Stimulation of Guanosine 3’,5’-Monophosphate- Phosphodiesterase Activity by Adrenocorticotropic Hormone-Activated Increase of Guanosine 3’,5’- Monophosphate in Isolated Adrenocortical Carcinoma Cells*
JEAN-PIERRE PERCHELLET AND RAMESHWAR K. SHARMA
Department of Biochemistry and Memphis Regional Cancer Center, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163
ABSTRACT. The decline of ACTH-stimulated cGMP to the basal level in isolated adrenocortical carcinoma cells was in- hibited by cyclic phosphodiesterase inhibitors. Time-course ex- periments showed that the ACTH-induced level of cGMP pre- ceded the activation of phosphodiesterase. Cells incubated with increasing concentrations of exogenous cGMP responded with a corresponding increase in the cGMP-phosphodiesterase activity. cAMP was 100-fold less effective than cGMP in the activation of cGMP-phosphodiesterase, indicating the nucleotide specific-
ity of enzyme activation. The activation of cGMP-phosphodies- terase by ACTH-induced cGMP or exogenous cGMP was rapid. In contrast to these observations, there was no activation of cAMP-phosphodiesterase by exogenous cAMP or cGMP. These results indicate that one mechanism by which isolated adreno- cortical carcinoma cells regulate the ACTH-induced increase in cGMP is the cyclic nucleotide control of the activity of its phosphodiesterase, thereby regulating cGMP degradation. (En- docrinology 105: 879, 1979)
P REVIOUS studies from this laboratory have dem- onstrated the important mediatory role of cGMP in ACTH-induced steroidogenesis in isolated rat adrenal cells (1-4; for a review see Ref. 5). Recently, evidence has been presented for the presence and characterization of cGMP-dependent protein kinase in bovine adrenal cortex (6). Thus, these studies have begun to reveal the molec- · ular mechanism by which cGMP regulates adrenal ste- roidogenesis.
One salient feature of the increase of cGMP in response to ACTH is that at submaximal steroidogenic concentra- tions of ACTH, there is a peak level of cGMP, but the level of the nucleotide sharply declines at near-maximal or maximal steroidogenic concentrations of the hormone (1-4). An influence of the cAMP system on this process has been ruled out, since the same phenomenon is ob- served in isolated adrenocortical carcinoma cells (7). In the latter cell system, the levels of cAMP were not increased by submaximal or near-maximal steroidogenic concentrations of ACTH (<100 LU). Furthermore, the
tumor cells did not have a detectable amount of cAMP- dependent protein kinase (8) or cAMP-phosphodiester- ase activity (9). Thus, one can use the adrenocortical carcinoma cell as a model system in elucidating the mechanism of regulation of cGMP levels in response to ACTH without interference by the cAMP system.
The present study was designed to investigate the possibility that one mode of controlling cGMP levels in these cells is by cGMP activation of its phosphodiester- ase.
Materials and Methods
Materials
Rat adrenocortical carcinoma 494 (10), maintained in our laboratory (11), was used as a source of the isolated adrenocor- tical carcinoma cells. The method of incubation of isolated adrenocortical carcinoma cells with ACTH, cGMP, and cAMP was as described earlier (11, 12). Adrenocortical carcinoma cells were isolated by trypsin digestion (11). In general, adrenocor- tical carcinoma cells corresponding to 30 mg tumor tissue (approximately 2 × 106 cells) were resuspended in 0.8 ml Krebs- Ringer-bicarbonate buffer, pH 7.4, containing 4% albumin and 0.2% glucose.
The incubation experiments in which cGMP and cGMP- phosphodiesterase activity were measured were conducted in quadruplicate; two of the samples were used for the determi- nation of cGMP and two were used for the phosphodiesterase
Received March 1, 1979.
Address requests for reprints to: Rameshwar K. Sharma, Ph.D., Department of Biochemistry, University of Tennessee Center for the Health Sciences, 894 Union, Memphis, Tennessee 38163.
