Further Studies on the Mechanisms Controlling Prostaglandin Biosynthesis in the Cat Adrenal Cortex: The Role of Calcium and Cyclic AMP SUZANNE G. LAYCHOCK, W. WARNER, AND R. P. RUBIN Department of Pharmacology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 ABSTRACT. In light of previous studies which have implicated prostaglandin (PG) formation as a link in ACTH-induced steroid production by isolated cat adrenocortical cells, experiments were carried out to provide additional information regarding the role of PGs in adrenal steroidogenesis and their interactions with calcium and cyclic AMP. Perfusion

cyclic AMP levels. In this same preparation, indomethacin completely blocked ACTH and NPSACTH facilitated PGF 2 Q and PGE2 release but failed to suppress steroid release markedly. Calciumdeprivation blocked PG and steroid release evoked by these two polypeptides, and depressed PG release elicited by monobutyryl cyclic AMP (bcAMP) without affecting steroid release. These experiments offer additional evidence to support the concept that PGs play a role in the mode of action of ACTH; however, they do not appear to be obligatory intermediates in the steroidogenic process. The importance of calcium in regulating PG formation is discussed with special regard for the idea that this cation has a direct action on the enzyme systems which control PG synthesis. (Endocrinology 100: 74, 1977)

of cat adrenal glands with Locke's solution plus

/3( l-24)-ACTH resulted in an immediate increase in PGF 2 Q release, which rapidly declined to basal levels after the stimulus was withdrawn. In contrast, maximal rates of steroid release were manifest some 30 min after removal of ACTH. ACTH and its onitrophenyl sulfenyl derivative (NPS-ACTH) increased PG (PGF2a and PGE2) and steroid release by trypsin-dispersed cat cortical cells, but NPSACTH, unlike ACTH, did not augment cortical

T

HE STEROIDOGENIC action of ACTH on the adrenal cortex appears to be mediated by complex interactions of several cellular events (1,2). A number of studies have established that calcium (3) and an adenylate cyclase-mediated increase in cyclic AMP (4) are important components of the steroidogenic response. Our recent studies also offer evidence that prostaglandins (PGs) may mediate ACTH-induced steroidogenesis (5,6). PGs not only enhance steroidogenesis in isolated adrenocortical cells (5), but ACTH augments the synthesis (7) and elicits a dose-related release (6) of PGE2 and PGF2a. This latter finding confirms an earlier study showing that ACTH enhances PGE and PGF synthesis in rat adrenal homogenates (8).

Received April 9, 1976. Supported by a grant from the National Institute of Health (AM-18066). SGL is an USPHS Predoctoral Fellow, supported by Pharmacology Training Grant GM-0711 awarded to the Department of Pharmacology, Virginia Commonwealth University, Medical College of Virginia.

Further elucidation of the role of PGs in corticosteroidogenesis must consider their interrelation with calcium and cyclic AMP. Toward this end, an investigation was conducted to ascertain whether the increases in cyclic AMP and PGs in response to ACTH occur independently or are inextricably linked. Such information may be gleaned with the aid of the o-nitrophenyl sulfenyl derivative of ACTH (NPS-ACTH), since it augments steroidogenesis in rat adrenal cells while circumventing large increases in cyclic AMP (9). In addition, the fact that the steroidogenic actions of PGE2 and cyclic AMP, unlike that of ACTH, are not completely dependent upon the availability of extracellular calcium (5) suggests that a portion of the calcium requirement for ACTHinduced steroidogenesis may reside in the ability of this cation to modify the synthesis, rather than the cellular actions of cyclic AMP and PG. While the role of calcium in the ACTH-activated adenylate cyclasecyclic AMP system is well established (cf. 10), a possible action of calcium on PG 74

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ADRENAL PROSTAGLANDIN SYNTHESIS

