- Email: [email protected]
Effects of prostaglandins on adrenal steroidogenesis in the rat
PROSTAGLANDINS
EFFECTS OF PROSTAGLANDINS ON ADRENAL STEROIDOGENESIS IN THE RAT H. Matsuoka, S. Y, Tan, and P, J. Mulrow Department of Medicine Medical College of Ohio C.S. #lo008 Toledo, Ohio 43699 ABSTRACT To elucidate the role of prostaglandins in adrenal steroidogengsis, we studied aldosterone and corticosterone responses to 3 x lo- y - 3 x 10-4 M of prostaglandin EE (PGEz), prosta landin Fza (PGFz,), prostacycrin (PGIz), and arachidonic acid (AAB in collagenase dispersed rat adrenal capsular and decapsular cells. Whereas adrenocorticotrophic hormone (ACTH) and angiotensin II (AII) stimulated aldosterone production in capsular cells and ACTH stimulated corticosterone production in decapsular cells in a dose dependent fashion, aldosterone and corticosterone production were not stimulated significantly by PGEB, PGFz,, PGIz, and AA. Although preincubation of dispersed adrenal cells with indomethacin (3 x 10m5 fj) markedly inhibited PGE2 synthesis, ACTH- and AII-stimulated aldosterone production and ACTH-stimulated corticosterone production were not attenuated despite prostaglandin blockade. These results indicate that prostaglandins are unlikely to play an important role in adrenal steroidogenesis, INTRODUCTION The role of orostaolandins in the reoulation of adrenal steroidogenesis has not been defined. In vitro and in vivo studies have yielded controversial results. bshzulati%r3) and non-stimuiation (4-6) have been reported. ACTH-stimulated steroidogenesis has been reported to be blunted by indomethacin (5, 7, 8), a known prostaglandin inhibitor, although these findings have been contradicted (6, 9). In most of these studies, aldosterone response to prostaglandins was not measured and prostaglandin blockade by indomethacin was not documented. Furthermore, the role of prostaglandins in AII-stimulated aldosterone production is still unknown. The present studies, therefore, were undertaken to evaluate the effect of prostaglandins (PGE2, PGFz,, and PGI2) and arachidonic acid on aldosterone and corticosterone production using rat collagenase dispersed adrenal capsular and decapsular cells, In addition, the effects of prostaglandin blockade by indomethacin on ACTH- and AIIstimulated steroidogenesis were also investigated, MATERIALS AND METHODS i3HlPGE2 (specific activity 160 Ci/mnole), and [3Hlcorticost-
FEBRUARY
1980 VOL. 19 NO. 2
291
PROSTAGLANDINS erone (specific activity 50 Ci/mmole) were obtained from Amersham. Unlabelled PGE2, PGE2a, and PG12 were kindly provided by Dr. J. E. Pike, The Upjohn Company, Kalamazoo, Michigan and indomethacin was from Merck Sha p & D hme Research Lab. Bovine serum albumin (BSA), synthetic (Asp!, Ileg ) AII, and porcine ACTH (11.65 IU/mg) were from Worthington Biochemical Corporation. Preparations of adrenal cells and stimulation of steroidogenesis, A modification of the procedures of Fredlund __t et al (10) and of Hanin s -et al. (11) was used. In brief, 16-20 Sprague-Dawley female rats 9 180-220 g) on normal sodium diet were used in each experiment, After decapitation, the adrenal glands were removed and separated into capsular and decapsular portions by incising the capsule and squeezing each adrenal gland. For dispersion of cells, each portion was minced into small pieces with scissors, washed with Medium 199 and incubated with collagenase (2 mg/ml of Medium 199) for 20 minutes at 37°C under 95% 02, 5% C02. To disperse capsular and decapsular cells, Medium 199 containing BSA (2 