Characteristics and Metabolism of (x1 Adrenergic Receptors in a Nonfusing Muscle Cell Line*

The BCIHl nonfusing muscle cell line possesses bind- ing sites for [3H]prazosin. These binding sites are typically a1 adrenergic receptors as shown by their greater affinity (3700-fold) for prazosin than for yohimbine. Both kinetic and equilibrium analyses indicated that [SHJprazosin interacted with only one category of independent binding sites with the following character- istics. KO = 0.13 f 0.01 m. B,, = 97 f 5 fmol/mg of protein corresponding to 25,000 sites/cell (n = 17). number of cells 50% a1 from New England Nuclear (Ref. NET 250). (-)-Adrenaline bitar- trate, (-)-noradrenaline hydrochloride, (-)-phenylephrine hydrochloride, yohimbine hydrochloride, and cycloheximide were from Sigma; phenoxybenzamine from Smith Kline and French, phentolamine hydrochloride from Ciba-Geigy, (+)-adrenaline bitartrate from Sterling-Winthrop Research Institute, clonidine from Boehringer In-gelheim, and prazosin hydrochloride from Pfizer.

Our knowledge of /3 adrenergic receptor properties and their coupling characteristics with the adenylate cyclase is mainly due to the use of two experimental tools, i.e. the obtention of highly labeled antagonists and agonists and the possibility of studying these systems in homogeneous cell populations such as avian erythrocytes (1)(2)(3)(4) and cell lines without (5-7) or with different genetic deletions (8,9). The investigation of a adrenergic receptors is more recent and, thus far, their characterization with labeled antagonists and agonists has only been done in whole tissues (10-14) and isolated platelets (15,16). Therefore, we looked for a cell line having both a adrenergic receptors and susceptibility to genetic manipulations. BCIHl cells were obtained from a tumor induced by nitrosoethylurea and were shown to possess many of the properties of muscle * This work was supported by LA 219, ATP 4149 from the Centre National de la Recherche Scientifique and ATP 58 78 90 from the Institut National de la Sante et de la Recherche Medicale. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$Recipient of a fellowship from the Deutscher Akademischer Austauschdienst. (17). In these cells, noradrenaline and acetylcholine are known to produce hyperpolarizing and depolarizing responses, respectively (17). We had previously shown that noradrenaline, through a adrenergic receptors, increases the permeability of the membrane of BC3Hl cells to K' ions (18). We show here that these cells contain highly specific a1 adrenergic receptors which can be labeled with [3H]-prazosin.
This cell line has also been shown to be a useful tool for studying the metabolism of acetylcholine receptors (19). It was, thus, of particular interest, in the same line, to investigate the metabolism of the aI adrenergic receptor, another intrinsic membrane protein. This study was possible since we possess phenoxybenzamine (POB)' which is an a adrenergic blocking agent with long lasting properties and has been extensively studied in biological and chemical experiments (20)(21)(22)(23)(24). Recently, it has been shown that POB irreversibly blocks the [3H]dihydroergocryptine binding to uterus membranes in uitro (23). The covalent interaction of POB with a adrenergic receptors is strongly suggested by the experiments of Guellaen et al. (22) who showed that [3H]POB binding on hepatic a adrenergic receptors is maintained after its solubilization with Lubrol PX. We, therefore, used POB for blocking al adrenergic receptors in intact cells in order to study the kinetics of receptor repopulation in BC3Hl cells.

