Characterization of Ectopic a-Adrenergic Binding Receptors of Adrenocortical Carcinoma Cells*
GOURI SHANKER 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 accompanying communication, we pro- vided evidence for the formation of cGMP via the activation of a-adrenergic receptors in isolated adrenocortical carcinoma cells. The investigations of Williams et al. have demonstrated that the compound [3H]dihydroergocryptine (DHE) can be used as a specific ligand for the direct characterization of a-adrenergic receptors. In the present communication, we have used this agent for the direct identification of a-adrenergic receptors on adrenocortical carcinoma cells. The results showed that the binding of [3H]DHE with the adrenocortical carcinoma cell receptors is rapid, reversible, and of high affinity, with an appar- ent dissociation constant of 0.55 x 10-9 M. Binding of [3H]DHE was a saturable process corresponding to 85 fmol of sites/2 × 106
cells. a-Adrenergic agonists and antagonists competed far more effectively for the binding receptors of DHE than the B-adre- nergic agents. Acetylcholine, a cholinergic agonist, was devoid of binding activity. No detectable binding of [3H]DHE with normal adrenal cell receptors was observed. These studies thus demon- strate ectopic production of a-adrenergic receptors in the adre- nocortical carcinoma cells. Taken together with the evidence provided in the accompanying paper, in which we have shown that catecholamines stimulate the tumor guanylate cyclase via a-adrenergic receptors, these studies present the first complete enzymatic and pharmacological analyses of adrenocortical car- cinoma «-adrenergic receptors and their relationship to the guanylate cyclase system. (Endocrinology 106: 1594, 1980)
S TUDIES at this laboratory have demonstrated the important mediatory role of cGMP in ACTH-in- duced steroidogenesis in isolated adrenal cells (1-6) (for a review, see Ref. 7). Epinephrine is ineffective in acti- vating guanylate cyclase in intact isolated fasciculata cells (8). In contrast, adrenocortical carcinoma 494 cells (9) respond to both ACTH and epinephrine with an increased elevation of cGMP, but the receptors mediating the response of ACTH and epinephrine are distinct (8). Evidence was also provided that the epinephrine-acti- vated increase in cGMP was mediated by @-adrenergic receptors. These results thus indicated an ectopic pro- duction of a-adrenergic receptors, which are coupled with the guanylate cyclase system and are not present in the normal adrenal cell.
In the present communication, we demonstrate the presence of a-adrenergic receptors on adrenocortical car- cinoma cells by direct binding techniques using the po- tent a-adrenergic antagonist, [3H]dihydroergocryptine ([3H]DHE). The latter compound has been shown to bind specifically with a-adrenergic receptors (10-12). Thus, these binding studies provide a direct method for
assessing whether the inappropriate epinephrine respon- siveness of guanylate cyclase in this adrenocortical car- cinoma cell is related to the ectopic formation of a- adrenergic receptors.
Materials and Methods
[3H]DHE (SA, 25.7 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA). Acetylcholine chloride, (-)epinephrine bitartrate, (-)phenylephrine hydrochloride, (-)norephinephrine hydrochloride, yohimbine hydrochloride, ergotamine tartrate, (-)isoproterenol hydrochloride, and (±)propranolol hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). Phentolamine hydrochloride and phenoxybenzamine hydrochloride were gifts from Ciba- Geigy and Smith, Kline, and French, respectively.
The isolated adrenocortical carcinoma 494 cells were pre- pared by trypsin digestion (13). In general, for each isolated adrenocortical cell preparation, 5.2 g tumor tissue were used, and 2 × 106 cells, representing 30-35 mg of adrenal tumor, were resuspended in 0.8 ml Krebs-Ringer bicarbonate (KRB) buffer, pH 7.4, containing 4% albumin and 0.2% glucose. DHE or other agents were dissolved in an appropriate volume of KRB buffer, pH 7.4.
[3H]DHE binding assays
The incubation mixture contained isolated adrenocortical carcinoma cells with 1 nM [3H]DHE in a total volume of 1 ml and was incubated at 37 C for the specified times. At the end of incubation, 1 ml ice-cold KRB buffer was added, and the diluted
* This work was supported by grants from the NCI (CA-16091) and the NSF (PCM-7800860).
reaction mixture was immediately filtered through a GF/C filter. Each tube was rinsed three times with 2 ml KRB buffer. The filter was dried and counted in 6 ml scintillation solution containing 4 g omnifluor/liter toluene. A tube containing the complete reaction mixture but with 10-3 M phentolamine served as an indicator of nonspecific binding, and all data were cor- rected for this value.
