Ectopic a-Adrenergic Mediated Accumulation of Guanosine 3’,5’-Monophosphate in Isolated Adrenocortical Carcinoma Cells*
JEAN-PIERRE PERCHELLETt AND RAMESHWAR K. SHARMA Department of Biochemistry and the Memphis Regional Cancer Center, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163
ABSTRACT. In the normal adrenal cell, epinephrine does not activate the rise of either cGMP or cAMP concentrations. In contrast, epinephrine activates the rise of cGMP but not cAMP concentrations in a concentration-dependent manner in isolated adrenocortical carcinoma cells. This effect was duplicated by the «-adrenergic agonist, phenylepherine, but was unaffected by the ß-adrenergic agonist, isoproterenol. The epinephrine-activated increase in cGMP was blocked by the a-adrenergic antagonist, phentolamine, but was not interfered with by the ß-adrenergic
antagonist, propranolol. Neither acetylcholine, a cholinergic ag- onist, nor exogenous calcium caused any increase in cGMP. The rise of cGMP maximally activated by ACTH was additive with that obtained with epinephrine. These results indicate that the adrenal neoplastic cell posseses ectopic a-adrenergic receptors, and that epinephrine causes a rise of the cGMP level through these receptors. The data, furthermore, suggest that the recep- tors for ACTH and epinephrine are distinct. (Endocrinology 106: 1589, 1980)
N ORMAL isolated adrenal cells (1-3) are markedly and specifically stimulated by microunit concen- trations of ACTH to form corticosterone. Adrenocortical carcinoma cells (4), in contrast, do not respond to this hormone with the activation of steroidogenesis (4, 5). An investigation has revealed various biochemical lesions both before and after the events leading to the cleavage of the cholesterol side chain (for a review, see Ref. 6). These metabolic lesions are responsible for the altered but unique ACTH-controlled system in these cells. These defects have been observed at the level of the plasma membrane (7, 8), adenylate cyclase (9, 10), and phospho- diesterase activity (11) and in the steroid biosynthetic transformations of (20S)-20-hydroxycholesterol (12, 13), pregnenolone, progesterone, and deoxycorticosterone to corticosterone (14). Specific studies designed to analyze the tumor adenylate cyclase in the intact cell have indi- cated that 10-50 AU ACTH do not raise the level of cAMP, in contrast to the normal adrenal cell, thus indi- cating a lesion in the tumor adenylate cyclase system (15). Accelerated hydrolysis of cAMP in the tumor can- not account for the lack of cAMP stimulation, since the
adrenocortical carcinoma cells used in these studies have undetectable cAMP phosphodiesterase activity (16). The absence of detectable normal cAMP-dependent protein kinase in the adrenal neoplasm (17) appears to be the reason for the lack of stimulation of corticosterone syn- thesis by exogenous steroidogenic concentrations of CAMP (4, 7). Instead, a novel protein kinase, AUT-P.K. 85, exists in the adrenocortical carcinoma (18). This homogenous protein binds cAMP and autophospho- rylates but is incapable of phosphorylating exogenous substrates, such as histones and casein. The result of this molecular change in the protein kinase is that cAMP is unable to propagate the steroidogenic signal.
In analyses of the cGMP component of the adrenal cell system, we have demonstrated that physiological concen- trations of ACTH (<20 uU) which raise cGMP levels in normal isolated adrenal cells (19-21) also raise cGMP levels in isolated adrenocortical carcinoma cells (22). This demonstrated an active hormonally dependent gua- nylate cyclase system in both normal and tumor cells, since neither possessed detectable cGMP phosphodies- terase activity (16) and both were also devoid of ACTH- degrading activity (23). Thus, the formation of cGMP was indicative of guanylate cyclase activity. In the pres- ent communication, we explored the possibility of an ectopic stimulation of guanylate cyclase by epinephrine in adrenocortical carcinoma cells. We were able to dem- onstrate the presence of such a guanylate cyclase system and to provide evidence that the formation of cGMP by
Received April 18, 1979.
Address requests for reprints to: Rameshwar K. Sharma, Ph.D., Department of Biochemistry, University of Tennessee Center for the Health Sciences, 894 Union Avenue, Memphis, Tennessee 38163.
* This work was supported by grants from the NCI (CA-16091) and the NSF (PCM-7800860).
+ Present address: McArdle Laboratory for Cancer Research, Uni- versity of Wisconsin, Madison, Wisconsin 53706.
epinephrine proceeds via «-adrenergic receptors without the obligatory involvement of calcium and that this was distinct from that of the ACTH-stimulated system. We provide further evidence that this a-adrenergic system does not exist in normal isolated adrenal cells.