mg/ml) was added to the collagenase-treated tissues, which was then drawn up repeatedly in a Pasteur pipette. The cell suspensions were then filtered through gauze to remove coarse materials into a 50 ml polyethylene centrifugation tube. These procedures were repeated several times, The dispersed cells were then pooled and centrifuged at 100 g for 15 minutes and cell pellets were resuspended in fresh Medium 199 containing BSA (2 mg/ml). After harvesting capsular and decapsular cells, these cell suspensions (averaging cell counts lOO,OOO/tube) were incubated in Medium 199 c (2 mg/ml) with various amounts 3 x 10-8 M to 3 x loAI1 (lo-!1 M to lo=4 M) and hours at 37TC under 95% 02, ml. Since PG12 is unstable in pH 7.4 solution (12), PG12 was dissolved in pH 9.3 Medium 199 and 10 ~1 of PG12 were added to incubation vials every 30 minutes during incubation, In the ir+;ethdcin .a1 experiments, the cells were preincubated for 10 minutes with 3 x 10-5 M of indomethacin an then capsular cells were incubatgd with two dEsgs of AII, 5 x 10-Qo M and 10-8 l$ and ACTH, 4 x lo- M and 4 x 10' M for 2 hours. Decapsular cells were incubated with-same dose of i?CTH for 2 hours. The maximal response doses of AI1 (lo-5 t$ and ACTH (4 x 10m6 l$ were also compared in each experiment. Radioimmunoassay After the incubation period, the cell suspension was centrifuged and the cells were discarded. Aldosterone was measured directly, or after extraction with methylenechloride by radioimmunoassay as reported by us previously (13). Corticosterone was measured directly using a highly specific antibody obtained from Endocrine Science. For PGE2 analysis, 2 ml of supernatant was pooled and assayed using a specific anti-PGE2 antibody obtained from the Pasteur
292
FEBRUARY 1980 VOL. 19 NO. 2
PROSTAGLANDINS
Institute, Paris, France according to the method previously reported from our laboratory (14). RESULTS ACTH- and AII-stimulated steroidogenesis Aldosterone concentrations measured by direct assay were comparable with the values obtained after methylenechloride extraction (r=0.91, p < 0.001, n=12). As shown in Fig. 1, significant increases dosterone and corticosterone production were produced by 4 x ;;$I M ACTH and maximum increase in aldosterone and corticosterone production was obtained at an ACTH concentration of 4 x 10-B M. The minimum AI1 concentration whit produced a significant increase of aldosterone production was lo-po M and the maximum increase was cells AI1 obtained at AI1 concentration of 10-8 M. In decapsular did not elicit a significant increase ';n corticosterone. Aldosterone ng/lOO.OOOCells 150-
ACTH
AII
l:I.Jg-J
0 t,
, lo-"
lo+
IO-'
lo-"
Peptlde
Fig. 1
Concentration
(hj)
Aldosterone response to ACTH and AI1 in adrenal capsular cells (left), and corticosterone response to ACTH in decapsular cells (right). Each point represents the mean f SE of 5-6 experiments.
Effects of Prostaglandins
PGE2,PGF2a,and AA were ineffective in stimulation of aldoster3ne and corticosterone production as shown in Fig. 2. PGI at a high lose (3 x 10-4 M) increased aldosterone from 34.0 f 4.5 (St) ng/ 100,000 capsular cells to 59.2 f 14.6 ng/lOO,OOO capsular cells, but :he results are not statistically significant (0.2 > p > 0.1).
FEBRUARY
1980 VOL. 19 NO. 2
293
PROSTAGLANDINS
Corticosterone
Aldosterone ng~lOO.OOOCapsular
ng/1OO,OOODecapsular
Cells
Cells
loo-
lo+
10-S Prostaglandins
Fig. 2
IO+
lo+
and Arachidonlc
Acid Concentration&)
lo+
1o-4
Effect of PGE2, PGF2a, PG12, and AA on steroidogenesis. point represents mean ? SE of 4-5 experiments.
