such additions were made 5 min prior to the termination of the second incubation. When indomethacin (100 pg/ml) or sodium meclofenamate (50 pg/ml) were used they were present throughout the initial and the second incubation. For the experiments examining the time course of Os-mediated increases in inner medullary PGE, production and cyclic AMP content, slices were preincubated at 0% 02, 5% COt2, 95% N, for 20 min. They were then transferred to flasks gassed with 95% 02, 5% CO,. At the times indicated in text (2, 5, 10, 15, and 20 min), the reaction was terminated and the medium analyzed for PGE, and the slice for cyclic AMP. Care was taken to gas the flasks during transfer with the appropriate gas mixture. All incubations were performed at 37°C with 100 cycle/min agitation in a Dubnoff metabolic incubator. At the end of this incubation period, the tissue was removed, homogenized, and extracted for cyclic AMP. The incubation medium was immediately frozen and stored at -35OC for later analys1s. Prostaglandin
Analysis
The incubation mediums were analyzed generally within 1 wk, although it was found that storage at -35°C for more than 1 mo did not alter the PG content. A trace amount of tritiated PGF2, or PGE, (approx.
LEVITT,
AND
DAVIS
1,000 counts/min or 3 pg) was added to each 0.5.ml aliquot of thawed medium. After adjustment to pH 3.03.5 with 1.0 N HCl, each aliquot was extracted twice with 1.5 ml of ethyl acetate. Combined extracts were evaporated to dryness at 40°C under N,. Dried extracts were dissolved in 0.5 ml of assay buffer at pH 7.0 consisting of 0.01 M sodium phosphate, 0.15 M sodium chloride, and 1% normal rabbit serum. An aliquot of 0.050 ml was removed to determine the recovery of PG during extraction (85-95%). Reported values were corrected for recovery. Additional aliquots were analyzed in duplicate to determine the PG content. Radioimmunoassay for PGE and PGF were performed as described previously (16). Antibody plus [“H]PG (2,000 counts/min, 5.0 pg) and PG standards or unknowns (0.05 ml) were incubated in 1 .O ml of pH 7.0 buffer at 4°C. After 4 h, goat antiserum was added to rabbit gamma globulins and incubation continued for 20 h at 4°C. The precipitate was collected by centrifugation at 3,000 rpm for 30 min, dissolved in 1.0 ml of 0.1 N NaOH, and counted. Standards and unknowns were routinely run in duplicate with the reproducibility between duplicate samples averaging 3.4%. Unknowns exhibited linearity with respect to sample dilutions and combinations of unknowns and standards were additive. Cross-reactivity of PGE antibody with PGFzu or PGD, and PGF antibody with PGE, and PGD, was approx. 1% at the 50% displacement level. Thus, the assay technique would distinguish among the three major classes of prostaglandins known to be produced by the kidney (5). The PGE antibody would not, however, distinguish PGE, from PGE, , nor would the PGF antibody distinguish PGF1, from PGF2,. However, it has been previously reported that only the 2 series prostaglandins are produced by the kidney (8, 12). Preliminary experiments utilizing radiolabeled PGE, indicated no detectable PGE, metabolism by the inner medulla, a finding consistent with previous reports (1). Therefore, it was assumed that under the conditions of these experiments the PGE and PGF antibodies were measuring PGE, and PGF2,, respectively. Logit transformations of the standard curves resulted in linear functions between 0.1 and 10 ng PG per tube, equivalent to 2.0200 ng PG/ml of incubation medium. Prostaglandin wet content is expressed as nanograms per milligram weight of tissue. Cyclic AMP Analysis Cyclic AMP was extracted
by homogenizing
slices in
0.5 ml of 50 mM sodium acetate buffer (pH 4.0) at 95°C (17). The homogenate was heated for 5 min at that temperature. Cooled homogenates were spun at 3,000 rpm for 10 min at 4°C. Cyclic AMP was determined in the supernatant by the protein-binding method of Gilman (13). Cyclic AMP recovery was monitored by addition of 2,000 counts/min of cyclic [ 3H]AMP to the extraction buffer. Each reported result was corrected for recovery and represents the mean t SE of three incubation samples, each analyzed in duplicate. Statistical differences were evaluated by Student t test for unpaired values.
