Metabolic Regulation of Steroidogenesis in Adrenocortical Carcinoma Cells of Rat
Effect of Adrenocorticotropin and Adenosine Cyclic 3’: 5’-Monophosphate on Corticosteroidogenesis
Rameshwar K. SHARMA
Laboratories of Endocrinology and Metabolism, Veterans Administration Hospital, and Department of Biochemistry, University of Tennessee, Medical Units, Memphis, Tennessee
(Received July 19/October 26, 1972)
The effect of adrenocorticotropin and cyclic AMP on the transformation of pregnenolone to corticosterone has been studied in adrenocortical carcinoma and isolated normal adrenal cells of rat. This has been carried out using both chemical and double labeling techniques. Adrenocortico- tropin or cyclic AMP did not shown any effect on the corticosteroidogenesis produced from preg- nenolone in the isolated adrenal cells whereas the synthesis of deoxycorticosterone and cortico- sterone was inhibited by adrenocorticotropin in adrenal tumor cells. A similar inhibitory effect of adrenocorticotropin was observed when progesterone was used as a precursor for corticosteroido- genesis in tumor cells but the incorporation of deoxycorticosterone into corticosterone was not inhibited by this substance. These studies, therefore, suggest that the site of inhibition of adreno- corticotropin on corticosterone synthesis in the tumor lies between progesterone and deoxy- corticosterone.
Cyclic AMP also inhibited the synthesis of corticosterone from pregnenolone but no inhibition of deoxycorticosterone synthesis was observed. Similar results were obtained with N6,2’-O-di- butyryl adenosine 3’:5’-monophosphate. It is suggested that the mechanism of inhibition of corticosteroidogenesis by adrenocorticotropin in the tumor may be different from that of cyclic AMP.
An isolated adrenocortical carcinoma 494 cell preparation was recently reported from these labora- tories which is useful in studying abnormalities in the hormonal regulation of steroidogenesis [1]. Using this preparation [1], it was demonstrated that corticosteroidogenesis, was not stimulated either by adrenocorticotropin or by adenosine 3’:5’-mono- phosphate (cyclic AMP) [1], whereas the isolated normal adrenal cell was found to be stimulated more than 100-fold [2] in response to this nucleotide. It was also reported from these laboratories that the tumor tissue has considerably lower cyclic AMP phosphodiesterase activity than normal adrenal tissue [3]. The lack of corticosteroidogenesis by these cells, therefore, cannot be explained on the basis of
Trivial Names and Abbreviations. Cyclic AMP, adenosine cyclic 3’:5’-monophosphate; cyclic Bu2AMP, N6, 2’-O-di- butyryl adenosine 3’:5’-monophosphate; pregnenolone, 5-pregnen-38-ol-20-one; prog sterone, 4-pregnen-3,20-dione; deoxycorticosterone, 21-hydroxy-4-pregnen-3,20-dione; de- oxycorticosterone acetate, deoxycorticosterone 21-acetate; corticosterone, 118, 21-dihydroxy-4-pregnen-3,20-dione.
their phosphodiesterase activity. Previous studies from these [1] and other laboratories [4,5] have shown that the route pregnenolone (I) → progesterone (II) → deoxycorticosterone (III) → corticosterone (IV) is at least partly intact in the adrenal tumor cells. During the course of the above investigation [1], preliminary observations had been made that adreno- corticotropin may inhibit the corticosteroidogenesis produced from pregnenolone. These observations prompted the study of the effect of adrenocortico- tropin and its messenger, cyclic AMP, on the bio- synthetic steps involved in the conversion of preg- nenolone to corticosterone.
MATERIALS AND METHODS
Tissue
Rats bearing adrenocortical carcinoma 494, a spontaneously occurring tumor discovered by Snell and Stewart [6], were used for these studies. Isolated
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adrenocortical carcinoma cells were prepared by the already published method [1].
The isolated adrenal cell preparation was made from the adrenals obtained from male Holtzman rats weighing 300-400 g, maintained on an ordinary diet, by the trypsin digestion method [2,7].