Calculation of equilibrium dissociation constants for com- peting adrenergic antagonists
A large number of adrenergic antagonists were used to in- vestigate their ability to displace [3H]DHE from its binding sites on isolated adrenocortical carcinoma cells. By using the following equation (14), the equilibrium dissociation constant (Ka) for each adrenergic agent was calculated from that con- centration which caused 50% of the maximum displacement (EC50) of [3H]DHE binding:
Ka = EC50/[1 + [DHE]/KDHE]
where [DHE] represents the concentration of [3H]DHE (1 nM), and KDHE represents the Ka for DHE (0.55 nm) obtained from Scatchard analysis.
Results
Binding affinity and number of sites
Figure 1 shows that the binding receptors of isolated adrenocortical carcinoma cells are saturated with in- creasing concentrations of [3H]DHE. Furthermore, a lin- ear relationship between the number of cells (1 × 106 to 4 × 106) filtered and [3H]DHE bound was found. Figure 2 is a Scatchard plot of the binding of DHE to the carcinoma cells. The plot of binding affinity demon-
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strates a single type of receptor site with a dissociation constant of 0.55 x 10-9 M. The number of binding sites (n) is determined from the intercept of the plot with the abscissa (n = 85 fmol/2 × 10-6 cells).
Kinetics of binding
The effect of the time of incubation on the binding of [3H]DHE to the isolated adrenocortical carcinoma cell receptors is shown in Fig. 3A. One can appreciate from this figure that half of the binding sites are saturated within 10 min, and maximal saturation appears to be approached within 60 min. Thus, this process is quite rapid. The rate of exchange between bound [3H]DHE and the a-adrenergic antagonist, phentolamine, may be seen in Fig. 4A. Most of the readily exchangeable isotope is released within the first 15 min of incubation in the presence of nonradioactive phentolamine, thus indicating that the dissociation rate is also rapid.
From the kinetics of binding and dissociation of [3H]- DHE with isolated adrenocortical carcinoma cell recep- tors, one can determine the equilibrium dissociation con- stant (Kd) (10). Since the concentration of [3H]DHE (1 nM) is far in excess of the concentration of binding sites (0.085 nm), the kinetics of [3H]DHE binding with the tumor cell membrane receptors can be considered to be a pseudo first order process. As the binding reaction is reversible in nature, it can be described by the equation In[Xeq/(Xeq - X)] = Kobt, where X represents the amount of [3H]DHE bound at each time interval (t), and Xeq represents the amount bound at equilibrium (0.08 nM). The plot of In[Xeq/(Xeq - X)] vs. time shows a
[‘H]-DHE Bound ( % Maximum)
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slope of 0.04 min-1 (Fig. 3B), which is the value of kob, the observed rate constant for the reversible pseudo first order reaction. The second order rate constant (kı) can be calculated from the equation kı = (Kob - k2)/[DHE], where k2 represents the rate constant for the reverse (dissociation) reaction (Fig. 4), and [DHE] is the [3H]- DHE concentration (1 nm) in the incubation mixture. The dissociation rate constant (k2) determined from the
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slope in Fig. 4B is 0.0106 min-1. The substitution of these parameters in the above equation for rate constant kı gives a value of 0.0294 × 109 M-1min~1. The ratio, k2:k] = 0.40 × 10-9 M, is the kinetically derived value of the Kd for the binding of [3H]DHE with the cell membrane receptors. This value is in excellent agreement with the Ka value determined by the Scatchard analysis (0.55 × 10-9 M).
Specificity of binding
An important feature of the binding sites for [3H]DHE is their specificity for a-adrenergic receptors. The data in
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Figs. 5 and 6, as well as that in Table 1, clearly demon- strates that «-adrenergic agonists and antagonists com- pete far more effectively for the binding receptors of DHE than the ß-adrenergic agonists and antagonists. For instance, phentolamine, an a-adrenergic antagonist, is 6 orders of magnitude more potent than the typical ß-adrenergic antagonist, propranolol. It is, however, al- most equipotent with other a-adrenergic agents, such as phenoxybenzamine, yohimbine, and ergotamine. A typi- cal a-adrenergic order of potency is observed in displacing the bound [3H]DHE from the cell receptors, with epi- nephrine > norepinephrine > phenylephrine > isopro- terenol.
