Life Sciences Vol. 17, pp . 2105-2.112 Printed in the U.S .A .
Pergamon Press
PROSTAGLANDIN SYNTHESIS IN RABBIT RENAL 14EDULLA Jurgen C. Frolich, Brian J . Sweetman, Keith Carr and John A . Oates Departments of Medicine and Pharmacology Vanderbilt University School of Medicine Nashville. Tennessee 37232 (Received in final form September 2, 1975 Summary Prostaglandin (PG) synthesis in rabbit renal medullary homogenates was investigated by gas chromatography-mass spectrometry utilizing two internal standards . The internal standards were added immediately after homogenization to an aliquot of the fresh homogenate . Another aliquot of the homDgenate was incubated and subsequently the internal standards were added. The internal standards were 3 .3 .4 .4 tetradeutero PGE2(d4- PGE2) for quantification of PGE2 and PGKI for quantification of PGA2 and 3,3,4,4 tetradeutero PGA the latter representing an in vitro dehydration product of d4-PGij generated during work 4_ pbT_t'W samples . In 6 experiments on 6 were rabbits levels of PGE2 4.4 t 1 .6 ug/g (mean :t SD) renal medulla increasing during Incubation to 74 .95 ± 6 .5 jjg/g (p < 0.01 .) PGA2 levels were 0 .03 1 0 .02 ug/9 in the non-incubated samples and did not increase during incubation . When PGA2 levels were corrected for the amount of PGA2 formed by it vitro dehydration from PGE2 as measured by the amount of d4-PU21JR~;irated to d4PGA2 during workup, PGA2 levels were indistinguishable from zero . The kidney has been known to exert an antihypertensive function independent of its excretory function since its transplantation into animals with renoprival hypertension led to the reversal of hypertension (1,2) . The search for a substance responsible for this function resulted in the discovery of renal extracts with antihypertensive properties (3) and further efforts led to the isolation of unsaturated, hydroxylated fatty acids from rabbit renal medulla (4) . Subsequently . prostaglandin E2 (PGE2), prostaglandin F (PGF ) and prostaglandin A2 (PGA2) were isolated from rabbit renal meduk and2ldentified by mass spectrometry (5,6) . PGE2 s and PGF2., undergo rapid metabolization and are removed almost completely on a Ingle passage through the lung (7) . By contrast PGA was found to cross-the pulmonary circulation largely unchanged (7) and ' t9erefore was thought to be involved in the antihypertensive incretory function of the kidney (8 .9 . 10) . More recently, estimation of PGA2 by newly developed radioimmunoassay methods suggested the presence of high level.s of PGA2 in rabbit renal medulla (11,12) . In contrast, Crowshaw utilizing renal medullary slices incubated with C 4-labelled arachidipUic acid, the precursor of PGE2 PGF c, and presumably . PGA2 detected only C"' labelled PGE2 and PGF2,, but not C11 labelled PGA2 (13) .
Part of this work was presented at the 48th Annual- Fall Meeting of the American Oil Chemists Society, Philadelphia, September 29, 1974 . 1105
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Vol. 17, No . 7
However. there were difficultie; in measuring the labelled C14 PGA2 since it co-chromatographed with other C14 labelled biosynthatic products . Reported levels of PGA2 in rabbit renal medulla differ by three-fold (6,14) . Part of this discrepancy may be due to the fact that PGE2 is readily converted to PGA2 especially under the acidic conditions commonly used for the extraction of PGs from biological matrices (5,6,11,12,14,23) . Thus, there is considerable uncertainty as to the levels and significance of PGA2 in rabbit renal medulla . In view of the important role that has been ascribed to PGA as a circulating hormone with vasodepressor properties of renal origin (8), we investigated renal medullary prostaglandin production in the rabbit in an experimental approach that allowed measurement of PGE2 and PGA2 by chemical methods and quantification of -the amount of PGE2 dehydrated to PGA2 in vitro during their isolation . Methods White, male Now Zealand rabbits were killed by a blow an the head and their kidneys excised immediately . The renal cortex was separated from the medulla, the medulla weighed (usually 0.6 g/kidney) and homogenized in 15 ml of Icecold phosphate buffer pH 7 .4 . The homogenate was split into two samples of equal size . To the first (control) sample were added the internal standards 3 .3,3 .4 tatradoutero-PSE2 (2 .5 pg) and PGAI (I jAg) as well as tritiated PGE and tritiated PGAI (Amershm-Searle . Arlington Heights/Ill . ; specific activity 60 and 15 C/m mole . respectively) for use as tracers during purification . Immediately thereafter . 