Chemicals
Adrenocorticotropin, a United States Pharmaco- peia standard, was purchased from United States Pharmacopeia. Each vial contained 1.5 IU adreno- corticotropin and was diluted to give the desired concentration in 0.2 ml of vehicle. Cyclic AMP and Nº,2’-O-dibutyryladenosine 3’:5’monophosphate (cyclic Bu2AMP) were purchased from Sigma Chemi- cal Company (St. Louis, Mo.) and Boehringer-Mann- heim (New York). All the other reagents were reagent grade and were obtained commercially.
Steroids
[4.14C]Pregnenolone (specific activity 50 mCi/ mmol), [7x-3H]pregnenolone (specific activity 5 Ci/ mmol), [4-14C]progesterone (specific activity 50 mCi/ mmol) and [7x-3H]progesterone (specific activity 5 Ci/mmol) were purchased from Amersham/Searle Corporation (Arlington Heights, Illinois). [4-14C]De- oxycorticosterone (specific activity 20-30 mCi/ mmol) and [1,2-3H2]deoxycorticosterone (specific activity 30-50 Ci/mmol) were purchased from New England Nuclear (Boston, Mass.).
Chromatography
Precoated silica-gel plates (Silica Gel F254, Brink- mann, New York) were used for thin-layer chromatog- raphy in the indicated solvent systems. Thin-layer plates were used for the final purification of deoxy- corticosterone and corticosterone. Chromatographi- cally homogeneous products were further checked for purity and identity by cocrystallization to constant specific activity and constant 3H/14C ratio.
Counting
Counting was carried out in the Nuclear-Chicago Mark II automatic liquid scintillation counter. The samples were dissolved in 15 ml of a scintillation solution of toluene containing 4 g of PPO and 100 mg of POPOP per 1. Counting efficiency for 3H and 14C under these conditions was 40% ± 1% and 68% ± 1%, respectively.
Method of Incubation
The method of incubation for adrenocorticotro- pin, cyclic nucleotides and nonradioactive steroids was that already described [1, 2]. In general, for each isolated adrenal cell preparation, adrenals from 16 rats were used, and the cells from each adrenal (approxi- mately 400000 cells) were resuspended in 0.8 ml of Krebs-Ringer bicarbonate buffer pH 7.4, containing 4% albumin and 0.2% glucose. Isolated adreno- cortical carcinoma cells were prepared from 1.30 to 1.40 g tissue and the cells (2 to 3 million cells) re- suspended in Krebs-Ringer bicarbonate buffer as mentioned above. Corticosterone was measured fluorometrically [8]. The experiments with radio- active precursors were conducted as follows. Incuba- tion was carried out in 3 teflon flasks. Each flask contained 20 ml isolated adrenal tumor cell suspen- sion [1] prepared from the 1.5 g adrenal tumor tissue or the same amount of cell suspension obtained from 32 adrenals of the rat [2]. In addition, to the appro- priate cell suspension, the first flask contained a mixture of a pCi of 14C-labeled steroid (pregnenolone, progesterone or deoxycorticosterone) and 20 uCi of corresponding 3H-labeled steroid (3H/14C ratio 20.00); the second flask contained 1 pCi of 14C-labeled steroid and the third flask 20 u.Ci of 3H-labeled steroid + adrenocorticotropin (200 µU/ml) or cyclic AMP (10 mM), or cyclic Bu2AMP (1 mM). The incubation was continued for 150 min and the reaction was stopped by the addition of 5 ml distilled water and 25 ml methylene chloride into each flask. The con- tents of the second flask which contained 14C-labeled steroid were mixed with the contents of the third flask which contained 3H-labeled steroid precursor + adrenocorticotropin, cyclic AMP or cyclic Bu2- AMP. The reaction mixtures of the two flasks were processed identically.
The methylene chloride extract from each flask was washed with 2×10 ml 5% sodium hydroxide and then with 2x 10 ml distilled water. The methy- lene chloride was dried over sulfate and filtered. The solvent was evaporated to dryness under nitro- gen and deoxycorticosterone and corticosterone iso- lated as described below.