Materials and Methods
The isolated adrenocortical carcinoma 494 cells were pre- pared by trypsin digestion (1, 4). The method of cell preparation in which the effect of calcium was evaluated was described earlier (24). Briefly, in this method, the cells were trypsin digested as usual (14) and resuspended in calcium-free Krebs- Ringer bicarbonate buffer (pH 7.4) containing 4% albumin, 0.2% glucose (KRB-GA), lima bean trypsin inhibitor, and 0.25 mM EGTA to ensure the removal of any residual calcium. The cells were incubated for 15 min and centrifuged at 100 x g for 45 min at 4 C. The cell pellet was suspended in 5 ml calcium-free KRB-GA, pelleted by centrifugation, as described above, then resuspended in an appropriate volume of calcium-free KRB- GA. The method of incubation with ACTH and other agents has already been described (4, 14). In general, for each isolated adrenocortical carcinoma cell preparation, 1.3 g tumor tissue were used, and the cells, representing 30-35 mg adrenal tumor (~2 × 106 cells), were resuspended in 0.8 ml Krebs-Ringer bicarbonate buffer, pH 7.4, containing 4% albumin and 0.2% glucose. Every incubation experiment was conducted in dupli- cate and repeated at least three times.
Extractions of cAMP and cGMP were performed as described previously (19, 21). The assay of cAMP was then accomplished by the method of Gilman (25) using the cAMP-binding protein isolated from bovine kidney (26). The cGMP level was deter- mined by the modified method (21) of Shibuya et al. (27), and certain samples were determined by RIA (28), as modified by Harper and Brooker (29).
Results
Effects of epinephrine on the formation of cGMP in isolated adrenal cells
In agreement with earlier findings (19-21), ACTH caused an increase in the level of cGMP, thus demon- strating an active hormonally dependent guanylate cy- clase system in normal fasciculata cells (Table 1). In contrast, epinephrine did not have any effect on the level of cGMP. This conclusion is further supported by the observation that epinephrine, when combined with ACTH at a concentration that causes maximal cGMP formation, did not produce an additive effect. These results demonstrate that the guanylate cyclase of normal isolated adrenal cells is unresponsive to epinephrine.
Effect of epinephrine on cGMP and cAMP accumulation in isolated adrenocortical carcinoma cells
Figure 1 shows that cGMP levels increased in response to epinephrine in a typical sigmoid concentration-re- sponse manner. Concentrations higher than 10 µM epi-
| Additionsª | Conc. | cGMP (% above con- trol) |
|---|---|---|
| Epinephrine | 10 µM | NS |
| ACTH | 5 μυ | 201 ± 44 |
| ACTH + Epinephrine | 184 ± 31 |
” The incubation system used was 2 × 106 normal isolated adrenal cells suspended in 0.8 ml KRB-GA; hormones were dissolved in 0.2 ml diluent. The total volume of incubation was 1 ml; the time of incubation was 10 min. Results are expressed as the average mean value of six separate determinations from three different experiments. The basal value of 3 pmol cGMP has been subtracted from the experimental results.
Cyclic AMP or Cyclic GMP (% above control)
120
80
40
T
0
3
0
5
5
a
5.
O
1
2.5 5 10 25
100
500
Epinephrine (UM)
nephrine caused a significant decline in the peak level of cGMP. In contrast, there was no change in the basal level of cAMP at any concentration of catecholamine used. These results indicate that the isolated adrenocor- tical carcinoma cells have a catecholamine-sensitive gua- nylate cyclase system. The decline in cGMP levels by higher concentrations of epinephrine could be due to the activation of cGMP-specific phosphodiesterase by cGMP. Recently, it was demonstrated that in these cells, an ACTH-activated rise of cGMP causes the concomitant stimulation of cGMP-specific phosphodiesterase (30).
Figure 2 shows the temporal correlation of the forma-
120
Cyclic AMP or Cyclic GMP (% above control)
80
40
O
5
ㅎ
O
1
2
5
15
60
Time (minutes)
tion of cGMP and cAMP in response to epinephrine (10 AM). Within 1 min, a significant rise in the level of cGMP was observed; this level peaked at 5 min and declined thereafter. In contrast, no change in the basal level of cAMP was observed in response to epinephrine. These results again confirm the observations discussed above that intact isolated adrenocortical carcinoma cells have an active epinephrine-dependent guanylate cyclase sys- tem but are devoid of epinephrine-activated adenylate cyclase.
Effects of a- and -adrenergic agonists on the formation of cGMP
Figure 3 shows that the a-adrenergic agonist phenyl- ephrine markedly increased the accumulation of cGMP, but in contrast, isoproterenol, a ß-adrenergic agonist, did not significantly change the basal level of cGMP. Fur- thermore, there was no additive accumulation of the cyclic nucleotide when the carcinoma cells were incu- bated with epinephrine (10 MM) and isoproterenol (10 UM). On the other hand, the epinephrine-stimulated rise of cGMP was reduced almost to the basal level by phen- tolamine, an a-adrenergic antagonist. The possibility that the a-adrenergic mediated rise of cGMP may be entirely via calcium was ruled out, since it was demonstrated that
Cyclic GMP (% above control)
160
120
80
40
O
ACTH
EPI
ACTH ISO PHENYL EPI
EPI +
+
+
EPI
PRO PHENTO
calcium alone (0-5 µM) does not cause an increase in the basal level (3.2 pmol) of this cyclic nucleotide (data not shown). However, it is very likely that, analogous to the situation in isolated adrenal cells, calcium is also one of the coupling factors of the cyclase system (24), but cal- cium by itself is unable to stimulate the adrenocortical guanylate cyclase. From these data, we conclude that a- adrenergic carcinoma cell receptors mediate the forma- tion of cGMP in response to epinephrine.
