GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

32, 330-340 (1977)

In Vitro ACTH Stimulation of Corticosterone Output in Relation to Cyclic Nucleotide Alterations in the Crocodilian (Caiman sclerops) Adrenal KENNETH

V. HONN

AND WALTER

CHAVIN

Departments of Radiology and Biology, Wayne State University, Detroit, Michigan 48202 Accepted March 9, 1977 As ACTH increases cyclic GMP (cGMP) levels in the crocodilian adrenal, the cyclic AMP (CAMP) mechanism of ACTH action in corticosterone output was evaluated in Caiman sclerops. In vitro production of CAMP and corticosterone was determined by radioimmunoassay (RIA). Control adrenals showed a significant increase in CAMP levels above that occurring in normal and zero-time adrenals. In addition, a basal level of corticosterone output was present. Porcine ACTH produced a log-dose depression of CAMP levels below the control level. This inhibitory action was shared by Caiman ACTH. Both porcine and Caiman ACTH significantly increased Caiman corticosterone output. In contrast to the reptilian response, rat adrenals used as reaction controls responded to both porcine and Caiman ACTH with increased CAMP levels and corticosterone output. The cAMP/cGMP ratios in Caiman adrenals pursuant to ACTH stimulation demonstrated that increased steroid output was coincident with the lowest nucleotide ratio. A high degree of correlation exists between total steroid output and nucleotide ratio: however, evidence suggests that additional factors may also be involved.

Considerable support is present for the concept that CAMP is a critical factor in the action of ACTH upon the adrenal cortex (Halkerston, 1975). Nevertheless, evidence also is present to indicate that other factors are involved in the action of ACTH upon the adrenal cortex (Kitabchi et al., 1974; Honn and Chavin, 1975a, 1976a, b; 1977a, b). These factors may supersede or modulate the role of CAMP (Honn and Chavin, 1976a). In addition to such factors, the cyclic nucleotides may have an interactive effeet upon cells, as an inverse relationship may develop between CAMP and cGMP (Honn and Chavin, 1975a; Whitley ef al., 1975). Thus, thephysiologicaleffectsofACTH in some species may not be attributable to either nucleotide but rather to simultaneous alterations in the levels of both nucleotides pursuant to ACTH stimulation.

The crocodilian adrenal is interesting in the above regard, for it responds to ACTH by elevation of cGMP levels (Honn and Chavin, 1974a, 1975a) in contrast to the ACTH elevation of CAMP levels in the mammalian adrenal (Grahame-Smith et al., 1967; Halkerston, 1975). In mammals, the elevation of CAMP levels is related to corticoid output, for the most part. However, the physiological significance of the nucleotide changes in relation to steroid output by the reptilian adrenal is unknown. As the crocodilians show an unusual adrenocortical cyclic nucleotide response to homologous and heterologous ACTH (Honn and Chavin, 1973, 1974b), the relationships of the cyclic nucleotides to glucocorticoid output were investigated in order to aid in the elucidation of the mechanisms of ACTH action. 330

Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.

