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Acth-induced prostaglandin biosynthesis from 3H-arachidonic acid by adrenocortical cells
ACTH-INDUCED PROSTAGLANDIN BIOSYNTHESIS FROM 3H-ARACHIDONIC ACID BY ADRENOCORTICAL CELLS S.G. Laychock* and R.P. Rubin Department of Pharmacology Virginia Commonwealth University Medical College of Virginia Richmond, Virginia 23298
ABSTRACT Prostaglandins biosynthesized from 3H-arachidonic acid by trypsin-dispersed cat adrenocortical cells were isolated by silicic acid and thin layer chromatography. PGE, PGF, and a third component with mobility properties indistinguishable from either PGA or PGB were identified both in cortical cell homogenates and incubation medium. Concentrations of ACTH (125-250~U) which stimulate steroidogenesis enhanced the conversion of labeled precursor to all three of these prostaglandins. These findings provide further evidence for the proposal that prostaglandins function as a critical link in ACTH-induced steroidogenesis.
ACKNOWLEDGMENTS Unlabeled prostaglandins were generously supplied by Dr. J.E. Pike, The Upjohn Company; Kalamazoo, Michigan and synthetic 81-24-ACTH by Ciba Pharmaceuticals; Summit, New Jersey. Tritiated prostaglandins and arachidonic acid were purchased from New England Nuclear; Boston, Massachusetts. This grant was supported by USPHS Research Grant AM-18066 (NIAMDD) and USPHS Pharmacology Training Grant GM-07111 awarded to the Department of Pharmacology, Virginia Commonwealth University, Medical College of Virginia.
*USPHS Predoctoral Fellow
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INTRODUCTION Adrenocorticotropin (ACTH) induces corticosteroidogenesis through a complex sequence of events which has been incompletely characterized (Garren et al., 1971). C ~ cium and cyclic AMP are thought to mediate certain of these events (cf. Rubin, 1974), although there is reason to believe that prostaglandins may also participate in this process, since prostaglandins of the E series (PGE) stimulate steroidogenesis in vitro in rat and bovine adrenal preparations (Flack et al., 1969; Saruta and Kaplan, 1971), as well as in isolated feline cortical cells (Warner and Rubin, 19~5). However, in order to assess more clearly the relationship of prostaglandins to the action of ACTH, the identity and metabolism of prostaglandins in the adrenal cortex must be elucidated; and heretofore, this information has been lacking (cf. Fiack, 1973). Accordingly, this report describes the isolation and identification of certain prostaglandins from isolated cat adrenocortical cell suspensions and demonstrates the ability of A C T H to enhance their conversion from radioactive precursor. MATERIALS
A N D METHODS
Cell Incubation and Extraction. Both adrenal glands were removed from male cats and the cortical cells dispersed with trypsin by the procedure described in detail elsewhere (Rubin and Warner, 1975). Dulbecco's Modified Eagle's Medium (GIBCO) (MEM), supplemented to contain ++ 2.5mM Ca , was the basic incubation medium employed. Following their dispersion, the cells (250,000/beaker) were incubated for 75 minutes at 37°C with 1.0 ml. MEM containing 3H-arachidonic acid (4~Ci) plus reduced glutathione (66~g/ml), in the absence or presence of ACTH. Control and stimulated cells were pooled separately, centrifuged at 3000 rpm for i0 minutes at 4°C and the cell pellets washed and recentrifuged in 0.5ml MEM. The supernatants were decanted, acidified to pH3 with IN HCl, and extracted twice each with 20ml ethyl acetate. The organic phase was evaporated i__nnva_cu 9. The cell pellets were homogenized in 4ml incubation medium (titrated to pH3 with formic Icid) and centrifuged for one hour at 20,000 rpm at 4°C. the resulting supernatant was decanted into extraction =ubes and the pellet was washed with 1 ml MEM (pH3) and
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centrifuged for ten minutes at 15,000 rpm. The combined supernatants were extracted twice with 15 ml of ethyl acetate: cyclohexane (2:1) and the pooled organic phases evaporated to dryness in vacuo. Chroma_~tographic Separation of Prosta~landins. The prostaglandins were separated by silicic acid chromatography by a modification of the method of Jaffe et al., 1973. The prostaglandin fractions were obtained by developing glass columns (0.5 x 30 cm) serially with 7 ml solvent 3 (benzene: ethyl acetate, 60:40) (PGAB fraction), 18 ml solvent 1 (benzene: ethyl acetate: methanol, 60:40:2) (PGE fraction), and 9 ml solvent 4 (benzene: ethyl acetate: methanol, 60:40:20) (PGF fraction). The fractions were evaporated in vacuo at 42 ° , and recovered from the evaporation flasks