Isolation of Deoxycorticosterone
The isolation of deoxycorticosterone was carried out on the above residue by thin-layer chromatog-
raphy (benzene: methanol, 18:1.5, by vol., develop- ed twice). A sample of authentic non-radioactive deoxycorticosterone was co-chromatographed, the deoxycorticosterone zone was detected under ultra- violet light and the area of radioactivity correspond- ing to that zone was extracted. The extract was repurified by thin-layer chromatography (benzene- ethyl acetate, 1:1, v/v, developed twice). 10 mg non- radioactive deoxycorticosterone was added and the product converted to the 21-acetate by treatment with acetic anhydride in the presence of the catalytic amount of p-toluenesulfonic acid [9]. The crude acetate was purified by thin-layer chromatography (as above) and crystallized from acetone-n-hexane to constant specific activity and 3H/14C ratio. The product was identified as 21-acetate of deoxycortico- sterone by comparison and a sample recrystallized.
On the basis of the 14C present in deoxycortico- sterone crystallized to constant specific activity and constant ratio the incorporation of pregnenolone into deoxycorticosterone in adrenal tumor cells was 8.4% (average of 4 experiments).
Isolation of Corticosterone
The isolation of corticosterone was carried out on the methylene chloride extract by thin-layer chromatography as for deoxycorticosterone. The purified corticosterone was diluted with non-radio- active corticosterone (5 mg) and crystallized several times from acetone- ligroin until the specific activity and 3H/14C ratio was constant.
On the basis of the 14C present in the corticoste- rone crystallized to constant specific activity and constant ratio, the incorporation of pregnenolone into corticosterone in tumor cells was 5.2% (average of 4 experiments).
RESULTS
Effect of Adrenocorticotropin, Cyclic AMP and Cyclic Bu,AMP on Corticosteroidogenesis Produced from Pregnenolone in Isolated Adrenal Cells
In order that the effect of adrenocorticotropin, cyclic AMP and cyclic Bu2AMP on the corticosterone produced from pregnenolone might be examined, the isolated adrenal cells were incubated with pregneno- lone in the presence and absence of these nucleotides. The results in Table 1 show that all these three sub- stances stimulated corticosteroidogenesis more than 30-fold, in confirmation of previous findings [2]. Pregnenolone was also effectively converted to corti- costerone; the incubations of pregnenolone with these three substances produced an approximately additive effect to that of pregnenolone. This is in keeping with the previous data showing that adrenocortico- tropin stimulates corticosterone synthesis from
| Additions | Corticosterone |
|---|---|
| ug/2 h | |
| Control | 0.053 |
| ACTH (100 p.U/ml) | 1.844 |
| Cyclic AMP (10 mM) | 1.703 |
| Cyclic Bu2AMP (1 mM) | 1.863 |
| Pregnenolone (63.2 p.M) | 2.162 |
| Cycloheximide (10 p.M) | 0.002 |
| Actinomycin (10 p.M) | 0.000 |
| Pregnenolone + cycloheximide | 1.910 |
| Pregnenolone + actinomycin | 2.010 |
| Pregnenolone + ACTH | 3.571 |
| Pregnenolone + ACTH + cycloheximide | 2.252 |
| Pregnenolone + ACTH + actinomycin | 3.419 |
| Pregennolone + cyclicAMP | 3.602 |
| Pregnenolone + cyclicAMP + cycloheximide | 2.298 |
| Pregnenolone + cyclicAMP + actinomycin | 3.285 |
| Pregnenolone + cyclic Bu2AMP | 3.722 |
| Pregnenolone + cyclicBu2AMP + cyclo- heximide | 1.889 |
| Pregnenolone + cyclic Bu2AMP + actino- mycin | 3.268 |
endogenous cholesterol present in the adrenal [2,7]. Cycloheximide, but not actinomycin, however, in- hibited the corticosteroidgenesis stimulated by either of the three substances. These results are in agree- ment with the concept that the rate-limiting step affected by adrenocorticotropin in the stimulation of corticosteroidogenesis lies before the formation of pregnenolone. These data further support the concept that the corticosteroidogenesis induced by these three substances is mediated through the regulation of adrenal protein synthesis.