EXPERIMENTAL PROCEDURES
Tissue CuZture-BC3H1 muscle cell line was a gift from Dr. J. Patrick, The Salk Institute, San Diego, CA. The cells were grown in Dulbecco-modified Eagle's medium containing 10% fetal calf serum, 50 IU/ml of penicillin G, and 50 pg/ml of streptomycin sulfate at 37 "C in an atmosphere of 5% COS and 95% air. The line was maintained in the exponential growth phase by passage every 4 days. Cells used for experimentation were plated in 90-mm diameter plastic tissue culture dishes at IO3 cells/cmz. They were used between 14 and 21 days after seeding and the medium was changed twice a week. The cells reached confluency 6 days after plating, but differentiated properties were fully expressed only after 10 days (17,19).
Preparation of Particulate Fractions-Cells were washed twice at room temperature with 0.15 M NaCl and scraped off with a rubber probe in 5 ml of 0.15 M NaCl/dish. They were centrifuged for 3 min at 400 X g and the pellet was homogenized at 0 "C in 5 mM Tris-HC1, pH 7.6, and 1 m~ MgC12 (about 3 . IO6 cells/ml) with a Teflon-glass homogenizer. The homogenate was diluted by one-half with the homogenization buffer and centrifuged for 15 min at 30,000 X g at 4 "C. The pellet was resuspended in 50 mM Tris-HC1, pH 7.6, and 10 mM MgC12 (about 1.5-IO6 cells/ml) filtered through a silk screen (150 /.un pore diameter) to remove some DNA filaments which had been formed during homogenization. It was centrifuged for 15 min at 30,000 X g at 4 "C. The final pellet was resuspended in 50 mM Tris-HC1, pH 7.6, and 10 mM MgClz at a protein concentration of about 1-2 mg/ml. The particulate fractions were kept at 0 "C and used for experiments within 3 h.
Measurements ~f[~HJPrazosin Binding-The particulate fraction (100-200pg) was incubated for 30 min at 30 "C in a solution containing 50 m~ Tris-HC1, pH 7.6, 10 mM MgCh, and 2. IO-" M to 2. IO-' M concentration of [3H]prazosin (1 ml total volume). Incubations were terminated by rapid filtration of the entire mixture through Whatman GF/B glass fiber filters and by washing three times with 5 ml of icecold incubation buffer. The filters were dried and counted in scintillation fluid. Figures show specific binding defined as the binding which is inhibited by M phentolamine. Incorporation of 1-Amino Acid Mixture-The cells were incubated 3 h at 37 "C in culture medium containing lo6 cpm of the amino acid mixture per culture dish (about 4 . IO6 cells). The incorporation was stopped by three washes with 10 ml of 0.15 M NaCl at room temperature. The proteins were precipitated with 5 ml of 10% trichloroacetic acid at 0 "C and collected by centrifugation 5 min at 1500 x g. The trichloroacetic acid-precipitable material was washed three times with 10% trichloroacetic acid at 0 "C and the final pellet was dissolved in 4 ml of 1 N NaOH/dish. 0.5 ml of this solution was counted in scintillation fluid.
Blockade of a Adrenergic Receptors with Phenoxybenzamine-The cells were incubated in 10 ml of Dulbecco-modified Eagle's medium/dish containing the appropriate concentration of POB for 10-15 rnin at 37 "C. The medium was then aspirated and the cells were washed three times at room temperature with 10 ml/dish of Dulbecco-modified Eagle's medium. To follow the reappearance of the a adrenergic receptors, the cells were placed in culture medium at 37 "C in an atmosphere of 5% COn and 95% air during varying times before the determination of the concentration of a adrenergic receptors. In each case, this concentration was the maximal binding of [3H] prazosin as determined by Scatchard analysis.
Material~-[~H]Prazosin (28 Ci/mmol) was purchased from the Radiochemical Centre, Amersham. 1-Amino acid mixture (3H-G) was from New England Nuclear (Ref. NET 250). (-)-Adrenaline bitartrate, (-)-noradrenaline hydrochloride, (-)-phenylephrine hydrochloride, yohimbine hydrochloride, and cycloheximide were from Sigma; phenoxybenzamine from Smith Kline and French, phentolamine hydrochloride from Ciba-Geigy, (+)-adrenaline bitartrate from Sterling-Winthrop Research Institute, clonidine from Boehringer Ingelheim, and prazosin hydrochloride from Pfizer. and dissociation rate constant, respectively) determined from the association binding curves obtained at three different ligand concentrations (Fig. 1D) allowed us to determine k-1 = 0.046 min" and k+l = 5.13 lo-@ M" min". The ratio of k-l/ k,, was thus equal to 0.09 nM, a value in good agreement with the KO determined at equilibrium (see above). The direct measurement of k-] gave a value of 0.035 min" (Fig. 1C).