* This work was supported by Grant PCM 7800860 from the NSF and Grant CA-16091 from the NCI.
activity. Other experiments were conducted in duplicate. Every experiment was repeated at least three times. Extraction (11) and measurement of cGMP levels were performed as described previously (3, 13).
cGMP-phosphodiesterase assay
After incubation of isolated adrenocortical carcinoma cells with appropriate agents, the reaction was stopped by the ad- dition of 2 ml ice-cold 20 mM Tris-HCl buffer, pH 7.5, and the tubes were centrifuged at 100 x g. The cell pellet was resus- pended in 0.5 ml of the same buffer, sonicated for 0.5 min at 0 C, and centrifuged at 2000 x g. The supernatant was passed through a Sephadex G-25 column (5 x 0.5 cm), and phospho- diesterase activity was eluted in the void volume (0.5 ml) of the column. cGMP-phosphodiesterase activity was measured by a previously described method (14) for cAMP-phosphodiesterase using [3H]cGMP as the substrate.
The assay mixture for cGMP-phosphodiesterase activity consisted of 40 mm Tris-HCI (pH 7.5), 1.8 mM MgSO4, 400 µM [3H]cGMP (approximately 100,000 dpm), and an appropriate aliquot of the enzyme in a total volume of 0.5 ml. The reaction was started by the addition of [3H]cGMP. Incubation was carried out at 30 C for 60 min. After 50 min, 0.05 mg Crotalus atrox snake venom in 0.05 ml 10 mm Tris-HCI (pH 7.5) was added to convert the 5’-GMP to guanosine, and the reaction mixture was incubated for an additional 10 min. A control tube without phosphodiesterase was included to correct for trace hydrolysis of cGMP by snake venom. At the end of the incu- bation, 0.05 ml ice-cold 10 mm guanosine were added. Guanosine formed during the reaction was separated from the unreacted cGMP by passing the reaction mixture through a QAE-Sepha- dex (Cl-) column and eluting the column (5 x 0.5 cm) with 4 ml distilled water. Radioactivity in the aqueous solutions was determined by counting each sample in 15 ml Aquasol in a LS- spectrometer model Isocap 300, (Searle Analytical Co., Arling- ton, Heights, IL).
Results
Figure 1 depicts the endogenous levels of cGMP formed in response to the varying concentrations of ACTH at 10- and 60-min intervals. The results show that after 10 min of incubation, 5 uU ACTH caused a peak net synthesis of cGMP, while at higher concentrations of the hormone, there was a significant decline in the cGMP level. At 60 min, there was no increase in the cGMP level at any of the concentrations of the hormone. These results confirm previous observations (1-4, 7) that high concentrations of ACTH cause a transient peak of cGMP; they also demonstrate that this process is time depend- ent. This observation is substantiated by the results depicted in Fig. 2, showing a peak level of cGMP within 2 min in the presence of 5 uU ACTH but a decline to the basal level after 60 min. However, when the cells were incubated with 5 LU ACTH in the presence of phospho- diesterase inhibitors, the level of cGMP remained ele-
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vated throughout the 60-min study interval (Fig. 2). This indicates that the degradation of cGMP by cGMP-phos- phodiesterase was suppressed by the enzyme inhibitors. That this phenomenon could be due to the indirect influence of cAMP-phosphodiesterase was ruled out, since no activation of cAMP-phosphodiesterase by ex- ogenous cAMP was observed (data not shown).
An important aspect of the time course of the forma- tion of cGMP in response to 5 uU ACTH in relation to activation of the phosphodiesterase is that an increase in the cGMP level precedes the activation of cyclic phos- phodiesterase (Fig. 3). Within 1 min there was a rise in the cGMP level without any corresponding change in the cGMP phosphodiesterase. Although the profiles of the two curves are the same, the increments and declines of cGMP levels always precede those of the corresponding phosphodiesterase activities. These results indicate that degradation of cGMP is being controlled by a correspond- ing activation of the cyclic nucleotide phosphodiesterase.