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OXYGEN
MODULATION
OF
RENAL
PROSTAGLANDIN
F541
PRODUCTION
RESULTS
There was an increase in the inner medullary production of both PGE, and PGFza with increasing oxygen concentration. Values for PGE, at 0, 5, 10, 20, and 95% were 1.2 t 0.2, 4.8 t 1.0, 9.9 t 1.6, 14.5 t 2.2, and 27.0 t 3.5 ng/mg wet tissue wt per 20 min, respectively. The values for PGF% were 0.2 t 0.01, 0.6 t 0.02, 1.2 t 0.2, and 2.5 t 0.3 ng/mg wet tissue wt per 20 min, respectively. While the values for PGF, were considerably less than those for PGE, the 0, concentrations which gave the half-maximal production of PGF2,, 16.8% or 160 PM, was approximately that of PGE2, 15.1% or 143 PM. The Vmax for PGE, and PGF2, was 25.6 and 3.0 ng/mg wet wt per 20 min, respectively (Fig. 1). The time course of the oxygen response at 95% O3 is illustrated in Fig. 2. A response was measurable within 2 min (the earliest time examined), and appeared to be maximal within 15 min. The time curve for PGFa was similar to that of PGE,. In the experiments in which 5% 0, was used in the preincubation phase, PGE, production was 6.8 t 0.5 ng/mg wet wt per 20 min when the gas phase of the second incubation was 5% 0’) and 21.6 t 2.0 when it was 95% 0,. There Was an effect of oxygen to increase cyclic AMP content in slices of inner medulla (Table 1). At 5% 0, the cyclic AMP content was 3.6 t 0.4 pmol cyclic AMP/ mg wet wt and this increased to 21 t 2 at 95% 0,. Figure 2 also contains the time curve for oxygen-induced increases in cyclic AMP content. The curve can
be seen to exhibit characteristics similar to the one for PGE,. As shown in Table 1, the responsiveness to vasopressin was maintained at the lower oxygen concentration. Cyclic AMP content at 95% 0, with vasopressin 3.5 x lo+ M (58 _+ 6 pmol cyclic AMP/mg wet wt) was not measurably different from the value at 5% O2 (45 t 6). Since the basal cyclic AMP content was lower at 5%, the relative cyclic AMP increase was actually greater at 5% than at 95% Oz. PGEz, 2.9 x 10m4 M, caused an increase in cyclic AMP content at 5% 0, but not at 95% 0, (Table 1). PGE*, however, increased cyclic AMP content at 95% O2 when sodium meclofenamate (50 pg/ml) 14 t 0.9 pmol cyclic AMP/ i
u
15
Of
2
5,
101 Time [minutes]
154 xi-
FIG. 2. Time course of O,-mediated increases in inner medullary PGE production and cyclic AMP content at 95% 0,. Slices were preincubated at 0% 0, for 20 min and then transferred to flasks with 95% 0, for indicated times. Values for prostaglandins and cyclic AMP are cumulative from 0 time to indicated time.
I
5
I
r
I
15
10
l/O
2
20
25
TABLE 1. Effects of prostaglandins, vasopressin, meclofenamate on inner medullary cyclic AMP content at high and low oxygen tension
Control AVP PGE, PGF, pm01 cyclic AMP/mg wet tissue wt
ImMl
95% 0, x: SE + Meclofenamate, i SE
l/O
2 WI
FIG. 1. Double reciprocal plots of effects of medium substrate (0,) on inner medullary PGE, (A) and PGF,, (B) production. Following preincubation at 0% 0, for 20 min, gas phase oxygen concentration was varied from 5 to 95% for 20 min.
and
5% 0, x SE + Meclofenamate, 2 SE
22 2
58 6
24 3
23 4
3.8 0.4
54 6
14 0.9
4.2 0.3
4.5 0.4
45 6
11 0.9
4.3 0.5
3.5 0.4
46 5
12 1.1
4.0 0.3
50 pg/ml
50 pglml
Slices were incubated at 5% 0, for 20 min as indicated to flasks containing 5% or 95% 0, vasopressin (AVP) (3.5 x 10m8 M), PGE, (2.9 (2.9 x 1O-4 M) were present during the last 5
and then transferred for 15 min. Arginine x 10e4 M), and PGF, min of incubation.
Downloaded from www.physiology.org/journal/ajprenal at American Univ of Beirut (193.188.128.021) on June 19, 2019.