ISSN 00166480

CAIMAN

MATERIALS

STEROID-NUCLEOTIDE

AND METHODS

A total of 242 young Caiman sclerops (34.5 a 6.5 g; x f SEM) were maintained and sacrificed as previously described (Honn and Chavin, 1975a). Paired adrenals were removed immediately, trimmed free of adherent tissue, weighed, diced, and placed in cold (O-4”) Krebs-Ringer bicarbonate buffer, KRBGA (pH 7.4, 200 mg of glucose/d1 and 0.5% bovine albumin fraction V). Each pair of diced adrenals was incubated (I ml of KRBGA; 30”; 95% 0, + 5% CO,) in a Dubnoff metabolic shaker for l-256 min. Controls were incubated in KRBGA. CAMP and corticosterone responses to chromatographically purified (Schwarz/Mann; 134.8 IUlmg) porcine ACTH (l1000 mIU/ml; l-256 min), CPH (Caiman pituitary homogenate), and 10 mM theophylline (l-32 min) alone or in combination with these hormone preparations were evaluated. In addition, adrenals were preincubated (4 min) in 1000 mIU of porcine ACTH or in CPH (588 ~1I-J of immunoreactive ACTH), removed, and placed in KRBGA theophylline (10 m&I) in addition to ACTH or CPH. All incubation intervals were terminated by quenching the incubates in liquid nitrogen. Normal CAMP levels were determined in adrenals removed, weighed, and quenched in liquid nitrogen immediately postdecapitation. Zero-time adrenals were removed from the reaction mixture (O4”), diced, and quenched, immediately prior to incubation. Pituitaries from 66 C. sclerops were removed, weighed, and homogenized in cold (O-4”) KRBGA (5 mg/ml). The ACTH contents of CPH (588 pIU of ACTHlml) as well as of serum of six Caiman 1203 2 22.4 (2 * SEM) fiU of ACTHlml] were determined by radioimmunoassay (Singley and Chavin, 1975). Two doses of CPH (64 and 588 ,uIU of ACTH/ml) were utilized. The CAMP and corticosterone responses of rat adrenals to porcine ACTH and to CPH were used as controls. Adrenals from 48 male Holtzman albino rats (134 f 1.17 g; 2 + SEM) sacrificed by decapitation were used. Rat adrenal zero-time CAMP levels were determined as above. Incubations were performed as described above except the incubation temperature was 37”. Adrenal CAMP levels were determined by radioimmunoassay (RIA) (Honn and Chavin, 1975a). The protein content of each adrenal incubate was determined (Honn and Chavin, 1975b) and data were expressed as picomoles of CAMP per milligram of adrenal protein. In addition, the cGMP data (Honn and Chavin, 1975a) were recalculated on the basis of adrenal protein content and expressed as picomoles of cGMP per milligram of protein. The corticosterone output of the adrenals was determined by RIA (Foster and Dunn, 1974) using a highly specific antibody (cross-reactivities: corticosterone, 100%; cortisol, 0.46%; cortisone, 0.5%; I&OH-corticosterone, 0.1%;

RELATION

331

aldosterone, 0.36%). Steroid data are expressed as nanograms of corticosterone per milligram of adrenal protein per milliliter of KRBGA. Data were subjected to the analysis of variance, linear regression analysis, and the Student distribution test for unpaired observations. A minimum of three adrenal pairs was utilized per datum point. Data are indicated as mean f SEM. Differences were accepted as significant when P < 0.05.

RESULTS The level of Cuiman adrenal CAMP at zero time (48 t 6 pmol/mg of protein) was not significantly different from the normal Caiman adrenal CAMP level (60 + 18 pmol/mg of protein). Therefore, zero-time CAMP levels represent baseline in vivo and in vitro levels. The control groups showed a rapid significant (P < 0.005) rise in CAMP levels above baseline levels (Fig. 1). The maximal control response (108 k 17 pmoYmg of protein) occurred early (4-8 min) and declined precipitously (14.3 + 2 pmol/mg of protein) below baseline CAMP levels at 32 min (P < 0.005). The control and baseline CAMP levels were not different at 256 min. ACTH at the lowest dose level used (1 mIU) did not significantly alter the CAMP response from that of the control groups at l-16 min (Fig. 1). However, the CAMP levels of the 1-mIU group were significantly higher than the controls, from 32 to 128 min, but were not significantly different from the baseline levels (Fig. 1). At 256 min, the I-mIU, control, and baseline CAMP levels were the same. The CAMP levels of the IO-mIU ACTH groups were depressed (57 -+ 18 pmol/mg of protein) at 8 min (Fig. 2). This depression was intermediate to the control and the lO@mIU ACTH CAMP levels. The lO@ and lOOOmIU ACTH groups did not significantly differ at any time interval studied (Figs. 1 and 3). Both doses of ACTH significantly depressed CAMP below baseline levels during the early (Fig. 3) and later time intervals studied (Fig. 1). In addition, I-100 mIU of ACTH produced a dose-dependent inhibi-

332

HONN

1632

AND CHAVIN M L-4

Control ACTH

/m/U

c-e

ACTH

10 mlU

+-a+ D- a ---

ACTH lOOmU ACTH 1000 m/U Zero time

256

128

64

TIME,mh FIG. 1. Temporal depression of Caiman adrenal CAMP levels by various doses of porcine ACTH. Zerotime adrenals were removed, placed in cold (O-4”) reaction mixture, and quenched (liquid N,) immediately prior to the initiation of the incubation series. Control adrenals were incubated (30”) in reaction mixture without added hormones and quenched at given time intervals.

tion of Caiman adrenal CAMP levels as early as 1 min (Fig. 4). CPH containing immunoreactive ACTH (588 ~IU/ml) significantly depressed Caiman adrenal CAMP levels (10.9 t 1.2 pmol/mg of protein) below those of the control (P < 0.025; 4 min). These depressed + 1= _-.