5 volumes of ethanol were added . The other sample was incubated at 370C for 10 min . and then the internal standards, tracers and ethanol were added in a fashion identical to the first sample . Both samples were initially washed with an equal volume of petroleum ether to remove neutral lipids . After adjustment of the pH to 3 .4 with formic acid . prostaglandins were extracted into-25 ml of chloroform . The chloroform phase was flash evaporated and immediately applied to an LM-20 column I x 13 cm (15) . Subsequent chromatography was performed on a I g SiO2 column with a discontinuous gradient of ethyl acetate toluene (15) . This column separates the PGE-group from PGA-group but not PGAI from PGA2 nor PGE2 from d4-PGE2 . The PGE fraction from this column was derivatized to the PGE-mothylester-r&thyloxime-bis-acetate (17) and the PGA-frar-tion to the PGA-mthylester-trimethylsilylether (18) . Analysis was accomplished essentially as described previously (17,18) with an LKB 9000 gas chi tograph-mats spectrometer (GC/MS) in the selected ion monitoring (SIM) mode coupled to a POP 12A on-line computer system (19) . PGEcompounds were analyzed on a column of 3 ft . length and 2 mM inner diameter packed with 1 .2% Dexsil on Supelcoport 100/200 and PGA-compound on a column of 6 ft ., 2 m I .d . packed with 1% Dexsil on jupelcoport 100/200 . Column temperature was 2500C and source temperature 250 C . helium flow rate approximately 20 ml/min, trap current 60 uA, ionizing voltage 32 .5 eV . For the analysis of PGE2 and d4-PGE2, the ions monitored were We 419 and m/e 423 and for PGA PGAI and d4-PGA2 ions We 349, m/e 351 and m/e 353, respectively were molored. been Since no d4-PGA had added . any measured d4-PGA2 reflected the extent of dehydration of 4-PGE2 in vitro . The purity of the d4-PGE2 was assured by isolating it on a 1 g SiO column (16) on the day prior to its use in order to remove any trace of d4 -PGA2 in the d -PGE2 . In addition . 50 ug of d4-PGE2 were spotted on a TLC plate together a fth I ug of PGA1 and tritiated PGA2 and developed in solvent system A 11 (20) . w The plate was scanned for radioactivity (Nuclear Chicago, Actigraph 111) and the zone containing the radioactive tracer scraped, eluted and derivatized . Analysis by GC/MS revealed that the amount of d4 -PGA2 in this zone represented < 0 .01% of the d4-PGE2 applied to the plate.
Vol . 17, No . 7
Prootaglanding A2 and E2 of Ronal Medulla
110 7
Calculation of results was accomplished as follows : The amount of PGE2 present In each sample was arrived at by Obtaining the central area (CA) ratio of m/e 419/423 . Since the amount of added d4-PGE2 was known, the mount of PGE2 could be calculated . . The amount of PGA2 was calculated as the CA ratio of m/e 349/351 . Since the amount of added PGAI was known . the amount of PGA2 could be calculated . It was ascertained separately that a given amount of PGAI would generate 2 .5 times the area on a SIM profile of mi/e 351 . than would an equimolar amount Of PGA2 on the m/e 349 profile . Hence, the calculated results reflect this factor . The amount of d4- PGA2 was calculated as the CA ratio of We 351 * 2 .5053 . The standard deviation of measurements at levels of PGE2 and PGA2 encountered in this study was < 4% . Results Ion profiles of a typical experiment are shown in Fig . 1-3 . Fig . I shows the profiles of m/e 419 and 423 of a control experiment and Fig . 2 the corresponding fraction after incubation . Incubation leads to a considerable increase for the area of ion m/e 419 . indicative of active PGE2 synthesis . Fig . 3 shows ion profiles of ions m/e 349, 351 and 353 corresponding to PGAg, PGAI and d4 - PGA2 . All three ions are clearly visible and there is an almost base-line separation of the m/e 351 profile from the profile of m/e 349 and 353 . This is of particular importance since PGAI also generates a .minor peak at m/e 349 . If the GC resolution of PGAI and PGA2 is incomplete, the m/e 349 profiles from the two different origins tend to merge into one profile with the production of misleading area ratio measurements . The results of 6 experiments are shown in Table 1 . FIG . I PGE2 in rabbit renal medullary homogenate, non-incubated . Original ion profile of ions m/e 419 (small peak) and We 423 (large peak) corresponding to PGE2-Me-NOX-bis-acetate and d4 -PGE2 -Me4VX-_bis - acetate, respectively . -
>m/e 423
_~_m/e 419
1108
Prostaglanding A2 and E2 of Renal Medulla
Vol . 17, No . 7
FIG . 2 PGE2 In rabbit renal medullary homogenate, Incubated. Same ions as Fig . 1 . Note increase in peak height of m/e 419 indicative of PGE2-synthesis . __m/e 423 ;-, M/e 419
FIG . 3 PGA2 in rabbit renal medullary homogenate, non-incubated . Original ion profiles of ions m/e 349, 351 and 353 corresponding to the mothylester-TMS derivatives of PGA2 , PGAI and d4- PGA2 . respectively .