synthesis needs to be explored. Accordingly, a consideration of the interactions of PGs, calcium, and cyclic AMP using trypsin dispersed feline cortical cells constitutes the basis of this study. Materials and Methods Materials Trypsin and lima bean trypsin inhibitor were purchased from Worthington Biochemical Corp.; bovine serum albumin (fatty acid free) and monobutyryl cyclic adenosine-3',5'-monophosphate were from Sigma Chemical Corp.; [5,6,8,11,12, 14,15-3H (N)] prostaglandin F 2a (SA-175 Ci/ mmol), [5,6,8,11,12,14,15-3H(N)] prostaglandin E2 (SA-117 Ci/mmol) and [3H (G)] adenosine3',5'-cyclic phosphate (SA-23 Ciymmol) were from New England Nuclear. Synthetic ACTH (/31-24) (Synacthen) generously supplied by Ciba Pharmaceuticals was the ACTH preparation employed in all experiments. Methods Preparation of the cortical cell suspension. Cats were injected with pentobarbital intraperitoneally, and both adrenals were removed and cortical cells dispersed with trypsin as previously described (11). Krebs bicarbonate buffer fortified with essential amino acids and vitamins was the basic medium employed. The constituents of this medium were identical to those contained in Modified Eagle Medium (MEM) (193G) (Grand Island Biological Company, Grand Island, New York). Following their dispersion, the cells (2.5 x 105/beaker) were incubated for 60-90 min at 37 C in 1.0 ml of medium containing 0.2% bovine serum albumin (BSA) plus 0.04% trypsin inhibitor. Stimulating agent and/or inhibitor was added as specified. In experiments concerned with calcium deprivation, calcium was omitted from the incubation medium and ethylene glycolbis-(/3-aminoethyl ether) N,N'-tetraacetic acid (EGTA) (0.4 mM) was added to chelate residual calcium. Following incubation, the suspension was centrifuged and the supernatant assayed for PGs or corticosteroids as described below. Adrenal perfusion. The left cat adrenal gland was perfused in situ according to the method of Douglas and Rubin (12). Perfusion was carried

75

out at room temperature with Locke's solution which had the following composition (mM); NaCl 154; KC1 5.6; CaCl2 2.0; MgCl2 0.5; NaHCO3 12; glucose 10. The perfusion medium was equilibrated with 95% oxygen and 5% carbon dioxide, and had a pH of 7.0. The rate of flow was maintained between 0.8-1.2 ml/min by regulation of the perfusion pressure and the addition of ACTH to the medium did not alter flow rate through the gland. The perfusate was collected in aliquots every 8 or 10 min from a cannula placed in the adrenolumbar vein. Prostaglandin analysis. Direct radioimmunoassay (RIA) of 300-400 /A aliquots of incubation medium, by a method described in detail elsewhere (13), provided a quantitative analysis of PGF 2a release. For PGE2 determinations, 200 /x\ of incubation medium was subjected to RIA using 50 (JL\ of a 1:500 dilution of a PGE antibody produced in rabbits against PGE2-bovine serum albumin which had been conjugated with ethyl chloroformate (14). Although PGEi was more than twice as effective as PGE2 in displacing [3H]-PGE2 from the PGE antibody, PGAl5 PGA2, PGBj, PGB2, PGF 2a , and 13,14dihydro PGE t showed negligible cross-reactivity (4% or less). The values obtained by direct assay of the incubation medium (299 ± 97 pg/2.5 x 105 cells) compared favorably with the RIA quantitation of these same samples after ether extraction and separation of the PGEs by thin layer chromatography (209 ± 106 pg) (n = 4). This indicates that the PG values obtained using this PGE antibody for direct RIA of incubation media are largely, if not entirely, PGE species. While the antiserum is not specific for one or another of the PGs of the E series, previous analytic techniques have established that PGE2, as opposed to PGEX, is the predominant PGE released by feline cortical cells (13); thus all PGE values are expressed as PGE2 equivalents. For estimation of intracellular PGs, isolated cortical cells which had been incubated 60-90 min in the presence or absence of ACTH and/or calcium were homogenized in 4 ml acidified Modified Eagle Medium (pH 3) and centrifuged for 15 min at 27,000 x g at 4 C. The resulting supernatant was extracted twice using either ethyl acetate:cyclohexane (2:1) or freshly distilled diethyl ether; extractions with either solvent system gave similar results. The extracts were dried in vacuo or under N2 and subjected to silicic acid chromatography (6) or, in the case of

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LAYCHOCK, WARNER AND RUBIN

76 500r

-.1900

1700 2 400

1500 o

1300 I o

300

1100 Z o 900 ° 200

700 ™

Endo i 1977 Vol 100 i No 1

which had been separated from the incubation medium by centrifugation. The cells were washed with Krebs bicarbonate buffer containing theophylline (0.5 mM) and were homogenized in 5% trichloroacetic acid containing [3H]-cyclic AMP. The homogenate was purified with Dowex-50 resin, lyophilized, and reconstituted in phosphate-citrate buffer (pH 6.3). One portion of the sample was counted in a liquid scintillation counter for determination of recovery; the remaining portion was assayed by the method of Steiner et al. (16).