Control ACTH ACTH Zero

IO* IO’ time

levels were not significantly different from the depression evoked by 100 or 1000 mIU of porcine ACTH. Theophylline, however, significantly (P < 0.005) elevated CAMP levels (43 2 3.6 pmoYmg of protein; 8 min) above CPH or porcine ACTH treatment (Fig. 5). No significant difference between

120

i---f

0

1

I

I

4

8

I2

I

16

TIME, min. FIG. 2. Intermediate depression of Caiman adrenal CAMP level (8 min) with 10 mIU of ACTH (IO’) compared to control and 100 mIU of ACTH (101).

FIG. 3. Depression of Caiman adrenal CAMP to lowest levels with 100 mIU of ACTH (102) and 1000 mIU of ACTH (103).

CAIMAN STEROID-NUCLEOTIDE

I

1 IO' ACT H , m/U/ml.

102

FIG. 4. ACTH dose-dependent depression of Cuiman adrenal CAMP levels at the I-min incubation interval. ACTH doses: l(l), lO(lV), and lOO(19) mIU.

RELATION

01

8

333

I6

24

32

TIME,mh FIG. 5. Depression of Caiman adrenal CAMP levels with CPH in contrast to CAMP levels following theophylline (IO mM) treatment.

significantly (P < 0.01) higher with 1000 theophylline-treated adrenals and their cor- mIU of ACTH compared to the other responding control was present at 8 min. ACTH doses used. ACTH, 10, 100, and Preincubation (4 min) in ACTH (CPH, 588 1000 mIU, significantly (P < 0.05, < 0.0005, and < 0.005, respectively) elevated PIU or 1000 mIU) abolished the subsequent CAMP increase (8 min) observed with corticosterone production above that of the theophylline alone (Fig. 6). However, at 16 control at 32 min (Fig. 7). Although not statistically significant, 1 mIU of ACTH inand 32 min these ACTH-theophylline groups were not significantly lower than creased steroid production 12% at 32 min. ACTH-induced corticotheophylline alone (Fig. 6). Although the The observed sterone production at 32 min was log-dose 16-min CAMP levels of the ACTHtheophylline groups were 30 and 137% related (Fig. 8). ACTH, 10-1000 mIU, conhigher than ACTH or CPH alone, respec- tinued to significantly (P < 0.0005) increase tively, they were not statistically sig- corticosterone production above that of the nificant. controls from 64 to 256 min, although the Control adrenal incubates demonstrated rate relative to control output diminished a steady basal corticosterone output during (Table 1). ACTH, 1 mIU, significantly (P < 0.01) elevated corticosterone production at the interval studied (l-256 min), increasing from 108 + 2.8 ng/mg of protein/ml of 64 and 128 min (Fig. 7); however, no sigKRBGA at 1 min to 340 + 8 ng/mg of nificant difference was present between I protein/ml of KRBGA at 256 min (Table 1). mIU of ACTH and controls at 256 min (TaPorcine ACTH (I- 1000 mIUlm1) did not ble I). Examination of the cAMP/cGMP (A/G) significantly elevate corticosterone producratios immediately prior to and after the tion above controls during 1-16 min. However, early (l-2 min) steroid output was first significant increase (32 min) in steroid