by resuspending them in 0.5 ml distilled absolute ethanol and quantitatively transferring them to assay tubes and drying them under N2 at 37 ° . The separation procedure was validated by adding tritiated PGE 1 and PGFI& to incubating cortical cells and extracting them in the same manner as the experilaental samples. With 3H-PGE1 extracts, 82% was recovered in the PGE fraction and less than 4% was recovered in either of the other two fractions. Sixty-six per cent of the 3H-PGFI~ was recovered in the PGF fraction, 15% in the PGE fraction, and 5% in the PGAB fraction. In other experiments, 3H-PGE was quantitatively converted to 3H-PGB (Zusman, 1972), with resultant recoveries after silicic acid chromatography of 65% in the PGAB fraction and 4% in the PGE fraction. Arachidonic acid residues were largely removed in the PGAB fraction. In preparation for thin layer chromatography, the sample residues were resuspended in absolute ethanol and spotted on silica gel G and developed in benzene: chloroform: acetone: methanol: acetic acid (20:20:5:5:1 by vol.). Standards were run in parallel and visualized with phosphomolybdic acid (Willis, 1970). A radioactive profile was obtained by scraping the plates into 1 cm zones, dissolving them into l0 ml Bray~ scintillation solution and counting them by scintillation spectrometry.
RESULTS Isolation and Identification of Prostaglandins. The extract of the cell homogenate was fractionated by silicic acid chromatography into three components, designated as the AB,
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E and F fractions (see Methods). The AB fraction was discarded since it contained the major portion of the free 3H-arachidonic acid. When the E fraction was subjected to thin layer chromatography, three major peaks of radioactivity were found (Fig. la); in contrast, the F fraction produced a single peak of radioactivity in the region of the plate where PGF migrates (Fig. ib). Two of the peaks identified in the E fraction corresponded to PGF (Rf 0.27-0.37) and PGE (Rf 0.46-0.57). The presence of a radioactive peak corresponding to PGF in the E fraction is ascribed to the finding that discernible amounts of standard PGF were recovered in this fradtion by column chromatography (see Methods). The third radioactive peak corresponded to the migration pattern of both PGA and PGB (Rf 0.77-0.85) (Fig. la). In order to ascertain whether biosynthesized prostaglandins were released from cortical cells, the incubation medium was extracted separately from cell homogenates and processed through silicic acid and thin layer chromatography. Total counts isolated from extracts of incubation media were some ten times higher than those isolated from cortical cell homogenates (compare Fig. 1 and 2), indicating that prostaglandins synthesized intracellularly are readily extruded into the extracellular fluid. Analysis of the E fraction revealed a peak of radioactivity which corresponded to that of 3H-PGE and a smaller peak with mobility properties similar to PGA and PGB (Fig. 2a). Thin layer chromatography of the F fraction isolated from incubation media produced a single peak of radioactivity in the PGF zone (Fig. 2b). A l t h o u g h the prostaglandins contained within the first two radioactive peaks were indistinguishable from PGF and PGE, the prostaglandins represented by the third radioactive peak could not be positively identified. However, the possibility was entertained that the immediate precursor of this prostaglandin was biosynthesized PGE rather than arachidonic acid, since PGE is a labile substance, which is readily converted to other derivatives, including PGA and PGB (Levine, 1973). This supposition was tested b y determining the fate of exogenous PGE after the several extraction and elution procedures. 3H-PGEI was added to the incubation medium containing cortical cells and then extracted and isolated in the same manner as the experimental samples. Figure 3 depicts the thh layer chromatogram of the resulting E fraction. A majority of the counts (57~) was localized
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Fig. i. Conversion of radioactive precursor into prostaglandins by isolated cat cortical cells. Trypsin-dispersed cells (250,000/beaker) were incubated in Dulbecco's MEM for seventy-five minutes in the presence of ACTH (125~U/ml) plus 3H-arachidonic acid (4~Ci/ml). Contents of six beakers were pooled, and following centrifugation, the prostaglandins in the pellet were extracted. The E fraction (A) and F fraction (B) were separated by silicic acid chromatography and run separately on thin layer plates with the resulting radioactive profiles.