To determine the specificity of activation of cGMP- phosphodiesterase by cGMP, isolated adrenocortical car-
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cinoma cells were incubated with varying concentrations of exogenous cGMP or cAMP, and the cGMP-phospho- diesterase activities were measured. The results in Fig. 4 indicate that cGMP is 100-fold more effective in stimu- lating cGMP-phosphodiesterase.
To study the time sequence of the activation of cGMP- phosphodiesterase by exogenously supplied cGMP, adre- nocortical carcinoma cells were incubated with 10 AM cGMP. Figure 5 shows that phosphodiesterase activation was quite rapid, reaching a peak within 1 min and declin- ing thereafter.
Discussion
The premise that cGMP plays a crucial role in the regulation of adrenal steroidogenesis by ACTH is based on the following observations.
Isolated rat fasciculata cells (12, 15, 16), devoid of cAMP-phosphodiesterase activity (17), are stimulated by physiological concentrations of ACTH to form cGMP with corresponding increases in protein kinease activity and corticosterone synthesis (1-4). Excellent temporal
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correlation is observed among cGMP formation, phos- phorylation, and corticosterone synthesis in response to as little as 5 uU ACTH (3). Thus, cGMP appears to be involved in the hormonal response through the cGMP- dependent protein kinase. The presence, characteriza- tion, and specificity of cGMP-dependent protein kinase from bovine adrenal cortex have been described (6). In contrast to the cAMP-dependent protein kinase (18), the bovine adrenal cGMP-dependent protein kinase is not dissociated into regulatory and catalytic subunits (6). Furthermore, the direct stimulatory effect of ACTH and cGMP on the transformation of cholesterol to corticos- terone has been demonstrated (19). Indirect evidence has been provided that the cycloheximide-sensitive transla- tional control of the hormone mediated by cGMP is at the entry of cytoplasmic cholesterol into the mitochon- dria (19). It has been further demonstrated that the cycloheximide-sensitive step is after the activation of protein kinase (2).
These studies (see above) thus provide strong evidence for the involvement of the cGMP component system in the process of adrenal steroidogenesis. One question, however, remained as to why there was only a transient peak of cGMP in response to physiological concentra-
tions of the hormone. The present studies with isolated adrenocortical carcinoma cells show that in accord with the previous results (1-4), a low concentration (5 uU) of ACTH causes a greater increase in the cGMP level than do higher concentrations of the hormone. Any influence of cAMP-phosphodiesterase on this event is minimal, since cAMP or cGMP do not activate cAMP-phospho- diesterase.
The decline of hormone-dependent cGMP levels is a time-dependent phenomenon. It is significant that within 30-60 min, the cGMP levels fall to the basal value. That this phenomenon is due to the activation of cGMP-phos- phodiesterase is indicated by the fact that phosphodies- terase inhibitors completely block the decline of the cGMP level. That the ACTH-stimulated formation of cGMP activates cGMP-phosphodiesterase is indicated by the finding that the increase in cGMP precedes the activation of cGMP-phosphodiesterase. At subsequent time intervals, the rise and fall of cGMP levels are closely followed by the corresponding changes in cGMP-phos- phodiesterase activity. This conclusion is confirmed by the fact that exogenous cGMP activates cGMP-phospho- diesterase in a concentration-dependent manner. The activation of cGMP-phosphodiesterase is specific for cGMP, since 100-fold more cAMP is needed to cause half-maximal activation of the enzyme. The time course of the activation of cGMP-phosphodiesterase by exoge- nous cGMP is comparable to that seen after the increase in cGMP generated in response to ACTH.
The molecular control by which cGMP controls its own catabolismis not clear at this time. To understand this mechanism requires kinetic studies on the purified enzyme. Two attractive possibilities are that this occurs by activation of the cGMP-dependent phosphorylation system or by the allosteric binding of cGMP with the phosphodiesterase. To our knowledge, this is the first report describing this type of hormonal control of regu- lation of cGMP levels in an endocrine cell. There is some evidence in support of this type of control in the cAMP system (20, 21).
Acknowledgment
We are thankful to Dr. W. Y. Cheung for his critical review of this manuscript.
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