334

HONN

AND CHAVIN

An ACTH dose-dependent relationship between decreasing A/G ratio and increasing corticosterone was evident (Fig. 8). Linear regression analysis (Fig. 9) revealed a highly significant correlation (r = 0.9 11) between a decreased A/G ratio and corticosterone output at 32 min. Regression analysis of A/G ratios on corticosterone at the other time intervals revealed a lack of correlation (Table 2) prior to the onset of steroid output (32 min). Following the 32-min interval the degree of correlation decreased as the A/G ratios increased (Table 2). Cairnan immunoreactive ACTH (CPH) at 64 and 588 $U of ACTH/ml significantly (P < 0.025 and P < 0.0005, respectively) increased corticosterone output above control corticosterone output at 16 min (Fig. 10). Corticosterone output in response to both doses of CPH continued to increase FIG. 6. Effect of theophylline (IO ti) upon throughout the period studied (Fig. 10). InACTH (1000 mIU or CPH)depressed CAMP levels in terestingly, the A/G ratios (8 min) for the the Caiman adrenal. low and high CPH doses were 27 and 0.96, production by porcine ACTH revealed a respectively. At 16 min these ratios debiphasic pattern with all ACTH doses creased to 1.5and 0.88, respectively, thereafter (32 min) increasing to 25 and 1.02, studied (Table 2). The A/G ratio decreased to a minimum at 32 min and increased respectively. thereafter (32-128 min). In general, the Theophylline, 10 mM, significantly (P < 0.005) increased corticosterone output (185 ratios decreased more rapidly (pre-32 min) rt 7.9 ng/mg of protein/ml of KRBGA) than their subsequent recovery (Table 2).

- 200 ii B

[lo;,, 8 0

0

I 16

,

,

I 32

I 64

, I 128

,I I 256

TIME,min. FIG. 7. Temporally ((16-256 16-256 min) increased Caiman graded doses of mammalian ACTH.

adrenal corticosterone

production

in response to

CAIMAN

STEROID-NUCLEOTIDE

RELATION

335

I 2 300k or : . 200-

-18

Q -B -6252 -

Y s: w iii 8 IOOi= 80 0

I

1

IO’

0

102

I03

AC T H , mfU/m/. FIG. 8. ACTH dose-dependent increase in Cuiman adrenal corticosterone simultaneous ACTH dose-dependent decreased cAMP/cGMP ratios.

above that of the controls at the 32-min interval. Corticosterone output by adrenals preincubated in ACTH or CPH and transferred to hormone + theophylline was highly variable and not significantly different from controls (l-32 min). However, the general trend was lower steroid output than with ACTH or CPH alone. In the absence of ACTH, rat adrenal control CAMP levels were not significantly different from zero-time levels. In both situations, however, these rat CAMP levels were significantly lower (P < 0.005) than Caiman adrenal zero-time and control CAMP levels during the interval studied (Fig. 11). Caiman immunoreactive ACTH (588 $U/ml significantly increased (P < 0.01, 8 min; P < 0.005, 16 min) rat adrenal CAMP h Vitro

I

-12

output at 32 min contrasted with

levels (36 k 10.5 pmoYmg of protein; 16 min) above that of the rat adrenal controls (Fig. 12). Both Caiman (588 @IU) and porcine (100 mIU) ACTH significantly (P < 0.005) increased rat adrenal corticosterone output (4 min) above the control output (Fig. 13). Steroid output continued to increase in response to both ACTH preparations during the interval (4-32 min) studied (Fig. 13). DISCUSSION

The existence of reptilian pituitary ACTH has been suggested previously by bioassay data (Licht and Bradshaw, 1969) and confirmed by radioimmunoassay of Caiman pituitary preparations (Honn and Chavin, 1975a). The present report, in addition,

TABLE I ACTH (I-1000 mIU/ml) STIMULATION OF CORTICOSTERONE BY ADRENALS OF Caiman sclerops“

OUTPUT

Time (min) ACTH (mIU/ml) 1000 100 10 1 Control 0 Corticosterone,

32 368 2 5.7 283 2 6.2 212 +- 34 153k 8 137 + 14

64 399 2 273 2 290 k 181 ” 141 k

128 11.3 14 36 12 6

634 + 449 4 4002 338 2 252 4

nanograms per milligram of adrenal protein per milliliter

256 36 27 15 16 17

7064 513 -t 451 -t 305 2 340 k

10 66 53 66 8

of KRBGA, x * SEM.