500 100

0—*8

18

28

38

48

58

300

Results

100 0

Temporal relation between PG and steroid release

MIN

ACTM

FIG. 1. Time course of PGF2a and corticosteroid release from the perfused cat adrenal gland. Left adrenal glands were perfused in situ with Locke's solution. ACTH (50 fxU/m\) was added for 8 min and perfusion was continued with ACTH-free Locke's solution for an additional 50 min. The perfusate was collected during the 8 min perfusion with ACTH and at 10 min intervals thereafter. Corticosteroids (solid line) were extracted from 1 ml aliquots and assayed by competitive protein binding. PGF 2a determinations (broken line) were made by direct radioimmunoassay of 400 fi\ aliquots of perfusate. All values are expressed as per cent of basal values obtained from perfusate collected during a 10 min interval immediately prior to exposure to ACTH. Each point represents the average rate of release (±SE) during the 8 or 10 min collection period from 4 different preparations. Mean values for basal PGF 2a and steroid release were 12 (±4) pg and 27 (±7) ng/min, respectively.

the ether extracts, to thin layer chromatography (13). Those thin layer zones corresponding to PGF and PGE were scraped and the PGs eluted with methanol. The eluates were dried under N2 and resuspended in sodium phosphate buffer (pH 7.4) for PG analysis by RIA. Steroid analysis. Corticosteroids, mainly in the form of cortisol, were extracted from 1 ml of supernatant or perfusate with 5 ml methylene chloride and assayed by competitive protein binding using transcortin from human plasma as the binding agent (15). Cyclic AMP analysis. Cyclic AMP determinations were carried out on isolated cortical cells

Initially, experiments using adrenocortical cell suspensions demonstrated that measurable increases in PG release occurred during the first 15-30 min of exposure to j8(l-24)-ACTH. However, earlier studies using this same preparation (Warner and Rubin, unpublished) had shown that increases in steroid release also occurred during these same time intervals; thus, isolated cortical cells were deemed not to be the optimal system for studying the temporal sequence of PG and steroid release. An alternative approach to this problem was to study the dynamics of PG and steroid release in the isolated intact perfused cat adrenal gland. As found in previous studies (17), the perfused feline adrenal displayed a protracted pattern of steroid release with maximum release occurring some 30-40 min following exposure to ACTH (Fig. 1). By contrast, PGF2a release rapidly increased during perfusion with ACTH. After the stimulus was removed, PGF2a release remained elevated over the next 10 min and then declined to basal levels (Fig. 1). In some experiments, corticosteroid secretion was still markedly increased some 90 min after ACTH removal, despite the fact that PG release had reverted to basal levels one hour before.

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77

ADRENAL PROSTAGLANDIN SYNTHESIS FIG. 2. The effect of ACTH and NPS-ACTH on steroid and adrenal cyclic AMP production by isolated cat adrenocortical cells. Equal numbers of cortical cells (2.5 x 105) were incubated at 37 C for 60 min in MEM, in the presence or absence of varying concentrations of ACTH (0.01-1 nut) or NPS-ACTH (323200 IIM). After the cells were separated from the medium by centrifugation, the pellet and supernatant were assayed for cyclic AMP and steroid, respectively. The vertical bars represent mean values (±SE) from 3 experiments, expressed as per cent of unstimulated control values. The average cyclic AMP and steroid values (±SE) of unstimulated cells were 15.2 ± 2.9 pmol and 28.7 ± 8.0 ng/2.5 x 105 cells, respectively.

1000

YJ\ |

CORTICOSIEROIO | CYCHC

AMP

800

600

Z

400

200 100

0.01

0.03 A C T H

Comparative effects of ACTH and NPSACTH on PG and cyclic AMP synthesis In light of the evidence that PG release may be an early event in the tropic action of ACTH, further studies were carried out to discern more clearly the relationship between PG and cyclic AMP synthesis during steroidogenesis by comparing the stimulant effects of ACTH and NPS-ACTH. Although the ACTH analogue was approximately one ten-thousandth as potent as ACTH, it elicited a dose-related facilitation of steroid release (Fig. 2). However, unlike ACTH, NPS-ACTH failed to induce a measurable increase in cortical cyclic AMP (Fig. 2). Since NPS-ACTH appeared to dissociate the early event of elevated cyclic AMP levels from steroid biosynthesis, it was of interest that both ACTH and NPS-ACTH enhanced the release of PGE 2 and PGF 2a (Figs. 3 and 4). Although the enhanced PG release elicited by both stimulants was completely suppressed by indomethacin, ACTH and NPS-ACTH still markedly stimulated steroid release (Fig. 3).

1.00

32

320

3200 n M

NPS-ACTH

Calcium deprivation and PG release Incubation of isolated feline cortical cells in a calcium-deprived medium containing EGTA completely suppresses the steroidogenic response to even high ACTH concentrations (5). This finding was confirmed in the present investigation (Fig. 4) which also extended the calcium dependency of ACTH stimulation to include PG release. Calcium deprivation reduced ACTH-evoked PGF 2a and PGE2 release to levels comparable to those obtained under basal conditions (Fig. 4). Likewise, calcium lack blunted the enhancement of both steroid and PG release elicited by NPS-ACTH (Fig. 4). By contrast, the steroidogenic action of the monobutyryl analogue of cyclic AMP (bcAMP) was not significantly diminished by calcium deprivation, although calcium lack blunted bcAMP facilitated PGE2 and PGF 2a release (Fig. 4). In order to ascertain that calcium deprivation was not irreversibly altering the response of cortical cells to ACTH, in 2 different experiments, cells were incubated for 60 min in calcium-deprived medium plus ACTH and then resuspended for the same

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ADRENAL PROSTAGLANDIN SYNTHESIS

78

I | COUT icosTEnoip PROS T A G L A N 0

I N

F,

400.