Each
Effects of indomethacin on ACTH- and AII-stimulated steroidogenesis Indomethacin inhibited adrenal PGE production from 145 pg/ 100,000 cells to 34 pg/lOO,OOO cells (79% inhibition) in capsular cells and from 650 pg/lOO,OOO cells to 74 pg/lOO,OOO cells (89% inhibition) in deca sular cells. Inhibition also appeared in the presence of 4 x lo-! M ACTH (from 276 pg/lOO,OOO cells to 78 pg/ 100,000 cells in capsiilarcells, 72% inhibition; from 756 pg/lOO,OOO cells to 100 pg/lOO,OOO cells in decapsular cells, 87% inhibition). Despite this inhibition, both aldosterone and corticosterone response to AI1 and ACTH were unaffected by indomethacin (Fig. 3).
294
FEBRUARY 1980VOL.19N0.2
PROSTAGLANDINS
Aldosterone
Corticosterone
Aldosterone
n~/lOO.OOOCapsular
Cells
“~/lOO.OOO Capsular
Cells
ng~lOO.OOO Decapsular
Cells
lndomethacin
Ol-
/ 10-10 10-g AlI@
Fig. 3
10-a
, 10-e
/ 10-e
ACTH(@
10“
, 10-g
1
10-s
10-7
ACTH(@
Effect of Indomethacin on AII- and ACTH-stimulated steroidogenesis. Each point represents mean f SE (n=5). DISCUSSION
The available data on prostaglandins and steroidogenesis are conflicting. Saruta -et al. (1) showed PGEl and PGE elicited aldosterone and corticosterone release from beef adrena? outer slices. Ellis et al. (15) reported recently that PG12 stimulated adrenal steroizg=esis in the cat. Stimulation of aldosterone and corticosterone by PGE2 in the rat have been reported (2, 3) but have not been confirmed by other studies (5, 6). -In vivo studies of BlairWest -et al, (4) found no direct action of PGEl on the sheep adrenal gland.
In our studies, aldosterone production was not stimulated by PGE2, PGF2 , or AA (the substrate precursor for prostaglandin) in rat adrena capsular cells. PG12 appeared to stimulate aldosterone prOd.KtioP at high concentration, but this was not statistically significant. Thus, our results suggest that prostaglandins do not stimulate steroidogenesis directly. Furthermore, both ACTH- and AII-stimulated steroidogenesis were not blocked by indomethacin pretreatment, despite a reduction of endogenous prostaglandin production. The failure of PG blockade to influence AI1 or ACTH action
FEBRUARY
1980 VOL. 19 NO. 2
295
PROSTAGLANDINS
upon steroidogenesis suggests that prostaglandins are not mediators of this action on adrenal steroidoqenesis. These results are in agreement with these of Lowry -2 et ai (6) and Vukoson et al, (9) who -7 also were unable to demonstrate an inhibitorv effect of lndomethacin on adrenal responsiveness to ACTH in the rat-adrenal gland. In man, we have shown that indomethacin-induced hypoaldosteronism was a consequence of hyporeninemia rather than a direct effect of the drug on adrenal steroidogenesis (16) and also reported cortisol production was unaffected by indomethacin (17). Golub -et al, (18) have shown that the infusion of PGAl into normal human volunteers led to a parallel rise in both plasma aldosterone concentration and plasma renin activity and they concluded that PGAl stimulated aldosterone production thru stimulating renin secretion. These results, however, contrast with a report of Laychock and Rubin (8) who claimed an inhibitory effect of indomethacin (3 x IO-5 l$ on adrenal corticosteroidogenesis in the rat. One possible explanation for our findings might be that the prostaglandin receptors of the adrenal are sensitive to collagenase dispersion, and therefore, would not respond to prostaglandins. Dazord -et al. (19) have demonstrated that human and ovine adrenal glands had prostaglandin receptors and these receptors were very sensitive to trypsin dispersion.
In conclusion, the failure of prostaglandins to stimulate steroidogenesis, and the failure of indomethacin to block ACTH- or AIIstimulated steroidogenesis indicate that prostaglandins play little, if any, role in the regulation of aldosterone and corticosterone production. ACKNOWLEDGEMENT This work was supported by USPHS, NIH grant # 5-ROI-HLl9229-03,
296
FEBRUARY
1980 VOL. 19 NO. 2
PROSTAGLANDJNS
REFERENCES 1.