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~ . Release of biosynthesized prostaglandins from isolated cortical cells. The experimental procedure for extracting and isolating prostaglandins in the incubation medium was essentially the same as described in Fig. i, except that following centrifugation the supernatant rather than the pellet was processed (see Methods).
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PROSTAGLANDINS to the E zone and only a very small amount (4%)was found in the F zone; however, 40% of the counts were recovered from the AB zone. By contrast, when a sample of 3H-PGEI, not processed through extraction and column chromatography, was analyzed by thin layer chromatography, a single peak of radioactivity in the E region was obtained. This indicates that prior to thin layer chromatography a portion of the PGE formed in the cell suspension is converted to a derivative with a polarity similar to PGA and PGB. The Effect of A C T H on Label Incorporatio n. Having identified PGE, PGF, and a third component with a polarity similar to PGA or PGB in cortical cell homogenates and incubation medium, experiments were then carried out to determine whether ACTH could enhance the conversion of arachidonic acid into one or another of these prostaglandins. When fractions from unstimulated cells were compared by thin layer analysis with their respective fractions obtained from stimulated cells, submaximal steroidogenic concentrations of ACTH (125-250~U) were found to augment the conversion of arachidonic acid into PGE and PGF (Fig. 4). The amount of label incorporated into PGF and PGE by cells exposed to ACT}{ was 158 and 193~/~respectively, of that incorporated by unstimulated cells. An ACTH-induced increase in the incorporation of label in the third zone was also observed (Fig. 4). Since this radioactive peak may represent degradation products of PGE (vide supra), the increase in PGE turnover induced by ACTH may be significantly greater than indicated by the radioactivity in the E zone alone. Analysis of thin layer chromatograms of radiolabeled prostaglandins extracted from the incubation medium suggested a trend in incorporation similar to that described for the cell homogenates, although quantitatively the effects were less striking. The amount of labeled PGE and PGF released by ACTH-stimulated cells was 121 (~22) and 125 (~4)% respectively, of that released by unstimulated cells (n=4). The rather small effect of ACTH on prostaglandin release observed by analysis of the incubation medium suggests that a portion of the newly-synthesized prostaglandin is metabolized intracellularly rather than released. DISCUSSION Implicating a potential intermediate in the action of a given hormone dictates that the substance under question
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Fig. 3. Isolation of radioactive prostaglandins from the medium bathing cortical cells following the addition of 3H-PGEI. Cells were incubated with ~-PGE~D2uCi/ml) for seventy-five minutes; the medium obtained from beakers was then processed for thin layer chromatography, with the resulting radioactive profile.
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Fig. 4. Radiochromatogram of ACTH-facilitated prosta~landin synthesis. Cortical cells were incubated with H-arachidonic a c i d w i t h or without ACTH. The contents of six beakers containing unstimulated and stimulated cells were pooled separately into two aliquots, and the prostaglandins of the cell homogenates were extracted and separated by silicic acid and thin layer chromatography. Radioactivity (cp~) in each zone of ACTH-treated cells is expressed as a per cent of the radioactivity in the corresponding zone of unstimulated cells. To simplify representation of the data, the chromatograms obtained from the E and F fractions were combined. Each mean value (~ SEM) was derived from four different cell preparations.