Specificity of [3X]Prazosin Binding
Sites-The agonist specificity of [3H]prazosin binding sites was very typical of an (Y adrenergic receptor. The order of specificity was (-)-adren- Fig. 2 A ) .
The binding was stereospecific since (-)-adrenaline was 16 times more potent than (+)-adrenaline. The slope of the Hill plot of the displacement curves was very close to one, indicating the absence of any heterogeneity of the sites for agonists. The antagonist specificity clearly indicated the al characteristics of the receptors labeled with [3H]prazosin. Prazosin, an a1 specific antagonist, was 3700 times more potent than yohimbine, an (~2 specific antagonist (25) (Fig. 2B). Note that phenoxybenzamine did not give a displacement curve parallel to those of other antagonists. This is due to the nonreversible interaction of phenoxybenzamine with the binding sites (see below).

Specific Irreversible Blockade of a1 Adrenergic Receptors by Phenoxybenzarnine on Intact Cells-Cells were incubated
for 10 min with increasing concentrations of POB (lo-' M, lo-@ M, 3 X lo-@ M, and W 7 M). After extensive washing of the cells, the particulate fractions were prepared and [3H]prazosin binding measured. As seen in Fig. 3A, POB reduced the total number of sites without affecting the dissociation constant of the remaining sites. At M concentration of POB, more than 95% of the binding sites were blocked and no Scatchard plot could be drawn. At 3 X M concentration of POB, the irreversible blocking action of POB was very rapid since it was maximal after 5 min (Fig. 3B). Note that the blocking action of POB was not increased even when incubation lasted for 30 min and despite the fact that free POB was still present. This is probably due to a rapid inactivation of the reactive aziridinium ion of free POB (24). This rapid inactivation of POB has also been reported by Williams and Leflrowitz (23). The POB action was receptor specific since it could be suppressed by phentolamine M) (Fig. 3B). as seen in Fig. 4 A . a1 Adrenergic receptor sites reappeared after only 3 h, and this reappearance was time dependent.

Reappearance of a1 Adrenergic Receptors in B C~H I Cells after an Irreversible
These newly detectable receptors had a KD similar to that of control cells which did not receive POB. When the receptor blockade was performed with a 10-fold higher concentration of POB M rather than M ) and tested 24 h later, the receptor reappearance was similar (Fig. 4B).
Determination of Synthesis and Degradation Rates of a , Adrenergic Receptors in BCJI, Cells-The entire time course of reappearance was studied as described in Fig. 4A. In each experiment, the cells reached confluency before the beginning of POB blockade, so that during the entire time course, the total number of cells and the total protein content per plate did not vary (Fig. 5A). Furthermore, the total number of a1 adrenergic receptors in control cells which did not receive POB was unchanged during the entire period of reappearance (Rss, concentration of receptor at steady state). It was equal to 107 k 5 fmol/mg at the beginning of the experiment and 103 2 5 fmol/mg of protein 7 days later (Fig. 5B).
In order to demonstrate that the receptor reappearance after irreversible blockade with POB truly represents newly synthesized molecules, we studied the reappearance in the presence of cycloheximide. Cycloheximide (1 pg/ml) blocked 86% of the total protein synthesis (Table I). After 24-h treatment with cycloheximide (1 pg/ml), the cells were still alive, since a 3-h period after elimination of cycloheximide was sufficient to allow 88% recovery of protein synthesis (Table I).
When cells were treated with POB (lo" M) and incubated thereafter with cycloheximide (1 pg/ml), the receptor reappearance was decreased by more than 95% (Fig. 5B). In addition, after this 24-h blockade of receptor reappearance by cycloheximide, the elimination of this protein synthesis inhibitor allowed cells to synthesize new receptors with approximately the same rate as before protein synthesis inhibition (Fig. 5B). Phenoxybenzamine M and M) did not affect protein synthesis (Table I).
If one makes the following simple hypotheses, already verified for acetylcholine (26) and insulin receptors (27), that 1) the receptor production is constant during the entire period  Then the repopulation kinetics is given by the difference between the production and the degradation of new receptors: giving when t + m [Rt] approaches r / k , which is, therefore, equal to [Rss]; [Rss] = r / k (2). Therefore, Equation 1 can be written (1 - e-") ( 3 ) . The logarithmic transformation (Log [Rss]/[Rss -Rt] = kt (4)) of the experimental repopulation curve confirms that the repopulation was a monoexponential process (Fig. 5C). The slope of the straight line obtained gave the value of the rate constant for degradation k = 0.03 h" (Fig. 5C)