336

HONN

cAMP/cGMP

AND

CHAVIN

TABLE 2 RATIOS IN Cniman sclerops ADRENALS AT INTERVALS FOLLOWING ACTH STIMULATION In Vitro Time (min)

ACTH (mIU/ml) 1000 100 IO I P

8

16

32

64

128

7.89 8.45 39.7 71.0 0.307

4.33 7.39 34.3 32.0 0.324

1.07 4.34 8.25 16.7 0.911

3.0 4.53 13.6 42.0 0.837

3.96 5.9 18.9 50.0 0.766

a Correlation coefficient of linear regression analysis (least-squares method) of A/G ratios on adrenal corticosterone output.

demonstrates circulating immunoreactive ACTH in the Cairnan. It is clear, therefore, that the hypophyseal hormone is present in the reptile. The failure of the initial reports to demonstrate a steroidogenic action of ACTH in reptilian adrenals (Phillips er al ., 1962; Gist and DeRoos, 1966; Macchi and Phillips, 1966; Nothstine et al., 1971) has been ascribed to the low temperatures

c p

Y=-11.96X r= 0.911

Q 400 Q

+ 343f

used (Licht and Bradshaw, 1969; Callard et al., 1975). Indeed, the subsequent reports demonstrating a positive ACTH effect in reptilian adrenal steroidogenesis were pei-formed at temperatures of 30” or higher (Leloup-Hatey, 1968; Licht and Bradshaw, 1969; Huang et al., 1969; Callard, 1975; Callard et al., 1975; Keung et al., 1975; Gist

34.5

F g# 300 . Y & 200 r zi 0

100

Li t3 0

0

J 4

0

12

01

16

cAMP/cGMP 9. Linear regression analysis (least-squares method) of cAMP/cGMP ratios on corticosterone at the 32-min incubation interval. Solid line, calculated estimate k standard error (dotted line); circle, observed data: r, correlation coefficient. FIG.

0

16

24

32

TIME, min. IO. Corticosterone production in response to two doses of C&man immunoreactive ACTH compared to KRBGA controls. Circle, CPH (588 pIU of ACTH/ml): square, CPH (64 pIU of ACTH/ml); triangle, KRBGA control. FIG.

STEROID-NUCLEOTIDE

CAIMAN

337

RELATION

RAT ADRENAL

------_ -ia’ 5 I0 30

4

8

12

16

TlME,mh. FIG. 11. Control CAMP levels in the Cuiman and the rat adrenals compared to each other and to their zero-time levels. Upper dashed line, Cairnan adrenal at zero time; lower dashed line, rat adrenal at zero time.

RAT ADRENAL

. !k a 0 IO control 9 i

+

O6

FIG. 12. Stimulation with CPH.

TIME,min. of rat adrenal CAMP levels

TI ME, min. FIG. 13. Temporal corticosterone production by the rat adrenal in response to mammalian (100 mILJ/ ml) and Caiman (588 ~IU/ml) ACTH.

and Kaplan, 1976). This evidence is supported by the present study which demonstrates the presence of a functional hypophyseal-adrenocortical axis in a reptile. Cuiman and rat adrenals demonstrate a basal, cumulative steroid output in vitro. Cuiman ACTH, like porcine ACTH, stimulates in vitro CAMP levels and corticosterone output in rat adrenals. In addition, Cuimun and mammalian ACTH produce a dose-related corticosterone output by Cuimun adrenals. Nevertheless, mammalian ACTH significantly depresses reptilian adrenal CAMP levels below that occurring in the unstimulated adrenal (Honn and Chavin, 1973). This inhibition is log-dose related and is shared by reptilian ACTH. However, a lower dose of the native hormone is required. The possibility of interspecies differences in potency is not surprising as the ACTH-adrenocortical cell receptor interaction appears to be sensitive to slight modifications in the ACTH mole-