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be found in the tissue and that some change~ ~n its metabolism be demonstrable during the action o~ the hormone. The present study shows that isolated adrenocortical cells are able to synthesize and release PGF and PGE from arachidonic acid. The additional finding that prostaglandin biosynthesis is enhanced by A C T H supports the concept that these unsaturated fatty acids play a role in A C T H - i n d u c e d steroidogenesis. A more specific identification of the radiolabeled prostaglandins remains to be made, but they are most likely PGE 2 and PGF2e, since arachidonic acid is thought to be the precursor of these prostaglandins, rather than of PGE 1 and PGFI ~ (Christ and van Dorp, 1972). Moreover, preliminary radioimmunoassay of cortical cell extracts demonstrated a much greater amount of PGF2e than PGFI~, when parallel assays were carried out using antibodies to both of these p r o s t a g l a n d i n s (Laychock and Rubin, unpublished observations). A third type of p r o s t a g l a n d i n with mobility properties similar to either PGA or PGB was also isolated. At least a portion of this prostaglandin was derived from PGE rather than directly from arachidonic acid, as evidenced by the fact that a portion of exogenous PGE added to incubating cortical cells was converted to a substance w i t h m o b i l i t y properties indistinguishable from the unidentified prostaglandin. But regardless of their specific identity, a p r e v i o u s l y - d e m o n s t r a t e d ability of prostaglandins (albeit in high concentrations) to elicit steroidogenesis (Warner and Rubin, 1975), and the present evidence for their enhanced turnover during A C T H - i n d u c e d steroidogenesis, provides strong support for the concept that prostaglandins function as an important link in the action of A C T H on the adrenal cortex. The nature of the roles of PGE and PGF in steroidogenesis is, of course, still unknown; however, some speculation may be warranted. PGE is the most potent of the prostaglandins in enhancing steroidogenesis in feline cortical cells (Warner and Rubin, 1975), so it may act as a positive modulator of steroidogenesis. On the other hand, the enhanced turnover of the very feebly-steroidogenic PGF may relate to its ability to participate in a feedback loop to inhibit steroidogenesis. A similar model, which has been p r o p o s e d for the regulation of steroidogenesis in ovarian tissue, also involves the p a r t i c i p a t i o n of cyclic AMP and cyclic GMP as respective facilitatory and inhibitory modulators of this process (Kuehl, 1974).
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PROSTAGLANDINS Although this study has provided some insight into the possible role of prostaglandins in controlling steroidogenesis, a closer functional relationship needs to be established to determine whether prostaglandins are indeed directly responsible for controlling steroidogenesis. A temporal and/or quantitative correlation should be established between alterations in endogenous prostaglandin levels and steroidogenesis during exposure to ACTH. An even more definitive experiment involves correlating the effects on steroidogenesis of inhibitors of prostaglandin synthesis with the specific inhibition of one or another of the prostaglandins. It is anticipated that such experiments, which are now in progress, will help to clarify the molecular picture of the processes which are required for full expression of the effects of ACTH.
REFERENCES Christ, E.J. and van Dorp, D.A. Comparative aspects of prostaglandin synthesis in animal tissues. Adv. in the Biosciences (Int. Conf. on Prostaglandins) 9: 35 (1972). Flack, J.D. The hypothalamus-pituitary-endocrine system. In: The Prostaglandins , P.W. Ramwell, ed., Plenum Press (New York), p. 327 (1973). Flack, J.D., Jessup, R. and Ramwell, P.W. stimulation of rat corticosteroidogenesis. 691 (1969).
Prostaglandin Science 163:
Garren, L.D., Gill, G.N., Masui, H. and Walton, G.M. On the mechanism of action of ACTH. Rec. Prog. Horm. Res. 2_~2: 421 (1971). Jaffe, B., Behrman, H.R. and Parker, C.W. Radioimmunoassay measurement of prostaglandins E, A and F in human plasma. J. Clin. Invest. 5_~2: 398 (1973). Kuehl, F. function.
Prostaglandins, cyclic nucleotides and cell Prostaglandins ~: 325 (1974).
Levine, L. Antibodies to pharmacologically active molecules: Specificities and some applications of antiprostaglandins. Pharmacol. Rev. 25: 293 (1973).
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Rubin, R.P. Calcium and the Secretory Process. Press (New York), p. 125 (1974).
Plenum
Rubin, R.P. and Warner, W. Nicotine-induced steroidogenesis in adrenocortical cells of the cat. Brit. J. Pharmacol. 53: 357 (1975). Saruta, T. and Kaplan, N.M. Adrenocortical steroidogenesis: The effects of prostaglandins. J. Clin. Invest. 5_~I: 2246 (1972). Warner, W. and Rubin, R.P. Evidence for a possible prostaglandin link in ACTH-induced steroidogenesis. Prostaglandins 9: 83 (1975). Willis, A.L. Simplified thin-layer chromatography of prostaglandins in biological extracts. Brit. J. Pharmacol. 40: 583P (1970). Zusman, R.M. Quantitative conversion of PGA or PGE to PGB. Prostaglandins ~: 167 (1972).