Saruta, T. and Kaplan, N.M. Adrenal steroidogenesis: The effects of prostaglandins. 3. Clin. Invest. 51: 2246, 1972.
2.
Flack, J.D. and Ramwell, P.W. A co:tiparison of the effects of ACTH, cyclic AMP, dibutyryl cyclic AMP and PGE on corticosteroidogenesis in vitro. Endocrinology 90: 371, '; 972.
3.
Sph't,A. and J6zan, S. Effect of prostaglandin E and A on steroidsynthesis by the rat adrenal gland. J. Ensocr. 6$: 55, 1975.
4.
Blair-West, J.R., Coghlan, J.P., Denton, D.A. et al. Effects of prostaglandin El upon the steroid secretion of the adrenal of the sodium deficient sheep. Endocrinology 88: 367, 1971.
5.
Gallant, S. and Brownie, A.C. The in vivo effect of indomethacin and prostaglandin E on ACTH and DBC AMP-induced steroidogenesis in hypophysectomize8 rats. Biochem. Biophys. Res. Commun. 55: 831, 1973.
6.
McMartin, C. and Peters,J. Properties of a simpliLowry, P.J., fied bioassay for adrenocorticotrophic activity using the steroidogenic response of isolated adrenal cells. J. Endocr. 59: 43, 1973,
7.
Warner, W. and Rubin, R.P. Evidence for a possible prostaglandin link in ACTH-induced steroidogenesis. Prostaglandins 9: 83, 1975.
8.
Laychock, S.G. and Rubin, R.P. Indomethacin-induced alterations in corticosteroid and prostaglandin release by isolated adrenocortical cells of the cat, Br. J. Pharmac. 57: 273, 1976.
9.
Vukoson, M.B., Kramer, R.E., Pope, M. et al. Failure of indomethacin to affect adrenal responsiveness to ACTH in vitro. Horm. Metab. Res. 8: 325, 1976.
10.
Fredlund, P., Saltman, S. and Catt, K.J. Aldosterone production by isolated adrenal glomerulosa cells: Stimulation by physiological concentrations of angiotensin II. Endocrinology 97: 1577, 1975.
11.
Haning, R., Tait, S.A.S. and Tait, J.F. In vitro effects of ACTH, angiotensins, serotonin and potassium on steroid output and conversion of corticosterone to aldosterone by isolated adrenal cells. Endocrinology 87: 1147, 1970.
12.
Cho, M.J. and Allen, M.A. Chemical stability of prostacyclin (PGI2) in aqueous solutions. Prostaglandins 15: 943, 1978.
FEBRUARY 1980VOL.19N0.2
297
PROSTAGLANDINS
13,
Tan, S.Y., Noth, R. and Mulrow, P.J. Direct non-chromatographic radioimmunoassay of aldosterone; Validation of a commercially available kit and observations on age-related changes in concentrations in plasma. Clin. Chem. 24: 1531, 1978.
14.
Impaired renal production Tan, S.Y., Sweet, P. and Mulrow, P,J. of prostaglandin E2: A newly identified lesion in human essential hypertension. Prostaglandins 15: 139, 1978.
15.
Ellis, E.F., Shen, J.C., Schrey, M.P. et al. Prostacyclin: A potent stimulator of adrenal steroidogenesis. Prostaglandins 16: 483, 1978.
16.
Tan, S.Y., Shapiro, R., France, R. et al. Indomethacin-induced prostaglandin inhibition with hyperkalemia: A reversible cause of hyporeninemic hypoaldosteronism. Ann. Int. Med. 90: 783, 1979.
17.
Inhibition of the renin-aldosterone Tan, S.Y. and Mulrow, P.J. response to furosemide by indomethacin. J. Clin. Endo, Metab. 45: 174, 1977.
18.
Golub, M.S., Speckart, P.F., Zia, P.K. et al. The effect of prostaglandin Al on renin and aldosterone in man. Cir. Res. 39: 574, 1976.
19.
Dazord, A., Morera, A.M., Bertrand, J. et al. Prostaglandin receptors in human and ovine adrenal glands: Binding and stimulation of adenyl cyclase in subcellular preparations. Endocrinology 95: 352, 1974.