338

HONN

AND

cule (Lefkowitz et al., 1970). Despite possible structural differences in reptilian and mammalian ACTH, the ability to stimulate CAMP and steroid output by the rat adrenal is retained. The difference between Caiman and rat adrenal CAMP responses, therefore, appears to be inherent to the adrenocortical cell of the given species and not to the ACTH molecule. This concept is supported by comparing the CAMP level in the control rat adrenal, which was not different from zero-time. However, in the a rapid increase in control Caiman, adrenal CAMP levels above zero-time levels occurs within 4-8 min. The only difference between zero-time (O-4”) and control conditions for the Caiman adrenal is incubation temperature (30”). Although a greater temperature disparity is present between zero-time (O-4”) and control (37”) rat adrenals, CAMP levels are not different. Theophylline inhibits phosphodiesterase activity and increases CAMP levels and steroid output by rat adrenals (Grahame-Smith et al., 1967). However, the theophylline effect upon Cuiman adrenal is not as dramatic, although sign&ant steroid output occurs. Possibly the theophylline effect upon CAMP is masked by the significant increase in control CAMP levels during the early incubation intervals. This is supported by the fact that theophylline significantly increases Cuiman adrenal cGMP levels when control cGMP levels are low (Honn and Chavin, 1975a). If the ACTH-evoked depression of Caimun adrenal CAMP is the result of adenylate cyclase inhibition, pretreatment of the Cuimun adrenal with ACTH should prevent any subsequent theophyllineinduced increase. Nevertheless, after an initial lag period (16 min), ACTH + theophylline elevates CAMP levels. These levels are higher than those produced by ACTH and are not different from those produced in response to theophylline. Thus, ACTH depression of CAMP levels may reflect the direct or indirect involvement of CAMP

CHAVIN

phosphodiesterase. In other systems (Rillema et al., 1973), hormonally depressed target organ CAMP levels are coincident with increased CAMP phosphodiesterase activity. Regardless of the mechanism init is clear that the current volved, hypothesis of the mechanism of ACTH action is not consistent with the evidence from this nonmammalian vertebrate. Coincident with data from the mammalian systems (Rubin et al ., 1973; Moyle et al., 1973; Kahnt et al., 1974; Sharma, 1974; Warner and Rubin, 1975) suggesting inconsistencies in the current ACTH-CAMP hypothesis, it is suggested that factors in addition to CAMP may be involved in the mechanism of action of ACTH on the adrenocortical cell. In this regard, the present data were compared with the cGMP response of Cuimun adrenals to ACTH (Honn and Chavin, 1975a). The CAMP and cGMP levels vary inversely in response to ACTH in both Caiman (Honn and Chavin, 1975a) and mammalian (Whitley et al., 1975) adrenals. CAMP levels increase while cGMP levels decrease in the mammalian adrenals, but the reverse response occurs in the Caiman adrenal. This species difference does not appear to be as divergent when the adrenal cyclic nucleotide ratio pursuant to ACTH stimulation is considered. Mammalian ACTH first increases Cuiman plasma corticosterone levels in vivo 30 min postinjection (Gist and Kaplan, 1976). A similar temporal corticosterone response (32 min) to mammalian ACTH occurs in vitro. This first significant burst in steroid output is correlated in vitro with the concomitant depressed cAMP/cGMP ratio. Significant corticosterone output by Cuiman adrenals in response to Caiman ACTH occurs earlier (16 min) than with mammalian ACTH (32 min). Significantly, the lowest cAMP/cGMP ratio in response to Cuimun ACTH occurs at 16 min while that following mammalian ACTH occurs at 32 min. Although there is a correlation between the actual cyclic nucleotide ratio (32 min) and the subsequent amount of cor-

CAIMAN

STEROID-NUCLEOTIDE

ticosterone output (i.e., lowest ratio with highest corticosterone output), additional factor(s) may be involved. For example, the cyclic nucleotide ratio (4.33; 16 min) in response to 1000 mIU of ACTWml is almost identical to the ratio (4.34; 32 min) in response to 100 mIU of ACTWml; however, no significant steroid output above control levels occurs in the former instance while significantly increased steroid output is observed in the latter. If the ratios are physiologically significant, it is possible that an additional factor(s) may be necessary before significant steroid output occurs. At this point it is necessary to discern between steroid release (output) and steroid synthesis. In most investigations of ACTH-adrenocortical cell interaction, the steroid output is assumed to be equivalent to steroidogenesis. However, these may represent separable events (Jaanus et al., 1970; Honn and Chavin, 1977~). In addition, steroid release appears to occur coincident with the synthesis and release (Rubin et al., 1974; Laychock and Rubin, 1974) of a specific protein factor and it requires calcium (Jaanus et al., 1970). Further, other factors (i.e., prostaglandins) may play a modulatory role (Honn and Chavin, 1976a; 1977a, b) in these events. Although a high degree of correlation exists between cyclic nucleotide ratios (32 min) and total steroid output, this does not represent a complete explanation for the events pursuant to ACTH-adrenocortical cell interaction. The role of CAMP as unitary second messenger in the mechanism of ACTH action must be expanded to include cGMP and, possibly, additional factors which may modulate (Honn and Chavin, 1976a, b; 1977a, b) the role of the cyclic nucleotides in the control of adrenal steroid biosynthesis and release. REFERENCES Callard, G. V. (1975). Control of the interrenal gland of the freshwater turtle Chrysemys picra in vivo and in vitro.