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ABSTRACT Prostaglandins biosynthesized from 3H-arachidonic acid by trypsin-dispersed cat adrenocortical cells were isolated by silicic acid and thin layer chromatography. PGE, PGF, and a third component with mobility properties indistinguishable from either PGA or PGB were identified both in cortical cell homogenates and incubation medium. Concentrations of ACTH (125-250~U) which stimulate steroidogenesis enhanced the conversion of labeled precursor to all three of these prostaglandins. These findings provide further evidence for the proposal that prostaglandins function as a critical link in ACTH-induced steroidogenesis.
ACKNOWLEDGMENTS Unlabeled prostaglandins were generously supplied by Dr. J.E. Pike, The Upjohn Company; Kalamazoo, Michigan and synthetic 81-24-ACTH by Ciba Pharmaceuticals; Summit, New Jersey. Tritiated prostaglandins and arachidonic acid were purchased from New England Nuclear; Boston, Massachusetts. This grant was supported by USPHS Research Grant AM-18066 (NIAMDD) and USPHS Pharmacology Training Grant GM-07111 awarded to the Department of Pharmacology, Virginia Commonwealth University, Medical College of Virginia.
*USPHS Predoctoral Fellow
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INTRODUCTION Adrenocorticotropin (ACTH) induces corticosteroidogenesis through a complex sequence of events which has been incompletely characterized (Garren et al., 1971). C ~ cium and cyclic AMP are thought to mediate certain of these events (cf. Rubin, 1974), although there is reason to believe that prostaglandins may also participate in this process, since prostaglandins of the E series (PGE) stimulate steroidogenesis in vitro in rat and bovine adrenal preparations (Flack et al., 1969; Saruta and Kaplan, 1971), as well as in isolated feline cortical cells (Warner and Rubin, 19~5). However, in order to assess more clearly the relationship of prostaglandins to the action of ACTH, the identity and metabolism of prostaglandins in the adrenal cortex must be elucidated; and heretofore, this information has been lacking (cf. Fiack, 1973). Accordingly, this report describes the isolation and identification of certain prostaglandins from isolated cat adrenocortical cell suspensions and demonstrates the ability of A C T H to enhance their conversion from radioactive precursor. MATERIALS
A N D METHODS
Cell Incubation and Extraction. Both adrenal glands were removed from male cats and the cortical cells dispersed with trypsin by the procedure described in detail elsewhere (Rubin and Warner, 1975). Dulbecco's Modified Eagle's Medium (GIBCO) (MEM), supplemented to contain ++ 2.5mM Ca , was the basic incubation medium employed. Following their dispersion, the cells (250,000/beaker) were incubated for 75 minutes at 37°C with 1.0 ml. MEM containing 3H-arachidonic acid (4~Ci) plus reduced glutathione (66~g/ml), in the absence or presence of ACTH. Control and stimulated cells were pooled separately, centrifuged at 3000 rpm for i0 minutes at 4°C and the cell pellets washed and recentrifuged in 0.5ml MEM. The supernatants were decanted, acidified to pH3 with IN HCl, and extracted twice each with 20ml ethyl acetate. The organic phase was evaporated i__nnva_cu 9. The cell pellets were homogenized in 4ml incubation medium (titrated to pH3 with formic Icid) and centrifuged for one hour at 20,000 rpm at 4°C. the resulting supernatant was decanted into extraction =ubes and the pellet was washed with 1 ml MEM (pH3) and
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centrifuged for ten minutes at 15,000 rpm. The combined supernatants were extracted twice with 15 ml of ethyl acetate: cyclohexane (2:1) and the pooled organic phases evaporated to dryness in vacuo. Chroma_~tographic Separation of Prosta~landins. The prostaglandins were separated by silicic acid chromatography by a modification of the method of Jaffe et al., 1973. The prostaglandin fractions were obtained by developing glass columns (0.5 x 30 cm) serially with 7 ml solvent 3 (benzene: ethyl acetate, 60:40) (PGAB fraction), 18 ml solvent 1 (benzene: ethyl acetate: methanol, 60:40:2) (PGE fraction), and 9 ml solvent 4 (benzene: ethyl acetate: methanol, 60:40:20) (PGF fraction). The fractions were evaporated in vacuo at 42 ° , and recovered from the evaporation flasks by resuspending them in 0.5 ml distilled absolute ethanol and quantitatively transferring them to assay tubes and drying them under N2 at 37 ° . The separation