.,mAi 351
M/6 349
349 351 353
Vol. 17, No . 7
1109
Prostaglandins A2 and E2 of Renal Medulla TABLE I Prostaglandin Synthesis in Rabbit Renal Medulla (ng/g wet weight) Control
Incubation
PGE2
4430 ± 1600
14950 ± 6500
PGA2 (measured)
34 .6
t
20
PGA2 (corrected)*
-6 .9
1
18
-8 .3 ± 55
NS
-0 .11
t
0 .4
-0 .17 ± 0 .4
NS
Calculated PGA2 as % of PGE2
150
t
166
p 0 .01 NS
* Corrected for the wAnWred in vitro dehydration of PGE2 The levels of PGE2 expressed in ug/g of tissue increased on incubation significantly from 4 .4 to 14 .95 . Simultaneously determined . PGA levels arrived at by use of the PGAI internal standard increased from 0 0341 to 0 .150 mg/g but this increase was not statistically significant . The amount of d4-PGA2 was calculated on the basis of the PGAI internal standard . It was thus possible to calculate the percentage of in vitro conversion of d4-PGE2 to d4-PGA2. This .percentage conversion, of courie, aTs -o applies to the in vitro conversion of PGE2 1:4~ PU2 . It therefore was possible to cor~ectNTues measured for PGA2 by sibtracting the amount of PGA2 that was generated by dehydration in vitro. The results of this subtraction are also shown in Table 1 . It is a~p _Wr_ ent that the levels of PGA2 after the appropriate correction of both the control and incubation experiments for in vitro generation of PGA2 are not different from each other nor from zero .
Di
Discussion The finding of Lee et al (5) of a compound in renal medulla called medullin with antihypertensive properties and its identification by mass spectrometry (6) led to the development of radioimmuno4ssays for PGA (12 .21) . With this method, PGA was detected in fetal (22) and adult human (12,23) as well as .rabbit kidney (11) . Since PGA had been shown in pharmacological studies in which it was administered intravenously to cause a reduction in blood pressure by a vasodilator action and an increase In renal sodium and fluid excretion (5,6,23,24 .25,26), it was speculated that It might function as an important physiological regulator of blood pressure and sodium balance (8) . Further support for this speculation was derived from the finding of specific binding sites for PGA in rabbit renal cortex (27) and an inhibition of PAH uptake by PGA (28) . This latter property was shared with the "natriuretic hormone" (29) and it was speculated that PGA2 might be responsible for this function (9) . In the initial report an the chemical identification Of PfA2 from rabbit kidney it was noted that very likely part of it represented an in vitro product generated during its isolation (6) . Further doubts to tFW ex-TRAnce of PGA as a biosynthetic product of renal medulla were raised by experiments in whfch labelled arachidonic acid was added to renal medullary slices and labelled PGA2 could not be identified (13) .