F542
ZENSER,
mg wet wt was added to the incubation medium. PGF2,, 2.9 x 10m4 M, had no effect on cyclic AMP content at either 5% 0, or 95% 0, in the presence of either indomethacin or meclofenamate. At the concentrations used both indomethacin and meclofenamate blocked the effect of 95% 0, to increase the production of PGE, (cl.0 ng/mg wet tissue), PGF, (~0.2 ng/mg wet tissue), and cyclic AMP (2.8 t 0.4 pmol/mg wet tissue). However, neither indomethacin nor meclofenamate blocked the increase in cyclic AMP content mediated by vasopressin (54 t 6 pmol/mg wet tissue). DBCXJSSION
The data indicate that in slices of rat inner medulla there is a relationship between ambient oxygen and production of both PGE, and PGF&. Analysis of that relationship estimates the half-maximal value for prostaglandin production as 143 PM 0, for PGE, and 160 PM for PGF2,. These oxygen concentrations correspond to P%‘s of 115 mmHg and 128 mmHg, respectively, and are just above the upper limits of the Po,‘s of the arterial blood in normal conditions. They are considerably higher than the PO& of 20-40 mmHg or approximately 3-5% O2 which are estimated to occur in the inner renal medulla during antidiuresis (2, 15). It has been proposed that intramedullary PO, is low because of the countercurrent exchange function of the blood flow to that area of the kidney. Of interest is the fact that the efficiency of the countercurrent exchange process is very sensitive to the rate of blood flow. Increases in flow rate decrease efficiency of exchange (16). A range of inner medullary PO& has been reported (3). The inner medulla contains a system, therefore, which could possibly meter the amount of oxygen available at the site of the prostaglandin synthetic system. Our data suggest that the effect of oxygen to stimulate PGE, production at 25 mmHg is approximately 20% of the half-maximal value. The theoretical maximal value for inner medullary PO, under physiological circumstances would be the oxygen concentration of the renal artery, approx. 100 mmHg. That value is approx. 86% of the half-maximal effect of oxygen on PGE, synthesis. In addition, increases in prostaglandin production produced by oxygen concentrations within the physiological range are associated with increases in medullary cyclic AMP content (Fig. 2). These in vitro findings are consistent with the concept that 0, plays a role in the physiological regulation of prostaglandin production by the inner medulla of the kidney. These experiments were designed to test the effect of oxygen on prostaglandin production. It has been postulated by Flower and Blackwell (12) that phospholipase A*, which provides arachidonic acid as substrate for the
LEVITT,
AND
DAVIS
cycle-oxygenase reaction, could exert a regulatory action in prostaglandin synthesis. It has been shown that hormones such as bradykinin and angiotensin II which increase prostaglandin production do so at least in part by increasing availability of arachidonic acid (10, 19). The experiments of Flower and Blackwell (12), and Danon et al. (10) were performed at 95% 02, a concentration of oxygen which would not be expected to seriously limit prostaglandin production. In our experiments an attempt was made to isolate oxygen as a variable. The data indicate that oxygen, in concentrations ambient to the inner medulla, can affect the rate of prostaglandin synthesis. These results are in no way incompatible with the concept or the results of Flower and Blackwell (12). Both substrates would be expected to be important in the determination of the rate of bimolecular reaction. The data do not distinguish among the various mechanisms by which oxygen might exert this effect. In general, it is possible that oxygen either increases the availability of the fatty acid precursor, arachidonic acid, of the prostaglandins or that it increases the catalytic efficiency of prostaglandin synthetase, a reaction which utilizes oxygen as a substrate. Oxygen has an effect of increasing cyclic AMP content in slices of inner medulla, but vasopressin-induced increases in cyclic AMP content were similar at low and high oxygen. Sodium meclofenamate decreased cyclic AMP content at 95% but not at 5% oxygen. That agent had no effect on vasopressin-induced increases in cyclic AMP content. Those observations combined with the fact that PGE, increased inner medullary cyclic AMP content at 5% but not at 95% oxygen and that PGF2, had no effect on cyclic AMP content at either oxygen concentration, suggests that the oxygen-induced increase in cyclic AMP content is mediated by endogethe ability of nously produced PGE,. Furthermore, PGE, to increase cyclic AMP content at 95% oxygen in the presence but not in the absence of inhibitors of prostaglandin syn .thetase suggests that it is possible to generate enough endogenous PGE, to exert a maximal effect on cyclic AMP generation. Whether or not there is a physiological process mediated by this series of reactions remains to be determined. The data, however, are all consistent with endogenously produced prostaglandins exerting a hormone-like action in the inner medulla. We thank Mrs. Maryanne B. Sandusky, Ms. Liz Newkirk, and Ms. Peggy Egan for their skilled technical assistance, and Mrs. Dorothy Nabors for secretarial assistance. Appreciation is expressed to Dr. John E. Pike for providing the prostaglandins, and to Dr. Charles M. Coleman for providing the prostaglandin antisera. This work was supported by the Veterans Administration. Received
for publication
25 March
1977.