procedure was validated by adding tritiated PGE 1 and PGFI& to incubating cortical cells and extracting them in the same manner as the experilaental samples. With 3H-PGE1 extracts, 82% was recovered in the PGE fraction and less than 4% was recovered in either of the other two fractions. Sixty-six per cent of the 3H-PGFI~ was recovered in the PGF fraction, 15% in the PGE fraction, and 5% in the PGAB fraction. In other experiments, 3H-PGE was quantitatively converted to 3H-PGB (Zusman, 1972), with resultant recoveries after silicic acid chromatography of 65% in the PGAB fraction and 4% in the PGE fraction. Arachidonic acid residues were largely removed in the PGAB fraction. In preparation for thin layer chromatography, the sample residues were resuspended in absolute ethanol and spotted on silica gel G and developed in benzene: chloroform: acetone: methanol: acetic acid (20:20:5:5:1 by vol.). Standards were run in parallel and visualized with phosphomolybdic acid (Willis, 1970). A radioactive profile was obtained by scraping the plates into 1 cm zones, dissolving them into l0 ml Bray~ scintillation solution and counting them by scintillation spectrometry.
RESULTS Isolation and Identification of Prostaglandins. The extract of the cell homogenate was fractionated by silicic acid chromatography into three components, designated as the AB,
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E and F fractions (see Methods). The AB fraction was discarded since it contained the major portion of the free 3H-arachidonic acid. When the E fraction was subjected to thin layer chromatography, three major peaks of radioactivity were found (Fig. la); in contrast, the F fraction produced a single peak of radioactivity in the region of the plate where PGF migrates (Fig. ib). Two of the peaks identified in the E fraction corresponded to PGF (Rf 0.27-0.37) and PGE (Rf 0.46-0.57). The presence of a radioactive peak corresponding to PGF in the E fraction is ascribed to the finding that discernible amounts of standard PGF were recovered in this fradtion by column chromatography (see Methods). The third radioactive peak corresponded to the migration pattern of both PGA and PGB (Rf 0.77-0.85) (Fig. la). In order to ascertain whether biosynthesized prostaglandins were released from cortical cells, the incubation medium was extracted separately from cell homogenates and processed through silicic acid and thin layer chromatography. Total counts isolated from extracts of incubation media were some ten times higher than those isolated from cortical cell homogenates (compare Fig. 1 and 2), indicating that prostaglandins synthesized intracellularly are readily extruded into the extracellular fluid. Analysis of the E fraction revealed a peak of radioactivity which corresponded to that of 3H-PGE and a smaller peak with mobility properties similar to PGA and PGB (Fig. 2a). Thin layer chromatography of the F fraction isolated from incubation media produced a single peak of radioactivity in the PGF zone (Fig. 2b). A l t h o u g h the prostaglandins contained within the first two radioactive peaks were indistinguishable from PGF and PGE, the prostaglandins represented by the third radioactive peak could not be positively identified. However, the possibility was entertained that the immediate precursor of this prostaglandin was biosynthesized PGE rather than arachidonic acid, since PGE is a labile substance, which is readily converted to other derivatives, including PGA and PGB (Levine, 1973). This supposition was tested b y determining the fate of exogenous PGE after the several extraction and elution procedures. 3H-PGEI was added to the incubation medium containing cortical cells and then extracted and isolated in the same manner as the experimental samples. Figure 3 depicts the thh layer chromatogram of the resulting E fraction. A majority of the counts (57~) was localized
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Fig. i. Conversion of radioactive precursor into prostaglandins by isolated cat cortical cells. Trypsin-dispersed cells (250,000/beaker) were incubated in Dulbecco's MEM for seventy-five minutes in the presence of ACTH (125~U/ml) plus 3H-arachidonic acid (4~Ci/ml). Contents of six beakers were pooled, and following centrifugation, the prostaglandins in the pellet were extracted. The E fraction (A) and F fraction (B) were separated by silicic acid chromatography and run separately on thin layer plates with the resulting radioactive profiles.