Editor: Harold Behrman Received 11/21/79, Accepted
298
l/15/80
FEBRUARY
1980 VOL. 19 NO. 2
EFFECTS OF PROSTAGLANDINS ON ADRENAL STEROIDOGENESIS IN THE RAT H. Matsuoka, S. Y, Tan, and P, J. Mulrow Department of Medicine Medical College of Ohio C.S. #lo008 Toledo, Ohio 43699 ABSTRACT To elucidate the role of prostaglandins in adrenal steroidogengsis, we studied aldosterone and corticosterone responses to 3 x lo- y - 3 x 10-4 M of prostaglandin EE (PGEz), prosta landin Fza (PGFz,), prostacycrin (PGIz), and arachidonic acid (AAB in collagenase dispersed rat adrenal capsular and decapsular cells. Whereas adrenocorticotrophic hormone (ACTH) and angiotensin II (AII) stimulated aldosterone production in capsular cells and ACTH stimulated corticosterone production in decapsular cells in a dose dependent fashion, aldosterone and corticosterone production were not stimulated significantly by PGEB, PGFz,, PGIz, and AA. Although preincubation of dispersed adrenal cells with indomethacin (3 x 10m5 fj) markedly inhibited PGE2 synthesis, ACTH- and AII-stimulated aldosterone production and ACTH-stimulated corticosterone production were not attenuated despite prostaglandin blockade. These results indicate that prostaglandins are unlikely to play an important role in adrenal steroidogenesis, INTRODUCTION The role of orostaolandins in the reoulation of adrenal steroidogenesis has not been defined. In vitro and in vivo studies have yielded controversial results. bshzulati%r3) and non-stimuiation (4-6) have been reported. ACTH-stimulated steroidogenesis has been reported to be blunted by indomethacin (5, 7, 8), a known prostaglandin inhibitor, although these findings have been contradicted (6, 9). In most of these studies, aldosterone response to prostaglandins was not measured and prostaglandin blockade by indomethacin was not documented. Furthermore, the role of prostaglandins in AII-stimulated aldosterone production is still unknown. The present studies, therefore, were undertaken to evaluate the effect of prostaglandins (PGE2, PGFz,, and PGI2) and arachidonic acid on aldosterone and corticosterone production using rat collagenase dispersed adrenal capsular and decapsular cells, In addition, the effects of prostaglandin blockade by indomethacin on ACTH- and AIIstimulated steroidogenesis were also investigated, MATERIALS AND METHODS i3HlPGE2 (specific activity 160 Ci/mnole), and [3Hlcorticost-
FEBRUARY
1980 VOL. 19 NO. 2
291
PROSTAGLANDINS erone (specific activity 50 Ci/mmole) were obtained from Amersham. Unlabelled PGE2, PGE2a, and PG12 were kindly provided by Dr. J. E. Pike, The Upjohn Company, Kalamazoo, Michigan and indomethacin was from Merck Sha p & D hme Research Lab. Bovine serum albumin (BSA), synthetic (Asp!, Ileg ) AII, and porcine ACTH (11.65 IU/mg) were from Worthington Biochemical Corporation. Preparations of adrenal cells and stimulation of steroidogenesis, A modification of the procedures of Fredlund __t et al (10) and of Hanin s -et al. (11) was used. In brief, 16-20 Sprague-Dawley female rats 9 180-220 g) on normal sodium diet were used in each experiment, After decapitation, the adrenal glands were removed and separated into capsular and decapsular portions by incising the capsule and squeezing each adrenal gland. For dispersion of cells, each portion was minced into small pieces with scissors, washed with Medium 199 and incubated with collagenase (2 mg/ml of Medium 199) for 20 minutes at 37°C under 95% 02, 5% C02. To disperse capsular and decapsular cells, Medium 199 containing BSA (2 mg/ml) was