Gen.

Comp.

Endocrinol.

25, 323-331.

Callard, G. V., Chan, S. W. C., and Callard,

I. P.

339

RELATION

(1975). Temperature effects on ACTH-stimulated adrenocortical secretion and carbohydrate metabolism in the lizard (Dipsosaurus dorsalis). .I. Comp.

Physiol.

99,

271-277.

Foster, L. B., and Dunn, R. T. (1974). Single-antibody technique for radioimmunoassay of cortisol in unextracted serum or plasma. Clin. Chem. 20, 36% 368. Gist, D. H., and DeRoos, R. (1966). Corticoids of the alligator adrenal gland and the effects of ACTH and progesterone on their production in v&o. Gen. Comp. Endocrinol. 7, 304-313. Gist, D. H., and Kaplan, M. L. (1976). Effects of stress and ACTH on plasma corticosterone levels in the caiman Caiman crocodilus. Gen. Comp. Endocrinol.

28, 413-419.

Grahame-Smith, D. G., Butcher, R. W., Ney, R. L., and Sutherland, E. W. (1967). Adenosine 3’,5’monophosphate as the intracellular mediator of the action of adrenocorticotropic homone on the adrenal cortex. J. Biol. Chem. 242, 5535-5541. Halkerston, I. D. K. (1975). Cyclic AMP and adrenocortical function. In “Advances in Cyclic Nucleotide Research” (P. Greengard and G. A. Robinson, eds.), Vol. 6, pp. 99-136. Raven Press, New York. Honn, K. V., and Chavin, W. (1973). In vitro inhibition of adrenal cyclic AMP by the mammalian ACTH in the crocodilian, Cuiman sclerops. Amer. Zoo/. 13, 1281. Honn, K. V., and Chavin, W. (l974a). Control of cyclic nucleotide levels in the reptilian adrenal. Endocrinology 94, Suppl. A-161. Honn, K. V., and Chavin, W. (1974b). Mechanism of ACTH action in the crocodilian adrenal (Caiman sclerops).

Fed.

Proc.

33,

205.

Honn, K. V., and Chavin, W. (1975a). ACTH control of adrenocortical c-GMP. Gen. Cump. Endocrinol. 26, 374-38 I. Honn, K. V., and Chavin, W. (1975b). An improved automated biuret method for the determination of microgram protein concentrations. Anal. Biothem.

68,

230-235.

Honn, K. V., and Chavin, W. (1976a). Prostaglandin modulation of the mechanism of ACTH action in the human adrenal. Biochem. Biophys. Res. Commun. 73, 164-170. Honn, K. V., and Chavin, W. (1976b). Role of prostaglandins in aldosterone production by the human adrenal. Biochem. Biophys. Res. Commun. 72, 1319-1326. Honn, K. V., and Chavin, W. (1977a). Antagonistic action of E and F series prostaglandins upon mineralocorticoid production by the human adrenal. Experientia 33, 398-400. Honn, K. V., and Chavin, W. (l977b). Temporal effects of ACTH, indomethacin and 7-oxa- 13prostynoic acid upon glucocorticoid production by the human adrenal. Experientia, in press.