It is also to be noted that the concentrations of ACTH and epinephrine which yield maximal cGMP production have an additive effect when combined. This indicates that the receptors for ACTH and a-adrenergic agonists are distinct in the isolated adrenocortical carcinoma cells.
Effect of a cholinergic antagonist on the epinephrine- stimulated rise of cGMP
To examine the possibility that the epinephrine-acti- vated increase of cGMP might also be mediated by cholinergic receptors, isolated adrenocortical carcinoma cells were incubated with the cholinergic agonist, acetyl- choline, with epinephrine and acetylcholine, or with the cholinergic antagonist atropine. The data in Table 2 show that acetylcholine alone neither increases the formation of cGMP nor potentiates the rise exhibited by epineph- rine. Furthermore, atropine does not inhibit the epineph- rine-activated rise of cGMP. These data thus demon- strate that there is no cholinergic receptor-mediated rise of cGMP.
Discussion
The present studies conducted with isolated normal adrenal and adrenocortical carcinoma cells show that
| Addition | Conc. (UM) | cGMP (% above control) |
|---|---|---|
| Acetylcholine | 10 | 0 |
| Atropine sulfate | 10 | 0 |
| Epinephrine | 10 | 95 ± 6 |
| + Acetylcholine | 100 ± 7 | |
| + Atropine sulfate | 86 ± 5 |
The conditions of the experiment were identical to those depicted in Fig. 1. The time of incubation was 10 min.
normal adrenal cells possess an ACTH-responsive gua- nylate cyclase, but this cyclase is unresponsive to epi- nephrine. In contrast, adrenocortical carcinoma 494 (31) cells respond to both ACTH and epinephrine with an increased elevation of cGMP, but the receptors mediating the responses to ACTH and epinephrine are distinct.
Evidence is also provided that the epinephrine-acti- vated increase in cGMP is mediated by a-adrenergic receptors. Furthermore, the adrenocortical carcinoma cells do not possess a cholinergic sensitive guanylate cyclase. Thus, these results indicate the ectopic produc- tion of a-adrenergic receptors which are coupled with the guanylate cyclase system and are not present in the normal adrenal cell.
These results are significant and novel, since previous findings have not demonstrated an epinephrine-activated rise in the concentration of cGMP mediated by a-adre- nergic receptors. These results assume further impor- tance since the a-adrenergic mediated rise of cGMP does not appear to require the mandatory involvement of calcium in adrenocortical carcinoma cells. To our knowl- edge, this is the first report in which an epinephrine- activated increase in cGMP has been demonstrated to proceed via «-adrenergic receptors and appears not to involve the intermediatory role of calcium. It is to be noted that we are not suggesting that calcium has no role in the activation of the guanylate cyclase component, but this role, as in the case of ACTH (24), could be such that other coupling factors are also needed for a functional guanylate cyclase system. Such information concerning this cell system is not available at this time. Nonetheless, this appears to be an example of another type of regula- tion of guanylate cyclase.
According to the original concept formulated by Murad and coworkers (32), it was thought that ß-receptors me- diate catecholamine-induced accumulation of cAMP. However, recent evidence has indicated that mammalian liver possesses both a- and ß-adrenergic receptors (33- 35). Evidence also has been provided that «-adrenergic activation of phosphorylase (36, 37) does not involve the formation of cAMP but occurs via an increase in cytosolic calcium ion. Recently, Asakawa et al. (38) have demon-
strated an epinephrine-activated rise of cGMP in isolated fat cells, but this rise was mediated by -adrenergic receptors. Original studies by George et al. (39), con- firmed by many investigators (40-45), implicated cGMP in cholinergic receptors in a variety of tissues.
A reason for the ectopic production of the membrane receptors demonstrated in this study cannot be provided at this time, but several speculative explanations can be offered. One possibility is that the early neoplastic lesion in the adrenal cell leads to dedifferentiation, but it is not known at this time whether the dedifferentiated adrenal cell has membrane characteristics similar to the malig- nant cell. This hypothesis could possibly be tested by examining the membrane receptors of the fetal adrenal cell. Another molecular explanation was proposed by Pitot (46). According to this concept, an alteration in the template stability of neoplastic cells could be the basis for the initial transformation event in neoplasia resulting in a molecular mask in the form of the neoplastic phe- notype imposed on a normal genotpye. If this is true, does such a molecular change in adrenal neoplasia lead to the ectopic but specific formation of a-adrenergic receptors? After the initial molecular malignant lesion, the cell needs to change its membrane receptor properties to express its neoplastic properties. If Pitot’s theory is true, then abnormal dedifferentiation could occur in par- allel with malignancy, resulting in a new phenotype (46).
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