as
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The present study utilized a novel approach to this problem by performing experiments which quantitatively determined the amount of in vitro generation of PGA2 from PGE2- Our results show that PGA is not presWt-rnmeisurable amounts in the intact kidney (control samplesf nor is it generated from endogenous precursors on incubation, even though during incubation active prostaglandin synthesis occurred as evidenced by the three-fold Increase in PGE2 during incubation . We therefore conclude that PGA2 is not a biosynthetic product of rabbit kidney . If PGA2 should be present as a circulating hormone (21,9) its origin would appear to be from sites other than the renal medulla . Acknowledgments Drs . Udo Axen and John Pike, The Upjohn Company, Kalamazoo . Michigan kindly supplied the prostaglandins used in this study . This study was supported by Grants #G1416431 and #HL16489 . The secretarial help of Ms . Evelyn Squires is gratefully acknowledged . Dr . Oates is the Joe and Morris Werthan Professor of Investigative Medicine . References E. E . MUIRNEAD, J. A. STIRMAN, and F. JONES . J . Clin . Invest . 39 :266281, 1960 . 2. E . E . MUIRHEAD, J . A . STIRMAN, W. LESCH and F . JOKES, Surg . Gynecol . Obstat . 103:673-686, 1956 . 3. E . E . MUIRHEAD . F. JONES and J . A. STIRMAN, J . Lab . Clin . Mod . 56 :167180, 1960 . 4. R. B . HICXLER, 0 . P. LAULER, C . A . SARAVIS, A. 1 . VAGNUCCI, G . STEINER, and G. W. THORN. Can . Ned . Ass . J. 90 :280-287SMITH . 1964 . , Circ . Res . 17 :57-77, 5 . J . 8 . LEE, J . COVIND, B. H . TAKMAN and E . R. 1965 . 6 . J . B. LEE, K . CROWSHAW . B. H . TAKMAN, K. A . ATTREP and J . Z. GOUGOUTAS, Biochem. J. 105:1251-1250, 1967 . 7 . J . C. McGIFF, N . A. .TERRAGNO, J . C . STRAND, J . C . LEE, A . J . LONIGRO and K. K . F. MG . Nature 223 :742-745, 1969 . 8. J . B. LEE, Prostaglandins in Cellular Biology, p . 399-442 Plenum Press . 1972 . 9 . J . B. LEE, Prostaglandins 1 :55-70, 1972 . 10 . J . B. LEE . The Prostaglandins, p . 133-187 Plenum Press, 1973 . 11 . A . A. ATTALLAN, J . B. LEE, Cire . Res . 33 :696-703, 1973 . 12 . V . K . VANCE, A. ATTALAH . A. PREZYNA and J . B . LEE, Prostaglandins 3 :647667, 1973 . 13 . K. CROWSHAW, Prostaglandins 3 :607-620, 1973 . 14 . E . G. DANIELS, J . W . HINMAN, B. E . LEACH and E. E . MUIRHEAD, Nature 215 : 1298-12" . 1967 . 15 . G. H . JOUVENAZ, D . H. NUGTEREN and D . A. VAN DROP, Prostaglandins 3 :175187, 1973 . 16 . M. BYGDEMAN and B . SANUELSSON, Clin . Chim . Acta 13 :465-474, 1966 . 17 . B. SAMUELSSON, M. HAMBERG and C . C . SWEELEY . Analyt . Biochem. 38 :301-304, 1970 . 18 . B. J . SWEETMAN, J . C . FROLICH and J . T. WATSON, Prostaglandins 3:75-87, 1973 . 19 . J . T . WATSON, D . R . PELSTER . B. J . SWEETMAN, J . C . FROLICH and J . A . OATES, Analyt . Chem . 45 :2071-2078, 1973 . 20 . K. GREEN and B. SANUELSSON, J. Lipid Res . 5 :117-120, 1965 . 21 . R. M . ZUSMAN, B. V . CJUJ)KLL, L . SPEROFF and H. R . BEHRMAN, Prostaglandins 2:41-53, 1972 . 22 . N . A. DAY, A. A . ATTALLAH and J . B . LEE . Prostaglandins 5 :491-494, 1974 . 23 . D . SPECTOR, R . M. ZUSMAN, B . V . CALDWELL and J . SPEROFF, Prostaglandins 6 :263-267, 1974 . 1.
Vol . 17 F No . 7 24 . 25 . 26 . 27 . 28 . 29 .