Effect of Adrenocorticotropin on the Transformation of Pregnenolone to Deoxycorticosterone and Corticosterone in Isolated Adrenal Cells
Studies carried out in the normal adrenal cells where one group of cells was incubated with [7x-3H]- pregnenolone + adrenocorticotropin and the other group with [4-14C]pregnenolone and the incubation solution mixed after terminating the incubation, showed (Table 2) no significant change in the 3H/14C ratios of the stimulated cells from that noted in the non-stimulated cells. This confirms the results mentioned in the previous section that adrenocortico- tropin has little effect on the conversion of pregneno- lone to corticosterone.
Table 2. Effect of adrenocorticotropin on the incorporation of [&H]pregnenolone into deoxycorticosterone and corticosterone in normal isolated adrenal cells
The 3H/14C ratios of the products (and their derivatives) obtained after the incubation of [4-14C, 7x-3H]pregnenolone with isolated adrenal cells. Incubation was carried out in 3 flasks containing 20 ml isolated adrenal cell preparation as mentioned in Materials and Methods. Flask 1 contained a mixture of [7x-8H]pregnenolone (20 pCi) + [4-14C]pregnen- olone (1 µCi) (3H/14C ratio 20.00); flask 2 contained [7xx-3H]- pregnenolone (20 pCi) + adrenocorticotropin (200 u.U/ml) and the flask 3 contained [4-14C]pregnenolone (1 (Ci). The incubation was for 2.5 h and the reaction was stopped by the addition of 25 ml methylene chloride to each flask. The contents of the second and third flask were mixed and deoxycorticosterone and corticosterone isolated as described in Materials and Methods
| Compound | Crystallization | *H/14C ratio of compound from | |
|---|---|---|---|
| Flask 1 | Flask 2 + Flask 3 | ||
| Deoxycortico- | Crude product | 19.06 | 18.20 |
| sterone acetate | 1st | 20.66 | 19.94 |
| Corticosterone | 1st | 19.40 | 12.66 |
| 2nd | 20.25 | 14.61 | |
| 3rd | 19.43 | 17.01 | |
| 4th | 19.40 | 17.55 | |
Effect of Adrenocorticotropin, Cyclic AMP and Cyclic BuzAMP on Corticosteroidogenesis Produced by Pregnenolone in Isolated Adrenocortical Carcinoma Cells
In order that the effect of adrenocorticotropin, cyclic AMP and cyclic Bu2AMP on corticosterone synthesis from pregnenolone could be compared in the normal adrenal with the adrenal tumor, the adrenocortical carcinoma cells were incubated with these agents in the presence and absence of preg- nenolone.
The results depicted in Table 3 demonstrate that these three substances do not stimulate any cortico- steroidogenesis in the tumor. The corticosteroido- genesis produced by pregnenolone was not additive with either adrenocorticotropin or cyclic AMP and cyclic Bu2AMP. Cycloheximide and actinomycin did not inhibit any corticosteroidogenesis. This supports our previous finding [1] that these three substances lack any apparent stimulatory effect on cortico- sterone synthesis in the adrenal tumor cells.
Effect of Adrenocorticotropin on the Transformation of Pregnenolone to Deoxycorticosterone and Corticosterone in Adrenocortical Carcinoma Cells
To compare the effect of adrenocorticotropin on the conversion of labeled pregnenolone to cortico- sterone in the normal adrenal with the adrenal tumor, this substance was incubated with [7xx-3H]pregneno- lone in the adrenocortical carcinoma cells. The results in Table 4 show that adrenocorticotropin, in contrast
Table 3. Corticosterone synthesis stimulated by adrenocortico- tropin and cyclic AMP with pregnenolone as precursor in adrenocortical carcinoma cells
Incubation system: adrenal tumor cell suspension 0.8 ml; reagents dissolved in 0.2 ml vehicle. Total volume of incuba- tion 1 ml. Results are the average of 4 observations. Control value has been subtracted from the experimental results. ACTH = adrenocorticotropin
| Additions | Corticosterone |
|---|---|
| ug/2 h | |
| Control | 0.054 |
| ACTH (100 p.U/ml) | 0.009 |
| Cyclic AMP (10 mM) | 0.000 |
| Cyclic Bu2AMP (1 mM) | 0.030 |
| Pregnenolone (63.2 p.M) | 0.178 |
| Cycloheximide (10 p.M) | 0.000 |
| Actinomycin (10 p.M) | 0.000 |
| Pregnenolone + ACTH | 0.168 |
| Pregnenolone + ACTH + cycloheximide | 0.169 |
| Pregnenolone + ACTH + actinomycin | 0.177 |
| Pregnenolone + cyclicAMP | 0.165 |
| Pregnenolone + cyclicAMP + cycloheximide | 0.191 |
| Pregnenolone + cyclicAMP + actinomycin | 0.190 |
| Pregnenolone + cyclic Bu2AMP + cyclo- heximide | 0.154 |
| Pregnenolone + cyclic Bu2 AMP + Actino- mycin | 0.178 |
| Compound | Crystallization | $H/14C ratio of compound in | |
|---|---|---|---|
| Flask 1 | Flask 2+ Flask 3 | ||
| Deoxycortico- | Crude product | 15.60 | 6.20 |
| sterone acetate | 1st | 16.80 | 6.39 |
| Corticosterone | 1st | 18.88 | 5.94 |
| 2nd | 17.93 | 5.72 | |
| 3rd | 17.72 | 5.74 | |
to the normal adrenal cells, strongly inhibits the incorporation of pregnenolone into desoxycortico- sterone and corticosterone.