300

200

100

~f£) ACTH

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Endo • 1977 Vol 100 • No 1

FIG. 3. The effects of indomethacin (INDO) on PG and steroid release evoked by ACTH and NPS-ACTH. Equal numbers of cortical cells were incubated for 90 min in MEM. ACTH (0.3 nM) (125 fiU) or NPS-ACTH (3200 nM) was added when required in the presence or absence of indomethacin (3 x 10"5 M). Steroid was extracted from 1 ml aliquots and assayed by competitive protein binding. PGF2Q determinations of the medium were made by direct radioimmunoassay, using 400 fA aliquots. Each vertical bar repre.... sents the mean value (±SE) from the number of experiments indicated at the base of the bar; all values are expressed as per cent of the corresponding unt,HJ" stimulated control samples in the presence or absence of indo-

methacin. Absolute basal steroid concentrations in the presence and absence of indomethacin were 31 ± 10 ng and 32 ± 7 ng, respectively. Absolute basal PGF 2a concentrations in the presence and absence of indomethacin were 246 ± 61 pg and 184 ± 42 pg, respectively. All values are per 2.5 x 105 cells.

time interval in calcium-containing medium plus ACTH. The average amount of PGF 2a and PGE 2 released by ACTH (250 /uU) in the absence of calcium was 212 and 105 pg, respectively, and subsequently, in the presence of calcium was 417 and 234 pg, respectively. A similar reversibility of the effects of calcium lack on steroid production was also demonstrable in these same experiments. Thus, the average amount of steroid released during and following calcium deprivation was 15 and 182 ng, respectively. Although variability from preparation to preparation somewhat limits statistical analysis of the data in terms of absolute concentrations, Table 1 gives the PGF 2a , PGE2, and steroid concentrations when data from different experiments were combined. Certain general conclusions can be drawn from these results which are not readily apparent from the data expressed as per cent of control. Although basal PGF 2a and PGE 2 levels were generally comparable, PGF 2a release was augmented to a greater extent by ACTH

and bcAMP than by NPS-ACTH, which appeared to enhance PGF 2a and PGE 2 release to a similar degree. Basal PG levels appeared slightly depressed by calcium deprivation, but the addition of stimulating agents generally failed to augment markedly PG release above these resting levels. Intracellular PG levels Although a host of studies have clearly established that PG release is the result of de novo PG synthesis (18), a few experiments were carried out to substantiate this fact in feline cortical cells by comparing the amount of PG remaining in cortical cells to the amount released to the medium following a 60 min exposure to 0.6 nM ACTH (250 /AU). The average amounts of PGF 2a (44 ± 19 pg) and PGE 2 (41 ± 32 pg) (n = 3) contained intracellularly was only about 15% of the amount of PGF 2a (309 ± 111 pg) and PGE 2 (278 ± 79 pg) released. Comparably low amounts of PGF 2a (36 ± 20 pg) and

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79

ADRENAL PROSTAGLANDIN SYNTHESIS PGE2 (55 ± 27 pg) were measured intracellularly in the absence of ACTH. In another experiment, cellular PG content was analyzed during calcium deprivation in order to ascertain whether intracellular levels were augmented in the absence of calcium. In this experiment, the cell PGE 2 and PGF 2a content after exposure to ACTH in the presence of calcium was 107 and 65 pg/2.5 x 105 cells, respectively, and in the absence of calcium was 84 and 12 pg, respectively.

TABLE 1. Effect of calcium deprivation on prostaglandin and steroid release PGF2( (pg)

• •

+ Ca - C a

+Ca

-Ca

+ Ca -Ca

Control

206 182 ± 5 5 ±50

170 125 ±30 ±30

14 ±2

14 ±2

ACTH

561 172 ± 145 ± 33

284 152 ± 6 5 ± 22

140 ±23

15 ±3

Control

204 134 ± 5 1 ±44

190 98 ±53 ± 29

26 ± 12

12 ±2

NPS-ACTH

313 152 ±59 ±60

311 94 ± 117 ±38

75

Further studies on the mechanisms controlling prostaglandin biosynthesis in the cat adrenal cortex: the role of calcium and cyclic AMP.

Further Studies on the Mechanisms Controlling Prostaglandin Biosynthesis in the Cat Adrenal Cortex: The Role of Calcium and Cyclic AMP SUZANNE G. LAYC...
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