Prostaglandins A2 and E2 of Panel Medulla
J . B . GROSS and F . C. BARTTER, Am . J . Physiol . 225 :218-224, 1973 . J . B . LEE, J . C . MCGIFF, H . KANNEGIESSER, Y . AYKENT, J . G . MUDD and T. F . FRAWLEY . Ann. Int . Ned . 74 :703-710 . 1971 S. J . LEE . J . G . JOHNSON, C. J . SMITH and F . E : HATCH, Kidney Internat . 1 :254-262, 1972 . A . A. ATTALLAH and J . B. LEE, Prostaglandins 4:703-709, 1973 . J . B . LEE and J . F . FERGUSON, Nature 222 :1185-1186 . 1969 . N. S . BRICKER . S . KLAHR and M . PUKERSON, Nature (London) 219 :1058-1059, 1968 .
Pergamon Press
PROSTAGLANDIN SYNTHESIS IN RABBIT RENAL 14EDULLA Jurgen C. Frolich, Brian J . Sweetman, Keith Carr and John A . Oates Departments of Medicine and Pharmacology Vanderbilt University School of Medicine Nashville. Tennessee 37232 (Received in final form September 2, 1975 Summary Prostaglandin (PG) synthesis in rabbit renal medullary homogenates was investigated by gas chromatography-mass spectrometry utilizing two internal standards . The internal standards were added immediately after homogenization to an aliquot of the fresh homogenate . Another aliquot of the homDgenate was incubated and subsequently the internal standards were added. The internal standards were 3 .3 .4 .4 tetradeutero PGE2(d4- PGE2) for quantification of PGE2 and PGKI for quantification of PGA2 and 3,3,4,4 tetradeutero PGA the latter representing an in vitro dehydration product of d4-PGij generated during work 4_ pbT_t'W samples . In 6 experiments on 6 were rabbits levels of PGE2 4.4 t 1 .6 ug/g (mean :t SD) renal medulla increasing during Incubation to 74 .95 ± 6 .5 jjg/g (p < 0.01 .) PGA2 levels were 0 .03 1 0 .02 ug/9 in the non-incubated samples and did not increase during incubation . When PGA2 levels were corrected for the amount of PGA2 formed by it vitro dehydration from PGE2 as measured by the amount of d4-PU21JR~;irated to d4PGA2 during workup, PGA2 levels were indistinguishable from zero . The kidney has been known to exert an antihypertensive function independent of its excretory function since its transplantation into animals with renoprival hypertension led to the reversal of hypertension (1,2) . The search for a substance responsible for this function resulted in the discovery of renal extracts with antihypertensive properties (3) and further efforts led to the isolation of unsaturated, hydroxylated fatty acids from rabbit renal medulla (4) . Subsequently . prostaglandin E2 (PGE2), prostaglandin F (PGF ) and prostaglandin A2 (PGA2) were isolated from rabbit renal meduk and2ldentified by mass spectrometry (5,6) . PGE2 s and PGF2., undergo rapid metabolization and are removed almost completely on a Ingle passage through the lung (7) . By contrast PGA was found to cross-the pulmonary circulation largely unchanged (7) and ' t9erefore was thought to be involved in the antihypertensive incretory function of the kidney (8 .9 . 10) . More recently, estimation of PGA2 by newly developed radioimmunoassay methods suggested the presence of high level.s of PGA2 in rabbit renal medulla (11,12) . In contrast, Crowshaw utilizing renal medullary slices incubated with C 4-labelled arachidipUic acid, the precursor of PGE2 PGF c, and presumably . PGA2 detected only C"' labelled PGE2 and PGF2,, but not C11 labelled PGA2 (13) .