Effect of Adrenocorticotropin on the Transformation of Progesterone to Deoxycorticosterone and Corticosterone in Adrenocortical Carcinoma Cells
Similarly, when the studies were carried out in adrenal tumor cells, adrenocorticotropin strongly inhibited progesterone incorporation into deoxy- corticosterone and corticosterone. The data shown in Table 5 suggest that the inhibitory effect of adrenocorticotropin lies between progesterone and deoxycorticosterone.
| Compound | Crystallization | 'H/14C ratio of compound in | |
|---|---|---|---|
| Flask 1 | Flask 2 + Flask 3 | ||
| Deoxycortico- | Crude product | 20.45 | 12.34 |
| sterone | 1st | 17.05 | 10.05 |
| acetate | 2nd | 18.18 | 10.55 |
| Corticosterone | 1st | 18.47 | 7.87 |
| 2nd | 19.78 | 8.25 | |
| 3rd | 20.12 | 8.27 | |
| Compound | Crystallization | 3H/14C ratio of compound in | |
|---|---|---|---|
| Flask 1 | Flask 2 + Flask 3 | ||
| Corticosterone | 1st | 6.77 | 13.73 |
| 2nd | 8.64 | 13.40 | |
| 3rd | 8.50 | 13.36 | |
| 4th | 8.40 | 13.29 | |
Effect of Adrenocorticotropin on the Transformation of Deoxycorticosterone to Corticosterone in Adrenocortical Carcinoma Cells
The results in Table 6 show that adrenocortico- tropin did not inhibit the incorporation of deoxy- corticosterone into corticosterone thus supporting the observation in the earlier section that the in- hibition due to adrenocorticotropin on corticosteroido- genesis produced by pregnenolone is between the step of progesterone to deoxycorticosterone.
Effect of Cyclic AMP on the Transformation of Pregnenolone to Deoxycorticosterone and Corticosterone in Adrenocortical Carcinoma Cells
In order that adrenocorticotropin might be com- pared with cyclic AMP in its effect upon the conver- sion of pregnenolone into deoxycorticosterone and corticosterone, adrenal tumor cells were incubated with labeled pregnenolone. The results in Table 7
| Compound | Crystallization | 'H/14C ratio of compound in | |
|---|---|---|---|
| Flask 1 | Flask 2 + Flask 3 | ||
| Deoxycortico- | Crude product | 17.79 | 15.67 |
| sterone | 1st | 17.41 | 15.91 |
| acetate | 2nd | 17.49 | 16.11 |
| Corticosterone | 1st | 19.36 | 14.86 |
| 2nd | 19.78 | 14.61 | |
| 3rd | 19.17 | 14.55 | |
Table 8. Effect of cyclic Bu2AMP on the incorporation of [3H]pregnenolone into deoxycorticosterone and corticosterone in adrenocortical carcinoma cells
The $H/14C ratios of the products (and their derivatives) obtained after the incubation of [4-14C, 7xx-3H]pregnenolone in adrenocortical carcinoma cells. Conditions of the experi- ment were similar to the experiment in Table 2. Flask 1 contained [4-14C, 7x-3H]pregnenolone; Flask 2 contained [7x-3H]pregnenolone and cyclic Bu2AMP; Flask 3 contained [4-14C]pregnenolone
| Compound | Crystallization | 'H/14C ratio of compound in | |
|---|---|---|---|
| Flask 1 | Flask 2 + Flask 3 | ||
| Deoxycortico- | Crude product | 15.49 | 14.52 |
| sterone | 1st | 15.61 | 14.61 |
| acetate | 2nd | 15.64 | 14.96 |
| Corticosterone | 1st | 17.77 | 12.36 |
| 2nd | 18.29 | 11.88 | |
| 3rd | 17.59 | 11.71 | |
show that although cyclic AMP induced a 24% inhibition of the incorporation of pregnenolone into corticosterone, no significant inhibition was obtained in the conversion of pregnenolone to deoxycortico- sterone.