DISCUSSION
BCBHl cells appear to be a useful model for studying a1 adrenergic receptors in an isolated cell line. The a1 adrenergic receptors can be easily labeled with [3H]prazosin. These receptors were typically of the al type, as shown by the fact that the affinity for prazosin was 3,700 times greater than the affinity for yohimbine (Fig. 2B) (25). Both kinetic and equilibrium analyses indicated that [3H]prazosin interacts with only one category of independent binding sites having a high affinity for [3H]prazosin (KO = 0.13 f 0.01 nM; n = 17). The total number of sites is 97 f 5 fmol/mg of protein corresponding to 25,000 -C 3,000 sites/cell.
It has been shown that at confluency, BCSHl cells exhibit some properties of muscle such as adenylate kinase and creatine phosphokinase activities, electrical excitability, and synthesis of acetylcholine receptors (17,19). The possibility of keeping BCsHl cells in a nondividing state at confluency for 7 days without variation in the number of receptors (see Fig.  5 ) makes this cell line particularly suitable for studying the a1 adrenergic receptor functions and biosynthesis.
POB was used for irreversibly blocking a1 adrenergic receptors in intact cells; this blockade was very rapid and concentration dependent (Fig. 3A). POB (lo-' M ) blocked 50% of a1 adrenergic receptors with no significant change in the apparent affinity of the free binding sites for [3H]prazosin (Fig. 3A). POB (10" M) blocked at least 95% of the entire a1 adrenergic population. This blockade was receptor specific since it was suppressed when a1 adrenergic receptors were saturated with phentolamine (Fig. 3B).
The reappearance of a1 adrenergic recept.ors after blockade with POB M ) was time dependent (Fig. 4). The new receptors had the same affinity as those of control cells (Fig.   4). In previous in vivo studies on POB's action in rats, a debate arose about the possibility that the long lasting effects of POB might be partly due to its being trapped in cells or membranes (20,21). The possibility that such a phenomenon might constitute a rate-limiting step for receptor reappearance was tested by blocking the a1 adrenergic receptors with M and M POB. Since after 24 h, the number of receptors which reappeared was the same for the two doses, we can exclude any interference of free POB with the time course of receptor reappearance (Fig. 4B).
The repopulation of a1 adrenergic receptors was due to the biosynthesis of new receptor molecules since it could be blocked by cycloheximide a t a concentration which inhibited 86% of protein synthesis without killing the cells (Fig. 5). Indeed, synthesis of a1 adrenergic receptors was reinitiated after washing out of cycloheximide (Fig. 5).
The time course of receptor repopulation was monoexpo-  (Fig. 5). This indicates that the following hypotheses are probably correct. 1) The production rate of a1 adrenergic receptors by cells is constant.
2) The degradation of receptors is proportional to the concentration of receptors in the cell. The semilogarithmic transformation of the time course of reappearance allowed the calculation of the rate constant for receptor degradation k = 0.03 h-'. The half-life of the a, adrenergic receptor was thus equal to 23 h. This value is identical with that described for acetylcholine receptors by Devreotes and Fambrough (26) in chick skeletal muscle in culture and is very similar to that generally observed for extrajunctional acetylcholine receptors (28). However, in BCaHl cells, Patrick et al. (19) reported a higher degradation rate constant (0.08 h-') for acetylcholine receptors. This difference in the degradation rate for a1 adrenergic receptors and acetylcholine receptors in the same cells is interesting.
In these same experiments, we were also able to estimate the production rate of a1 adrenergic receptors. This was equal to 3.2 fmol/mg of protein/h. In the study of acetylcholine receptors, several authors (19,26) have shown that in addition to surface membrane receptors labeled with a-bungarotoxin, there are hidden receptors, some of which are precursor receptors. We did not find such a heterogeneous population of receptors in our study. However, in contrast to a-bungarotoxin, we are not sure that phenoxybenzamine is unable to enter the cell and block precursor receptors.
During the course of these experiments with 10" M POB and even with M POB, we noted that there was a small percentage of receptors (always less than 5%) which remained in the particulate fraction of cells. It is possible that these receptors are indeed precursors, but this question remains to be investigated in more detail.
The possibility of a simple method for measuring both the rate of production and the rate of degradation of al adrenergic receptors should provide new insight into the problem of modulation of these receptors under different hormonal and pharmacological situations.