Part of this work was presented at the 48th Annual- Fall Meeting of the American Oil Chemists Society, Philadelphia, September 29, 1974 . 1105
1106
Prostaqlandins A2 and E2 of Renal Medulla
Vol. 17, No . 7
However. there were difficultie; in measuring the labelled C14 PGA2 since it co-chromatographed with other C14 labelled biosynthatic products . Reported levels of PGA2 in rabbit renal medulla differ by three-fold (6,14) . Part of this discrepancy may be due to the fact that PGE2 is readily converted to PGA2 especially under the acidic conditions commonly used for the extraction of PGs from biological matrices (5,6,11,12,14,23) . Thus, there is considerable uncertainty as to the levels and significance of PGA2 in rabbit renal medulla . In view of the important role that has been ascribed to PGA as a circulating hormone with vasodepressor properties of renal origin (8), we investigated renal medullary prostaglandin production in the rabbit in an experimental approach that allowed measurement of PGE2 and PGA2 by chemical methods and quantification of -the amount of PGE2 dehydrated to PGA2 in vitro during their isolation . Methods White, male Now Zealand rabbits were killed by a blow an the head and their kidneys excised immediately . The renal cortex was separated from the medulla, the medulla weighed (usually 0.6 g/kidney) and homogenized in 15 ml of Icecold phosphate buffer pH 7 .4 . The homogenate was split into two samples of equal size . To the first (control) sample were added the internal standards 3 .3,3 .4 tatradoutero-PSE2 (2 .5 pg) and PGAI (I jAg) as well as tritiated PGE and tritiated PGAI (Amershm-Searle . Arlington Heights/Ill . ; specific activity 60 and 15 C/m mole . respectively) for use as tracers during purification . Immediately thereafter . 5 volumes of ethanol were added . The other sample was incubated at 370C for 10 min . and then the internal standards, tracers and ethanol were added in a fashion identical to the first sample . Both samples were initially washed with an equal volume of petroleum ether to remove neutral lipids . After adjustment of the pH to 3 .4 with formic acid . prostaglandins were extracted into-25 ml of chloroform . The chloroform phase was flash evaporated and immediately applied to an LM-20 column I x 13 cm (15) . Subsequent chromatography was performed on a I g SiO2 column with a discontinuous gradient of ethyl acetate toluene (15) . This column separates the PGE-group from PGA-group but not PGAI from PGA2 nor PGE2 from d4-PGE2 . The PGE fraction from this column was derivatized to the PGE-mothylester-r&thyloxime-bis-acetate (17) and the PGA-frar-tion to the PGA-mthylester-trimethylsilylether (18) . Analysis was accomplished essentially as described previously (17,18) with an LKB 9000 gas chi tograph-mats spectrometer (GC/MS) in the selected ion monitoring (SIM) mode coupled to a POP 12A on-line computer system (19) . PGEcompounds were analyzed on a column of 3 ft . length and 2 mM inner diameter packed with 1 .2% Dexsil on Supelcoport 100/200 and PGA-compound on a column of 6 ft ., 2 m I .d . packed with 1% Dexsil on jupelcoport 100/200 . Column temperature was 2500C and source temperature 250 C . helium flow rate approximately 20 ml/min, trap current 60 uA, ionizing voltage 32 .5 eV . For the analysis of PGE2 and d4-PGE2, the ions monitored were We 419 and m/e 423 and for PGA PGAI and d4-PGA2 ions We 349, m/e 351 and m/e 353, respectively were molored. been Since no d4-PGA had added . any measured d4-PGA2 reflected the extent of dehydration of 4-PGE2 in vitro . The purity of the d4-PGE2 was assured by isolating it on a 1 g SiO column (16) on the day prior to its use in order to remove any trace of d4 -PGA2 in the d -PGE2 . In addition . 50 ug of d4-PGE2 were spotted on a TLC plate together a fth I ug of PGA1 and tritiated PGA2 and developed in solvent system A 11 (20) . w The plate was scanned for radioactivity (Nuclear Chicago, Actigraph 111) and the zone containing the radioactive tracer scraped, eluted and derivatized . Analysis by GC/MS revealed that the amount of d4 -PGA2 in this zone represented < 0 .01% of the d4-PGE2 applied to the plate.