Effect of Cyclic Bu2AMP on the Transformation of Pregnenolone to Deoxycorticosterone and Cortico- sterone in Adrenocortical Carcinoma Cells
Since previous studies [2] have shown that cyclic Bu2AMP is a much better stimulator of cortico- steroidogenesis than normal cyclic AMP, studies were conducted in the presence of this nucleotide on the conversion of [7xx-3H]pregnenolone into de-
oxycorticosterone and corticosterone. As shown in Table 8, the results were almost identical to those obtained with cyclic AMP. Although cyclic Bu2- AMP inhibited the incorporation of pregnenolone into corticosterone, no appreciable inhibition was obtained in the conversion of pregnenolone into deoxycorticosterone.
DISCUSSION
It has been well documented that the conversion of cholesterol to pregnenolone is the rate-limiting step in steroidogenesis which is stimulated by adreno- corticotropin [10,11,12]. The effect of this hormone on the transformation of pregnenolone to cortico- sterone is, however, a subject of controversy [12, 13,14]. The results depicted here, together with the radioactive experiments, confirm the observation that adrenocorticotropin has no effect on the conver- sion of pregnenolone to either deoxycorticosterone or corticosterone in a normal isolated adrenal cell.
The results obtained with the tumor, however, were entirely different. Adrenocorticotropin inhibited the incorporation of pregnenolone in both deoxy- corticosterone and corticosterone. Since the synthesis of deoxycorticosterone and corticosterone from pro- gesterone was also inhibited, it is suggested that the inhibitory effect of adrenocorticotropin lies before the formation of deoxycorticosterone. That this was indeed the case is supported by the fact that the incorporation of deoxycorticosterone into cortico- sterone was not inhibited by adrenocorticotropin.
Since the hormonal effect of adrenocorticotropin in the adrenal is mediated via the formation of cyclic AMP [15,16,17], the effect of this nucleotide on the incorporation of pregnenolone was investigated. Interestingly, cyclic AMP produced much less inhibi- tion of the transformation of pregnenolone to cortico- sterone. Of greater interest, however, was the finding that cyclic AMP in contrast to adrenocorticotropin had little effect of the transformation of pregnenolone to deoxycorticosterone. To confirm this observation and to eliminate the discrepancy of the two effects due to lesser permeability and solubility of cyclic AMP in the tissues, cyclic Bu2AMP was used to study the incorporation of pregnenolone to cortico- sterone. Since the experiments with the latter nucleo- tide gave virtually the same results as those obtained with cyclic AMP it is suggested that the mode of inhibition of adrenocorticotropin and cyclic AMP on the regulation of corticosteroidogenesis from pregnenolone to corticosterone is different in the rat adrenal tumor. The idea that other nucleotide(s) are involved as an intermediate(s) in the mechanism of adrenocorticotropin action, although an attractive possibility, remains only speculative at this stage.