added to the collagenase-treated tissues, which was then drawn up repeatedly in a Pasteur pipette. The cell suspensions were then filtered through gauze to remove coarse materials into a 50 ml polyethylene centrifugation tube. These procedures were repeated several times, The dispersed cells were then pooled and centrifuged at 100 g for 15 minutes and cell pellets were resuspended in fresh Medium 199 containing BSA (2 mg/ml). After harvesting capsular and decapsular cells, these cell suspensions (averaging cell counts lOO,OOO/tube) were incubated in Medium 199 c (2 mg/ml) with various amounts 3 x 10-8 M to 3 x loAI1 (lo-!1 M to lo=4 M) and hours at 37TC under 95% 02, ml. Since PG12 is unstable in pH 7.4 solution (12), PG12 was dissolved in pH 9.3 Medium 199 and 10 ~1 of PG12 were added to incubation vials every 30 minutes during incubation, In the ir+;ethdcin .a1 experiments, the cells were preincubated for 10 minutes with 3 x 10-5 M of indomethacin an then capsular cells were incubatgd with two dEsgs of AII, 5 x 10-Qo M and 10-8 l$ and ACTH, 4 x lo- M and 4 x 10' M for 2 hours. Decapsular cells were incubated with-same dose of i?CTH for 2 hours. The maximal response doses of AI1 (lo-5 t$ and ACTH (4 x 10m6 l$ were also compared in each experiment. Radioimmunoassay After the incubation period, the cell suspension was centrifuged and the cells were discarded. Aldosterone was measured directly, or after extraction with methylenechloride by radioimmunoassay as reported by us previously (13). Corticosterone was measured directly using a highly specific antibody obtained from Endocrine Science. For PGE2 analysis, 2 ml of supernatant was pooled and assayed using a specific anti-PGE2 antibody obtained from the Pasteur
292
FEBRUARY 1980 VOL. 19 NO. 2
PROSTAGLANDINS
Institute, Paris, France according to the method previously reported from our laboratory (14). RESULTS ACTH- and AII-stimulated steroidogenesis Aldosterone concentrations measured by direct assay were comparable with the values obtained after methylenechloride extraction (r=0.91, p < 0.001, n=12). As shown in Fig. 1, significant increases dosterone and corticosterone production were produced by 4 x ;;$I M ACTH and maximum increase in aldosterone and corticosterone production was obtained at an ACTH concentration of 4 x 10-B M. The minimum AI1 concentration whit produced a significant increase of aldosterone production was lo-po M and the maximum increase was cells AI1 obtained at AI1 concentration of 10-8 M. In decapsular did not elicit a significant increase ';n corticosterone. Aldosterone ng/lOO.OOOCells 150-
ACTH
AII
l:I.Jg-J
0 t,
, lo-"
lo+
IO-'
lo-"
Peptlde
Fig. 1
Concentration
(hj)
Aldosterone response to ACTH and AI1 in adrenal capsular cells (left), and corticosterone response to ACTH in decapsular cells (right). Each point represents the mean f SE of 5-6 experiments.
Effects of Prostaglandins
PGE2,PGF2a,and AA were ineffective in stimulation of aldoster3ne and corticosterone production as shown in Fig. 2. PGI at a high lose (3 x 10-4 M) increased aldosterone from 34.0 f 4.5 (St) ng/ 100,000 capsular cells to 59.2 f 14.6 ng/lOO,OOO capsular cells, but :he results are not statistically significant (0.2 > p > 0.1).
FEBRUARY
1980 VOL. 19 NO. 2
293
PROSTAGLANDINS
Corticosterone
Aldosterone ng~lOO.OOOCapsular
ng/1OO,OOODecapsular
Cells
Cells
loo-
lo+
10-S Prostaglandins
Fig. 2
IO+
lo+
and Arachidonlc
Acid Concentration&)
lo+
1o-4
Effect of PGE2, PGF2a, PG12, and AA on steroidogenesis. point represents mean ? SE of 4-5 experiments.