Table 1 depicts the half-maximal inhibitory concentra- tions (EC50) of various agonists and antagonists in com- peting for [3H]DHE-binding sites. While a-adrenergic agents competed successfully for the [3H]DHE receptors, ß-adrenergic agents and cholinergic agents were either far less potent or completely devoid of binding activity. These data thus indicate the presence of typical @-adre- nergic receptors on the tumor cells.
Table 2 shows the values of Ka for the interaction of each competing adrenergic antagonist with the [3H]- DHE-binding site, as calculated from the equation of Cheng and Prusoff (14), described in detail in Materials
| Agent“ | EC50 (M) |
|---|---|
| Phentolamine | 3.5 × 10-9 |
| Phenoxybenzamine | 5.0 × 10-9 |
| (-)Epinephrine | 8 × 10-7 |
| Phenylephrine | 8.5 × 10-6 |
| (-)Isoproterenol | 6 × 10-3 |
| (±)Propranolol | 1 × 10-3 |
| Yohimbine | 8.5 × 10-9 |
| Ergotamine | 5.5 × 10-9 |
| (-)Norepinephrine | 6 × 10-6 |
” Acetylcholine does not compete for [3H]DHE binding.
| Adrenergic antagonist | Ka of inhibition of [H]DHE binding (nM) |
|---|---|
| DHE | 0.55 |
| Phentolamine | 1.25 |
| Phenoxybenzamine | 1.80 |
| Ergotamine | 1.96 |
| Yohimbine | 3.00 |
| (±)Propranolol | >105 |
In the presence and absence of various concentrations of the adre- nergic antagonists, the cells were incubated with [3H]DHE, and from the results the equilibrium dissociation constants for the various com- peting ligands were calculated, as described in Materials and Methods.
and Methods. Table 2 and Fig. 6 indicate that in contrast to an excellent potency of the a-adrenergic antagonists in competing for [3H]DHE-binding sites, ß-adrenergic antagonists like propanolol competed for these sites only at a very high concentraton. Various other a-adrenergic antagonists, viz phenoxybenzamine, ergotamine, and yohimbine, were almost as potent as phentolamine in inhibiting [3H]DHE binding. Thus, these results clearly indicate that the tumor cells possess specific catechola- mine-binding «-adrenergic receptors.
Discussion
In the present report, we used [3H]DHE to characterize a-adrenergic receptors in adrenocortical carcinoma cells. The use of [3H]DHE as a specific marker for a-adrenergic receptors has been validated in several tissues (10-12). Cell-binding studies using radioligand has also been re- ported by other investigators (15). Binding of [3H]DHE was a saturable process, corresponding to 85 fmol DHE bound/2 × 106 cells at 37 C. Ka for the binding of [3H]- DHE with its a-receptor-binding sites was 0.55 x 10-9 M. These studies thus indicate that the adrenocortical carcinoma cells possess a finite number of physiologically significant «-adrenergic receptors, and that these recep- tors are undectable in the normal fasciculata cell. The binding was displaced by a-antagonists. The a-adrenergic antagonists, phentolamine, phenoxybenzamine, and er- gotamine, were potent inhibitors of [3H]DHE binding, whereas the potent ß-specific adrenergic agents (-)iso- proterenol and (+)propanolol have affinities 4-5 orders of magnitude lower for the a-receptors.
Our previous studies demonstrated that the guanylate cylcase system of the neoplastic cell, in contrast to the normal adrenal guanylate cyclase, responds to epineph- rine via the @-adrenergic receptors (8). Together with the present results, these data provide the first complete enzymatic and pharmacological analyses of adrenocorti- cal carcinoma a-adrenergic receptors and their functional role in the guanylate cyclase system. Recently, a novel autophosphorylating protein kinase, AUT-PK 85, unde- tectable in the normal adrenal gland, has been reported in the adrenocortical carcinoma cell (16). The present studies indicate a novel receptor change associated with malignant transformation. It may well be that this tumor is a classic example of the altered genetic expression in neoplasia. It is expected that further study of this neo- plastic cell model should not only provide a better un- derstanding of the molecular mechanisms involved in
adrenal neoplasia but should also contribute to a better understanding of the regulation of guanylate cyclase.
Acknowledgment
We appreciate the excellent technical assistance of Joan Sharma.
References
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