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~ . Release of biosynthesized prostaglandins from isolated cortical cells. The experimental procedure for extracting and isolating prostaglandins in the incubation medium was essentially the same as described in Fig. i, except that following centrifugation the supernatant rather than the pellet was processed (see Methods).
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PROSTAGLANDINS to the E zone and only a very small amount (4%)was found in the F zone; however, 40% of the counts were recovered from the AB zone. By contrast, when a sample of 3H-PGEI, not processed through extraction and column chromatography, was analyzed by thin layer chromatography, a single peak of radioactivity in the E region was obtained. This indicates that prior to thin layer chromatography a portion of the PGE formed in the cell suspension is converted to a derivative with a polarity similar to PGA and PGB. The Effect of A C T H on Label Incorporatio n. Having identified PGE, PGF, and a third component with a polarity similar to PGA or PGB in cortical cell homogenates and incubation medium, experiments were then carried out to determine whether ACTH could enhance the conversion of arachidonic acid into one or another of these prostaglandins. When fractions from unstimulated cells were compared by thin layer analysis with their respective fractions obtained from stimulated cells, submaximal steroidogenic concentrations of ACTH (125-250~U) were found to augment the conversion of arachidonic acid into PGE and PGF (Fig. 4). The amount of label incorporated into PGF and PGE by cells exposed to ACT}{ was 158 and 193~/~respectively, of that incorporated by unstimulated cells. An ACTH-induced increase in the incorporation of label in the third zone was also observed (Fig. 4). Since this radioactive peak may represent degradation products of PGE (vide supra), the increase in PGE turnover induced by ACTH may be significantly greater than indicated by the radioactivity in the E zone alone. Analysis of thin layer chromatograms of radiolabeled prostaglandins extracted from the incubation medium suggested a trend in incorporation similar to that described for the cell homogenates, although quantitatively the effects were less striking. The amount of labeled PGE and PGF released by ACTH-stimulated cells was 121 (~22) and 125 (~4)% respectively, of that released by unstimulated cells (n=4). The rather small effect of ACTH on prostaglandin release observed by analysis of the incubation medium suggests that a portion of the newly-synthesized prostaglandin is metabolized intracellularly rather than released. DISCUSSION Implicating a potential intermediate in the action of a given hormone dictates that the substance under question
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Fig. 3. Isolation of radioactive prostaglandins from the medium bathing cortical cells following the addition of 3H-PGEI. Cells were incubated with ~-PGE~D2uCi/ml) for seventy-five minutes; the medium obtained from beakers was then processed for thin layer chromatography, with the resulting radioactive profile.
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Fig. 4. Radiochromatogram of ACTH-facilitated prosta~landin synthesis. Cortical cells were incubated with H-arachidonic a c i d w i t h or without ACTH. The contents of six beakers containing unstimulated and stimulated cells were pooled separately into two aliquots, and the prostaglandins of the cell homogenates were extracted and separated by silicic acid and thin layer chromatography. Radioactivity (cp~) in each zone of ACTH-treated cells is expressed as a per cent of the radioactivity in the corresponding zone of unstimulated cells. To simplify representation of the data, the chromatograms obtained from the E and F fractions were combined. Each mean value (~ SEM) was derived from four different cell preparations.