Of interest may also be the fact that the experi- ments with the radioactive precursors in the tumor
cells indicate that adrenocorticotropin markedly inhibits the synthesis of deoxycorticosterone and corticosterone (Tables 4 and 5). The nonradioactive experiments, using pregnenolone in “substrate” concentrations in which corticosterone synthesis was measured fluorometrically, however, show no inhibi- tion of corticosteroidogenesis by adrenocorticotropin. This lack of inhibition when corticosterone is mea- sured fluorometrically, together with the radioactive precursor experiments, suggest that steroid(s) other than corticosterone may be synthesized in the tumor which are indistinguishable from it.
Of interest may also be our results obtained with cycloheximide in normal adrenal cells. Since, cyclo- heximide inhibited the steroidogenesis stimulated only by adrenocorticotropin or cyclic AMP and cyclic Bu2AMP and not that produced by pregneno- lone, it supports the earlier concept formulated by other investigators [15,16,17] and supported from our laboratories [2] that the mechanism of adreno- corticotropin regulation over steroidogenesis is via the formation of cyclic AMP and this nucleotide in turn exerts its activity by the translation of long- lived RNA [2,18, 19, 20].
Finally, it may be mentioned that the adreno- cortical carcinoma cell preparation [1] and normal adrenal cell preparation [2,7] offer potential tools to study the control mechanisms in intact cell. The fact that pregnenolone can be transformed to cortico- sterone shows that the transport mechanisms for the incorporation of the precursors and their sub- sequent metabolism exist in the whole cell. To date such studies have only been possible either in the adrenal slices or in the cell-free system [13, 14, 15, 18].
The author thanks Dr James Brush for stimulating discussions during the course of this investigation. Excellent technical assistance of Mrs. Lynda Sutliff is also apprecia- ted. This work was supported by Part-I and Part-II designated research funds of the United States Veteran Administration.
REFERENCES
1. Sharma, R. K. & Hashimoto, K. E. (1972) Cancer Res. 32, 666.
2. Kitabchi, A. E. & Sharma, R. K. (1971) Endocrinology, 88, 1109.
3. Sharma, R. K. (1972) Cancer Res. 32, 1734.
4. Johnson, D. F., Snell, K. C., Francois, D. & Heftmann, E. (1961) Acta Endocrinol. 37, 329.
5. Ney, R. L., Hochella, N. J., Grahme-Smith, D. G., Dex- ter, R. N. & Butcher, R. W. (1969) J. Clin. Invest. 48, 1733.
6. Snell, K. C. & Stewart, H. L. (1959) J. Natl. Cancer. Inst. 22, 1119.
7. Sayers, G., Swallow, R. L. & Giordano, N. D. (1971) Endocrinology, 88, 1063.
8. Glick, D., Redlich, D. V. & Levine, S. (1964) Endocrino- logy, 74, 653.
9. Sharma, R. K., Doorenbos, N. J. & Bhacca, N. S. (1971) J. Pharm. Sci. 60, 1677.
10. Stone, D. & Hechter, O. (1954) Arch. Biochem. Biophys. 51, 457.
11. Billair, R. B. & Eik-Nes, K. B. (1965) Biochim. Biophys. Acta, 104, 503.
12. Karaboyas, G. C. & Koritz, S. B. (1965) Biochemistry, 4, 462.
13. Karaboyas, G. C. & Koritz, S. B. (1965) Biochim. Bio- phys. Acta, 100, 600.
14. Koritz, S. B. & Kumar, A. M. (1970) J. Biol. Chem. 245, 152.
15. Haynes, R. C., Jr., Koritz, S. B. & Pèron, F. G. (1959) J. Biol. Chem. 234, 1421.
16. Lefkowitz, R. J., Roth, J., Pricer, W. & Pastan, I. (1970) Proc. Natl. Acad. Sci. U. S. A. 65, 745.
17. Schimmer, B. P., Ueda, K. & Sato, G. H. (1968) Bio- chem. Biophys. Res. Commun. 32, 806.
18. Davis, W. W. & Garren, L. D. (1968) J. Biol. Chem. 243, 5153.
19. Garren, L. D. (1968) Vitam. Horm. 26, 119.
20. Garren, L. D., Gill, G. N., Masui, H. & Walton, G. M. (1971) Recent Prog. Horm. Res. 27, 433.
R. K. Sharma Department of Biochemistry University of Tennessee Medical Units Memphis, Tennessee 38104, U.S.A.