Vol . 17, No . 7
Prootaglanding A2 and E2 of Ronal Medulla
110 7
Calculation of results was accomplished as follows : The amount of PGE2 present In each sample was arrived at by Obtaining the central area (CA) ratio of m/e 419/423 . Since the amount of added d4-PGE2 was known, the mount of PGE2 could be calculated . . The amount of PGA2 was calculated as the CA ratio of m/e 349/351 . Since the amount of added PGAI was known . the amount of PGA2 could be calculated . It was ascertained separately that a given amount of PGAI would generate 2 .5 times the area on a SIM profile of mi/e 351 . than would an equimolar amount Of PGA2 on the m/e 349 profile . Hence, the calculated results reflect this factor . The amount of d4- PGA2 was calculated as the CA ratio of We 351 * 2 .5053 . The standard deviation of measurements at levels of PGE2 and PGA2 encountered in this study was < 4% . Results Ion profiles of a typical experiment are shown in Fig . 1-3 . Fig . I shows the profiles of m/e 419 and 423 of a control experiment and Fig . 2 the corresponding fraction after incubation . Incubation leads to a considerable increase for the area of ion m/e 419 . indicative of active PGE2 synthesis . Fig . 3 shows ion profiles of ions m/e 349, 351 and 353 corresponding to PGAg, PGAI and d4 - PGA2 . All three ions are clearly visible and there is an almost base-line separation of the m/e 351 profile from the profile of m/e 349 and 353 . This is of particular importance since PGAI also generates a .minor peak at m/e 349 . If the GC resolution of PGAI and PGA2 is incomplete, the m/e 349 profiles from the two different origins tend to merge into one profile with the production of misleading area ratio measurements . The results of 6 experiments are shown in Table 1 . FIG . I PGE2 in rabbit renal medullary homogenate, non-incubated . Original ion profile of ions m/e 419 (small peak) and We 423 (large peak) corresponding to PGE2-Me-NOX-bis-acetate and d4 -PGE2 -Me4VX-_bis - acetate, respectively . -
>m/e 423
_~_m/e 419
1108
Prostaglanding A2 and E2 of Renal Medulla
Vol . 17, No . 7
FIG . 2 PGE2 In rabbit renal medullary homogenate, Incubated. Same ions as Fig . 1 . Note increase in peak height of m/e 419 indicative of PGE2-synthesis . __m/e 423 ;-, M/e 419
FIG . 3 PGA2 in rabbit renal medullary homogenate, non-incubated . Original ion profiles of ions m/e 349, 351 and 353 corresponding to the mothylester-TMS derivatives of PGA2 , PGAI and d4- PGA2 . respectively .
.,mAi 351
M/6 349
349 351 353
Vol. 17, No . 7
1109
Prostaglandins A2 and E2 of Renal Medulla TABLE I Prostaglandin Synthesis in Rabbit Renal Medulla (ng/g wet weight) Control
Incubation
PGE2
4430 ± 1600
14950 ± 6500
PGA2 (measured)
34 .6
t
20
PGA2 (corrected)*
-6 .9
1
18
-8 .3 ± 55
NS
-0 .11
t
0 .4
-0 .17 ± 0 .4
NS
Calculated PGA2 as % of PGE2
150
t
166
p 0 .01 NS
* Corrected for the wAnWred in vitro dehydration of PGE2 The levels of PGE2 expressed in ug/g of tissue increased on incubation significantly from 4 .4 to 14 .95 . Simultaneously determined . PGA levels arrived at by use of the PGAI internal standard increased from 0 0341 to 0 .150 mg/g but this increase was not statistically significant . The amount of d4-PGA2 was calculated on the basis of the PGAI internal standard . It was thus possible to calculate the percentage of in vitro conversion of d4-PGE2 to d4-PGA2. This .percentage conversion, of courie, aTs -o applies to the in vitro conversion of PGE2 1:4~ PU2 . It therefore was possible to cor~ectNTues measured for PGA2 by sibtracting the amount of PGA2 that was generated by dehydration in vitro. The results of this subtraction are also shown in Table 1 . It is a~p _Wr_ ent that the levels of PGA2 after the appropriate correction of both the control and incubation experiments for in vitro generation of PGA2 are not different from each other nor from zero .
Di
Discussion The finding of Lee et al (5) of a compound in renal medulla called medullin with antihypertensive properties and its identification by mass spectrometry (6) led to the development of radioimmuno4ssays for PGA (12 .21) . With this method, PGA was detected in fetal (22) and adult human (12,23) as well as .rabbit kidney (11) . Since PGA had been shown in pharmacological studies in which it was administered intravenously to cause a reduction in blood pressure by a vasodilator action and an increase In renal sodium and fluid excretion (5,6,23,24 .25,26), it was speculated that It might function as an important physiological regulator of blood pressure and sodium balance (8) . Further support for this speculation was derived from the finding of specific binding sites for PGA in rabbit renal cortex (27) and an inhibition of PAH uptake by PGA (28) . This latter property was shared with the "natriuretic hormone" (29) and it was speculated that PGA2 might be responsible for this function (9) . In the initial report an the chemical identification Of PfA2 from rabbit kidney it was noted that very likely part of it represented an in vitro product generated during its isolation (6) . Further doubts to tFW ex-TRAnce of PGA as a biosynthetic product of renal medulla were raised by experiments in whfch labelled arachidonic acid was added to renal medullary slices and labelled PGA2 could not be identified (13) .