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be found in the tissue and that some change~ ~n its metabolism be demonstrable during the action o~ the hormone. The present study shows that isolated adrenocortical cells are able to synthesize and release PGF and PGE from arachidonic acid. The additional finding that prostaglandin biosynthesis is enhanced by A C T H supports the concept that these unsaturated fatty acids play a role in A C T H - i n d u c e d steroidogenesis. A more specific identification of the radiolabeled prostaglandins remains to be made, but they are most likely PGE 2 and PGF2e, since arachidonic acid is thought to be the precursor of these prostaglandins, rather than of PGE 1 and PGFI ~ (Christ and van Dorp, 1972). Moreover, preliminary radioimmunoassay of cortical cell extracts demonstrated a much greater amount of PGF2e than PGFI~, when parallel assays were carried out using antibodies to both of these p r o s t a g l a n d i n s (Laychock and Rubin, unpublished observations). A third type of p r o s t a g l a n d i n with mobility properties similar to either PGA or PGB was also isolated. At least a portion of this prostaglandin was derived from PGE rather than directly from arachidonic acid, as evidenced by the fact that a portion of exogenous PGE added to incubating cortical cells was converted to a substance w i t h m o b i l i t y properties indistinguishable from the unidentified prostaglandin. But regardless of their specific identity, a p r e v i o u s l y - d e m o n s t r a t e d ability of prostaglandins (albeit in high concentrations) to elicit steroidogenesis (Warner and Rubin, 1975), and the present evidence for their enhanced turnover during A C T H - i n d u c e d steroidogenesis, provides strong support for the concept that prostaglandins function as an important link in the action of A C T H on the adrenal cortex. The nature of the roles of PGE and PGF in steroidogenesis is, of course, still unknown; however, some speculation may be warranted. PGE is the most potent of the prostaglandins in enhancing steroidogenesis in feline cortical cells (Warner and Rubin, 1975), so it may act as a positive modulator of steroidogenesis. On the other hand, the enhanced turnover of the very feebly-steroidogenic PGF may relate to its ability to participate in a feedback loop to inhibit steroidogenesis. A similar model, which has been p r o p o s e d for the regulation of steroidogenesis in ovarian tissue, also involves the p a r t i c i p a t i o n of cyclic AMP and cyclic GMP as respective facilitatory and inhibitory modulators of this process (Kuehl, 1974).
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PROSTAGLANDINS Although this study has provided some insight into the possible role of prostaglandins in controlling steroidogenesis, a closer functional relationship needs to be established to determine whether prostaglandins are indeed directly responsible for controlling steroidogenesis. A temporal and/or quantitative correlation should be established between alterations in endogenous prostaglandin levels and steroidogenesis during exposure to ACTH. An even more definitive experiment involves correlating the effects on steroidogenesis of inhibitors of prostaglandin synthesis with the specific inhibition of one or another of the prostaglandins. It is anticipated that such experiments, which are now in progress, will help to clarify the molecular picture of the processes which are required for full expression of the effects of ACTH.
REFERENCES Christ, E.J. and van Dorp, D.A. Comparative aspects of prostaglandin synthesis in animal tissues. Adv. in the Biosciences (Int. Conf. on Prostaglandins) 9: 35 (1972). Flack, J.D. The hypothalamus-pituitary-endocrine system. In: The Prostaglandins , P.W. Ramwell, ed., Plenum Press (New York), p. 327 (1973). Flack, J.D., Jessup, R. and Ramwell, P.W. stimulation of rat corticosteroidogenesis. 691 (1969).
Prostaglandin Science 163:
Garren, L.D., Gill, G.N., Masui, H. and Walton, G.M. On the mechanism of action of ACTH. Rec. Prog. Horm. Res. 2_~2: 421 (1971). Jaffe, B., Behrman, H.R. and Parker, C.W. Radioimmunoassay measurement of prostaglandins E, A and F in human plasma. J. Clin. Invest. 5_~2: 398 (1973). Kuehl, F. function.
Prostaglandins, cyclic nucleotides and cell Prostaglandins ~: 325 (1974).
Levine, L. Antibodies to pharmacologically active molecules: Specificities and some applications of antiprostaglandins. Pharmacol. Rev. 25: 293 (1973).
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Rubin, R.P. Calcium and the Secretory Process. Press (New York), p. 125 (1974).
Plenum
Rubin, R.P. and Warner, W. Nicotine-induced steroidogenesis in adrenocortical cells of the cat. Brit. J. Pharmacol. 53: 357 (1975). Saruta, T. and Kaplan, N.M. Adrenocortical steroidogenesis: The effects of prostaglandins. J. Clin. Invest. 5_~I: 2246 (1972). Warner, W. and Rubin, R.P. Evidence for a possible prostaglandin link in ACTH-induced steroidogenesis. Prostaglandins 9: 83 (1975). Willis, A.L. Simplified thin-layer chromatography of prostaglandins in biological extracts. Brit. J. Pharmacol. 40: 583P (1970). Zusman, R.M. Quantitative conversion of PGA or PGE to PGB. Prostaglandins ~: 167 (1972).
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