340

HONN

AN D CHAVIN

Honn. K. V.. and Chavin. W. (1977~). In vitas temporal CAMP and cortisol responses to ACTH by the normal human adrenal gland. Acta Endocrinol.. in press. Huang, D. P., Vinson, G. P., and Phillips, J. G. (1969). The metabolism of pregnenolone and progesterone by cobra adrenal tissue in t+o and the effect of ACTH on product yield-time curves. Gen. Comp. Endocrinol. 12, 637-643. Jaanus, S. D., Rosenstein, M. J., and Rubin, R. P. (1970). On the mode of action of ACTH on the isolated perfused adrenal gland. J. Physiol. 209, 539-556. Kahnt, F. W., Milani, A., Steffen, H., and Neher, R. (1974). The rate-limiting step of adrenal steroidogenesis and adenosine 3’,5’-monophosphate Eur. J. Biochem. 44, 243-250. Keung, W. M., Chiu, K. W., and Kong, Y. C. (1975). Steroidogenesis in Varunus adrenals in vitro. Comp. Biochem. Physiol. 51B, 307-308. Kitabchi, A. E., Nathans, A. H., James, P., Bowyer, F., Wilson, D. B., and Kitchell, L. C. (1974). Cyclic 3’,5’-GMP (cGMP) as possible mediator of steroidogenic action of ACTH at physiologic concentrations. Endocrinology 94, Suppl. A-160. Laychock, S. G., and Rubin, R. P. (1974). Isolation of ACTH-induced protein from adrenal perfusate. Steroids 24, 177-184. Letkowitz, R. J., Roth, J., Pricer, W., and Pastan, I. (1970). ACTH receptors in the adrenal: Specific binding of ACTH-rZ51 and its relation to adenyl cyclase. Proc. Nat. Acad. Sci. USA 65, 745-752. Leloup-Hatey, J. (1968). Controle corticotrope de la corticosteroidogenese interrenalienne chez les vertebres inferieurs (reptiles, teleosteens). Comp. Biochem. Physiol. 26, 997-1013. Licht, P., and Bradshaw, S. D. (1969). A demonstration of corticotropic activity and its distribution in the pars distalis of the reptile. Gen. Comp. Endocrinol. 13, 226-235. Macchi, I. A., and Phillips, J. G. (1966). In virro effect of adrenocorticotropin on corticoid secretion in the turtle, snake, and bullfrog. Gen. Comp. Endocrinol. 6, 170-182.

Moyle, W. R.. Kong, Y. C.. and Ramachandran. J. ( 1973). Steroidogenesis and cyclic adenosine 3’.5’-monophosphate accumulation in rat adrenal cells. J. Biol. Chem. 248, 2409-2417. Nothstine. S. A., Davis, J. 0.. and DeRoos. R. M. (1971). Kidney extracts and ACTH on adrenal steroid secretion in a turtle and a crocodilian. Amer. J. Physiol. 221, 726-732. Phillips, J. G., Jones, C., and Bellamy, D. (1962). Biosynthesis of adrenocortical hormones by adrenal glands of lizards and snakes. J. Endocrinol. 25, 233-237. Rillema, J. A., Kostyo, J. L., and Gimpel, L. P. (1973). Inhibition of metabolic effects of growth hormone by various inhibitors of cyclic nucleotide phosphodiesterase. Biochim. Biophys. Acta 297, 527-539. Rubin, R. P., Jaanus, S. D., Carchman, R. A., and Puig, M. (1973). Reversible inhibition of ACTHinduced corticosteroid release by cycloheximide: Evidence for a unidentified cellular messenger. Endocrinology 93, 575-580. Rubin, R. P., Sheid, B., McCauley, R., and Laychock, S. (1974). ACTH-induced protein release from the perfused cat adrenal gland: Evidence for exocytosis? Endocrinology 95, 370-378. Sharma, R. K. (1974). Metabolic regulation of steroidogenesis in isolated adrenal cells of rat. Effect of actinomycin D on c-GMP-induced steroidogenesis. Biochem. Biophys. Res. Commun. 59,992-1004. Singley, J. A., and Chavin, W. (1975). The adrenocortical-hypophyseal response to saline stress in the goldfish, Carassius aurafus L. Comp. Biochem. Physiol. SlA, 749-756. Warner, W., and Rubin, R. P. (1975). Evidence for a possible prostaglandin link in ACTH-induced steroidogenesis. Prostaglandins 9, 83-95. Whitley, T. H., Stowe, N. W., Ong, S. H., Ney, R. L., and Steiner, A. L. (1975). Control and localization of rat adrenal cyclic guanosine 3’,5’-monophosphate. J. Clin. Invest. 56, 146-154.