REFERENCES 1. ANGGARD,
E., S. 0. BOHMAN, J. E. GRIFFIN III, C. LARSSON, AND A. B. MAUNSBACH. Subcellular localization of the prostaglandin system in the rabbit renal papilla. Acta Physiol. Scud. 84: 231-246, 1972. 2. APERIA, A. C., AND A. A. LIEBOW. Implications of urine p0, for renal medullary blood flow. Am. J. Physiol. 206: 499-504, 1964. 3. AUKLAND, K., AND J. KROG. Influence of various factors on
urine oxygen tension in the dog. Actu Physiol. Stand. 52: 350365, 1961. 4. BERLINER, R. W., AND C. M. BENNETT. Concentration of urine in the mammalian kidney. Am. J. Med. 42: 777-789, 1967. 5. BLACKWELL, G. J., R. J. FLOWER, AND J. R. VANE. Some characteristics of prostaglandin synthesizing system in rabbit kidney microsomes. Biochim. Biophys. Acta 398: 178-190, 1975.
Downloaded from www.physiology.org/journal/ajprenal at American Univ of Beirut (193.188.128.021) on June 19, 2019.
OXYGEN
MODULATION
OF
RENAL
PROSTAGLANDIN
F543
PRODUCTION
6. CAVALU), T. Fine structural localization of endogenous peroxidase activity in inner medullary interstitial cells of the rat kidney. Lab. Invest. 31: 458-464, 1974. 7. CROWSHAW, K. The incorporation of [ 1-14C]arachidonic acid into lipids of rabbit renal slices and conversion to prostaglandins E, and F,. Prostaglandins 3: 607-620, 1973. 8. CROWSHAW, K., J. C. MCGIFF, J. C. STRAND, A. J. LONIGRO, AND N. A. TERRAGNO. Prostaglandins in dog renal medulla. J. Pharm. Pharmacol. 22: 302-304, 1970. 9. CROWSHAW, K., AND J. Z. SZLYK. Distribution of prostaglandins in rabbit kidney. Biochem. J. 116: 421-424, 1970. 10. DANON, A., L. C T. CHANG, B. J. SWEETMAN, A. S. NIES, AND J. A. OATES. Synthesis of prostaglandins by the rat renal papilla in vitro. Biochim. Biophys Acta 388: 71-83, 1975. 11. DERUBERTIS, F. R., T. V. ZENSER, P. A. CRAVEN, AND B. B. DAVIS. Modulation of the cyclic AMP content of rat renal inner medulla by oxygen: possible role of local prostaglandins. J. Clin.. Invest. 58: 1370-1378, 1976. 12. FLAIWER, R. J., AND G. J. BLACKWELL. The importance of phospholipase A, in prostaglandin biosynthesis. Biochem. Pharmacol. 25: 285-291, 1976. 13. GILMAN, A. G. A protein binding assay for adenosine 3’:5’-cyclic
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monophosphate. Proc. NatZ. Acad. Sci. US 67: 305-312, 1970. LEE, J. B., K. CROWSHAW, B. H. TAKMAN, K. A. ATTREP, AND J. 2. G~UGOUTAS. The identification of prostaglandins Ez, F, and A, from rabbit kidney medulla. Biochem. J. 105: 1251-1260, 1967. RENNIE, D. W., R. B. REEVES, AND J. R. PAPPENHEIMER. Oxygen pressure in the urine and its relation to intrarenal blood flow. Am. J. Physiol. 195: 120-132, 1958. WIRZ, H., AND R. DIRIX. Urinary concentration and dilution. In: Handbook of PhysioZogy. Renal PhysioZogy, edited by J. Orloff and R. W. Berliner. Washington, D.C.: Am. Physiol. Sot. 1973, p. 415-430. ZENSER, T. V., AND B. B. DAVIS. Mechanism of inhibition of organic acid transport in rabbit renal cortex by cyclic AMP. MetaboZism 25: 1137-1142, 1976. ZENSER, T. V., M. J. LEVITT, AND B. B. DAVIS. Effects of 0, and solute concentration on PGE and PGF production by rat kidney. Prostaglandins 13: 143-53, 1977. ZUSMAN, R. M., AND H. R. KEISER. Prostaglandin E, biosynthesis by rabbit renomedullary interstitial cells in tissue culture. J. BioZ. Chem. 252: 2069-2071, 1977.
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