Each
Effects of indomethacin on ACTH- and AII-stimulated steroidogenesis Indomethacin inhibited adrenal PGE production from 145 pg/ 100,000 cells to 34 pg/lOO,OOO cells (79% inhibition) in capsular cells and from 650 pg/lOO,OOO cells to 74 pg/lOO,OOO cells (89% inhibition) in deca sular cells. Inhibition also appeared in the presence of 4 x lo-! M ACTH (from 276 pg/lOO,OOO cells to 78 pg/ 100,000 cells in capsiilarcells, 72% inhibition; from 756 pg/lOO,OOO cells to 100 pg/lOO,OOO cells in decapsular cells, 87% inhibition). Despite this inhibition, both aldosterone and corticosterone response to AI1 and ACTH were unaffected by indomethacin (Fig. 3).
294
FEBRUARY 1980VOL.19N0.2
PROSTAGLANDINS
Aldosterone
Corticosterone
Aldosterone
n~/lOO.OOOCapsular
Cells
“~/lOO.OOO Capsular
Cells
ng~lOO.OOO Decapsular
Cells
lndomethacin
Ol-
/ 10-10 10-g AlI@
Fig. 3
10-a
, 10-e
/ 10-e
ACTH(@
10“
, 10-g
1
10-s
10-7
ACTH(@
Effect of Indomethacin on AII- and ACTH-stimulated steroidogenesis. Each point represents mean f SE (n=5). DISCUSSION
The available data on prostaglandins and steroidogenesis are conflicting. Saruta -et al. (1) showed PGEl and PGE elicited aldosterone and corticosterone release from beef adrena? outer slices. Ellis et al. (15) reported recently that PG12 stimulated adrenal steroizg=esis in the cat. Stimulation of aldosterone and corticosterone by PGE2 in the rat have been reported (2, 3) but have not been confirmed by other studies (5, 6). -In vivo studies of BlairWest -et al, (4) found no direct action of PGEl on the sheep adrenal gland.
In our studies, aldosterone production was not stimulated by PGE2, PGF2 , or AA (the substrate precursor for prostaglandin) in rat adrena capsular cells. PG12 appeared to stimulate aldosterone prOd.KtioP at high concentration, but this was not statistically significant. Thus, our results suggest that prostaglandins do not stimulate steroidogenesis directly. Furthermore, both ACTH- and AII-stimulated steroidogenesis were not blocked by indomethacin pretreatment, despite a reduction of endogenous prostaglandin production. The failure of PG blockade to influence AI1 or ACTH action
FEBRUARY
1980 VOL. 19 NO. 2
295
PROSTAGLANDINS
upon steroidogenesis suggests that prostaglandins are not mediators of this action on adrenal steroidoqenesis. These results are in agreement with these of Lowry -2 et ai (6) and Vukoson et al, (9) who -7 also were unable to demonstrate an inhibitorv effect of lndomethacin on adrenal responsiveness to ACTH in the rat-adrenal gland. In man, we have shown that indomethacin-induced hypoaldosteronism was a consequence of hyporeninemia rather than a direct effect of the drug on adrenal steroidogenesis (16) and also reported cortisol production was unaffected by indomethacin (17). Golub -et al, (18) have shown that the infusion of PGAl into normal human volunteers led to a parallel rise in both plasma aldosterone concentration and plasma renin activity and they concluded that PGAl stimulated aldosterone production thru stimulating renin secretion. These results, however, contrast with a report of Laychock and Rubin (8) who claimed an inhibitory effect of indomethacin (3 x IO-5 l$ on adrenal corticosteroidogenesis in the rat. One possible explanation for our findings might be that the prostaglandin receptors of the adrenal are sensitive to collagenase dispersion, and therefore, would not respond to prostaglandins. Dazord -et al. (19) have demonstrated that human and ovine adrenal glands had prostaglandin receptors and these receptors were very sensitive to trypsin dispersion.
In conclusion, the failure of prostaglandins to stimulate steroidogenesis, and the failure of indomethacin to block ACTH- or AIIstimulated steroidogenesis indicate that prostaglandins play little, if any, role in the regulation of aldosterone and corticosterone production. ACKNOWLEDGEMENT This work was supported by USPHS, NIH grant # 5-ROI-HLl9229-03,
296
FEBRUARY
1980 VOL. 19 NO. 2
PROSTAGLANDJNS
REFERENCES 1.