as
1110
ProStaqlanAinS A2 and E2 of Renal Medulla
Vol . 17, No . 7
The present study utilized a novel approach to this problem by performing experiments which quantitatively determined the amount of in vitro generation of PGA2 from PGE2- Our results show that PGA is not presWt-rnmeisurable amounts in the intact kidney (control samplesf nor is it generated from endogenous precursors on incubation, even though during incubation active prostaglandin synthesis occurred as evidenced by the three-fold Increase in PGE2 during incubation . We therefore conclude that PGA2 is not a biosynthetic product of rabbit kidney . If PGA2 should be present as a circulating hormone (21,9) its origin would appear to be from sites other than the renal medulla . Acknowledgments Drs . Udo Axen and John Pike, The Upjohn Company, Kalamazoo . Michigan kindly supplied the prostaglandins used in this study . This study was supported by Grants #G1416431 and #HL16489 . The secretarial help of Ms . Evelyn Squires is gratefully acknowledged . Dr . Oates is the Joe and Morris Werthan Professor of Investigative Medicine . References E. E . MUIRNEAD, J. A. STIRMAN, and F. JONES . J . Clin . Invest . 39 :266281, 1960 . 2. E . E . MUIRHEAD, J . A . STIRMAN, W. LESCH and F . JOKES, Surg . Gynecol . Obstat . 103:673-686, 1956 . 3. E . E . MUIRHEAD . F. JONES and J . A. STIRMAN, J . Lab . Clin . Mod . 56 :167180, 1960 . 4. R. B . HICXLER, 0 . P. LAULER, C . A . SARAVIS, A. 1 . VAGNUCCI, G . STEINER, and G. W. THORN. Can . Ned . Ass . J. 90 :280-287SMITH . 1964 . , Circ . Res . 17 :57-77, 5 . J . 8 . LEE, J . COVIND, B. H . TAKMAN and E . R. 1965 . 6 . J . B. LEE, K . CROWSHAW . B. H . TAKMAN, K. A . ATTREP and J . Z. GOUGOUTAS, Biochem. J. 105:1251-1250, 1967 . 7 . J . C. McGIFF, N . A. .TERRAGNO, J . C . STRAND, J . C . LEE, A . J . LONIGRO and K. K . F. MG . Nature 223 :742-745, 1969 . 8. J . B. LEE, Prostaglandins in Cellular Biology, p . 399-442 Plenum Press . 1972 . 9 . J . B. LEE, Prostaglandins 1 :55-70, 1972 . 10 . J . B. LEE . The Prostaglandins, p . 133-187 Plenum Press, 1973 . 11 . A . A. ATTALLAN, J . B. LEE, Cire . Res . 33 :696-703, 1973 . 12 . V . K . VANCE, A. ATTALAH . A. PREZYNA and J . B . LEE, Prostaglandins 3 :647667, 1973 . 13 . K. CROWSHAW, Prostaglandins 3 :607-620, 1973 . 14 . E . G. DANIELS, J . W . HINMAN, B. E . LEACH and E. E . MUIRHEAD, Nature 215 : 1298-12" . 1967 . 15 . G. H . JOUVENAZ, D . H. NUGTEREN and D . A. VAN DROP, Prostaglandins 3 :175187, 1973 . 16 . M. BYGDEMAN and B . SANUELSSON, Clin . Chim . Acta 13 :465-474, 1966 . 17 . B. SAMUELSSON, M. HAMBERG and C . C . SWEELEY . Analyt . Biochem. 38 :301-304, 1970 . 18 . B. J . SWEETMAN, J . C . FROLICH and J . T. WATSON, Prostaglandins 3:75-87, 1973 . 19 . J . T . WATSON, D . R . PELSTER . B. J . SWEETMAN, J . C . FROLICH and J . A . OATES, Analyt . Chem . 45 :2071-2078, 1973 . 20 . K. GREEN and B. SANUELSSON, J. Lipid Res . 5 :117-120, 1965 . 21 . R. M . ZUSMAN, B. V . CJUJ)KLL, L . SPEROFF and H. R . BEHRMAN, Prostaglandins 2:41-53, 1972 . 22 . N . A. DAY, A. A . ATTALLAH and J . B . LEE . Prostaglandins 5 :491-494, 1974 . 23 . D . SPECTOR, R . M. ZUSMAN, B . V . CALDWELL and J . SPEROFF, Prostaglandins 6 :263-267, 1974 . 1.
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