Saruta, T. and Kaplan, N.M. Adrenal steroidogenesis: The effects of prostaglandins. 3. Clin. Invest. 51: 2246, 1972.
2.
Flack, J.D. and Ramwell, P.W. A co:tiparison of the effects of ACTH, cyclic AMP, dibutyryl cyclic AMP and PGE on corticosteroidogenesis in vitro. Endocrinology 90: 371, '; 972.
3.
Sph't,A. and J6zan, S. Effect of prostaglandin E and A on steroidsynthesis by the rat adrenal gland. J. Ensocr. 6$: 55, 1975.
4.
Blair-West, J.R., Coghlan, J.P., Denton, D.A. et al. Effects of prostaglandin El upon the steroid secretion of the adrenal of the sodium deficient sheep. Endocrinology 88: 367, 1971.
5.
Gallant, S. and Brownie, A.C. The in vivo effect of indomethacin and prostaglandin E on ACTH and DBC AMP-induced steroidogenesis in hypophysectomize8 rats. Biochem. Biophys. Res. Commun. 55: 831, 1973.
6.
McMartin, C. and Peters,J. Properties of a simpliLowry, P.J., fied bioassay for adrenocorticotrophic activity using the steroidogenic response of isolated adrenal cells. J. Endocr. 59: 43, 1973,
7.
Warner, W. and Rubin, R.P. Evidence for a possible prostaglandin link in ACTH-induced steroidogenesis. Prostaglandins 9: 83, 1975.
8.
Laychock, S.G. and Rubin, R.P. Indomethacin-induced alterations in corticosteroid and prostaglandin release by isolated adrenocortical cells of the cat, Br. J. Pharmac. 57: 273, 1976.
9.
Vukoson, M.B., Kramer, R.E., Pope, M. et al. Failure of indomethacin to affect adrenal responsiveness to ACTH in vitro. Horm. Metab. Res. 8: 325, 1976.
10.
Fredlund, P., Saltman, S. and Catt, K.J. Aldosterone production by isolated adrenal glomerulosa cells: Stimulation by physiological concentrations of angiotensin II. Endocrinology 97: 1577, 1975.
11.
Haning, R., Tait, S.A.S. and Tait, J.F. In vitro effects of ACTH, angiotensins, serotonin and potassium on steroid output and conversion of corticosterone to aldosterone by isolated adrenal cells. Endocrinology 87: 1147, 1970.
12.
Cho, M.J. and Allen, M.A. Chemical stability of prostacyclin (PGI2) in aqueous solutions. Prostaglandins 15: 943, 1978.
FEBRUARY 1980VOL.19N0.2
297
PROSTAGLANDINS
13,
Tan, S.Y., Noth, R. and Mulrow, P.J. Direct non-chromatographic radioimmunoassay of aldosterone; Validation of a commercially available kit and observations on age-related changes in concentrations in plasma. Clin. Chem. 24: 1531, 1978.
14.
Impaired renal production Tan, S.Y., Sweet, P. and Mulrow, P,J. of prostaglandin E2: A newly identified lesion in human essential hypertension. Prostaglandins 15: 139, 1978.
15.
Ellis, E.F., Shen, J.C., Schrey, M.P. et al. Prostacyclin: A potent stimulator of adrenal steroidogenesis. Prostaglandins 16: 483, 1978.
16.
Tan, S.Y., Shapiro, R., France, R. et al. Indomethacin-induced prostaglandin inhibition with hyperkalemia: A reversible cause of hyporeninemic hypoaldosteronism. Ann. Int. Med. 90: 783, 1979.
17.
Inhibition of the renin-aldosterone Tan, S.Y. and Mulrow, P.J. response to furosemide by indomethacin. J. Clin. Endo, Metab. 45: 174, 1977.
18.
Golub, M.S., Speckart, P.F., Zia, P.K. et al. The effect of prostaglandin Al on renin and aldosterone in man. Cir. Res. 39: 574, 1976.
19.
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Editor: Harold Behrman Received 11/21/79, Accepted
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FEBRUARY
1980 VOL. 19 NO. 2