Further Evidence that Desensitization of fl-Adrenergic-sensitive Adenylate Cyclase Proceeds in T w o Steps MODIFICATION OF THE COUPLING AND LOSS OF P-ADRENERGIC RECEPTORS*

In c6 glioma cells adenylate cyclase activation by a P-adrenergic agonist is a Michaelis-Menten function of receptor occupancy as expected in the collision-cou- pling model. The apparent affinity of the receptor-ag-onist complex for the adenylate cyclase ( K ) is equal to 0.24 X [RT], [RT] being the maximal receptor concentration. When c6 glioma cells were incubated with lo-’ M isoproterenol for 3 h, there was a 45.4 f 2.7% (n = 10) reduction in the number of total r3HJDHA binding sites and the maximal adenylate cyclase activation dropped by 57 f 3.4% (n = 6). The affinity of the /3-adrenergic receptors for both agonists and antagonists did not change during desensitization. However, the apparent affinity constant for isoproterenol stimulation of the adenylate cyclase (Kaapp) rose from 3.9 +- 1.0 X lo-* M (n = 5) to 1.1 f 0.2 X M (n = 5). This shift is to be expected in all systems in which coupling between receptor occupancy and adenylate cyclase stimulation is not linear and in which either the number of coupled receptors decrease or the K constant is modified. Double reciprocal plotting of equiactive isoproterenol concentrations in control and desensitized adenyl-

show that coupling changes during desensitization. This change consists in a 3.7-fold decrease in the apparent affinity of the agonist-receptor complex for the enzymes. Two hypotheses are proposed to explain this reduction.
The assumption that desensitization involves two different steps, an alteration in the coupling and a loss of &adrenergic receptors, is substantiated further by the following observations: the loss of hormonal responsiveness preceded the loss of binding sites and long term treatment with cycloheximide or lowering the temperature to 4°C eliminate the loss of binding sites but not the decline in hormonal responsiveness.
Cells that respond to specific hormones possess regulation mechanisms allowing the control of their degree of responsiveness. Thus, exposure of cells to a hormone reduces their * This work was supported by LA 219 and ATP 4149 from Centre National de la Recherche Scientifique and ATP 58 78 90 from 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. response to subsequent exposure (desensitization). On the other hand, prevention of hormone-receptor interaction, either by suppressing the hormone or adding an antagonist, enhances the response (supersensitivity). Generally, in systems in which the hormonal effect is mediated by CAMP, broken cell preparations of desensitized cells show a decrease both in adenylate cyclase stimulation by the agonists and in the density of their specific receptor sites (1). Lefkowitz and co-workers suggested that catecholamine-induced desensitization of P-adrenergic sensitive adenylate cyclase mainly resulted from the loss of P-adrenergic receptors (1).
The relationship between the reduction in the adenylate cyclase response to a ,&adrenergic agonist and the loss of specific ,&adrenergic receptors shows a lack of correlation between the two (2-5).
Recent results by Su et al. (2) indicated that the decline in P-adrenergic-sensitive adenylate cyclase of desensitized system might be a complex phenomenon. The loss of isoproterenol-stimulated adenylate cyclase preceded the loss of /Iadrenergic receptors but, after 24 h desensitization, both activities were reduced by the same percentage. The same discrepancy was also reported by Shear et al. (4). They found that, in S 49 lymphoma cells, membranes prepared after 2% h desensitization showed a 70% reduction in adenylate cyclase stimulation by isoproterenol, corresponding to a 35% drop in the number of binding sites.
Nevertheless, in order to compare these parameters, two points should be kept in mind: 1) Binding capacity and adenylate cyclase activity must be measured after the same incubation period and in the presence of the same components, especially as regards nucleotides, since these are known to influence binding of agonists (6-7). 2) It is essential to consider that the relationship between receptor occupancy and adenylate cyclase stimulation (coupling function) is not necessarily linear. For example, in ADH (8,9) and P-adrenergic-sensitive adenylate cyclase systems (10-12), a 10% occupancy of receptor sites results in adenylate cyclase activities of 80% and 20% of maximally stimulated adenylate cyclase respectively.
An important question is whether the loss of P-adrenergic receptors is the cause or a consequence of the P-adrenergicsensitive adenylate cyclase desensitization.
In the present work we attempted to answer this question by studying the relationship between the loss in P-adrenergic receptors and the reduction in adenylate cyclase responsiveness in C6 glioma cells, with special reference to Points 1 and 2 mentioned above. We also tried to modify this relationship by two treatments which have been shown to alter desensitization, namely changes in the temperature (13) and inhibition of protein synthesis (for review see Ref. 14). pCi) and [cy-3ZP]ATP (1 to 2 pCi) were added after 8 min of incubation, and the reaction was stopped 5 min later. For binding measurements, [3H]DHA was added at the beginning of incubation and the reaction was terminated 10 rnin later by adding 1 ml of cold (4OC) washing medium containing 50 m M Tris-HC1, pH 8, and 20 m~ MgClZ. Samples were fdtered through GF/C Whatman filters. Filters were then washed and the bound radioactivity determined by scintillation counting. Specific binding was defined as the difference between the amount of r3H]DHA bound in the absence (total binding) and the presence (nonspecific binding) of 10 p~ unlabeled alprenolol.

MATERIALS AND METHODS
For desensitization experiments, isoproterenol M, except otherwise mentioned) was added to the culture medium (HAM F 10 supplement with 10% fetal calf serum). Isoproterenol M) was added every 90 min throughout incubation. Cells were then washed four times with 0.9% NaCl and particulate fractions prepared as previously described (10). We verified that 90 min after addition of isoproterenol ( W 5 M), the concentration of this agonist was still sufficient for maximal stimulation of CAMP production. Thus, another addition of M isoproterenol did not raise further the level of intracellular cAMP ( 5 0 0 pmol/mg of protein). The basal intracellular concentration of cAMP was 5 pmol/mg of protein. Measurements were made by radioimmunoassay.
Adenylate Cyclase Assay in Neuroblastoma Cells Neuroblastoma cells were grown on Falcon Petri dishes in MEM (Eagle's minimal essential medium) with Earle's salts, supplemented with 10% fetal calf serum. Cells were incubated for 4 days at 37°C in a humidifled atmosphere (90% air, 10% COz).
Membrane Preparation-After removal of the culture medium, cells were suspended in 0.9% NaCl at 4"C, centrifuged at 300 X g for 5 min, and washed three times in the same medium. After the third centrifugation, cells were lysed by resuspension for 15 min in hypotonic buffer composed of 5 m~ Tris-HC1 (pH 8) and 1 m~ MgClZ.
Lysates were disrupted in a Dounce homogenizer (20 strokes). After partial purification according to the method of Brunton et al. (15), membranes were suspended in the hypotonic buffer and used immediately.
Adenylate Cyclase Assay-Adenylate cyclase was assayed at 3OoC in a 50-4 incubation medium containing 25 m M Tris-HC1, pH 8, 0. pCi of r3H]cAMP, and 5 pg of bovine serum albumin. Membranes were incubated for 10 min, the reaction was stopped and cyclic AMP isolated as previously described (10).

Calculations
The isoproterenol dissociation constant for ,&adrenergic receptors (KO*) was determined experimentally: it was calculated from the isoproterenol concentration that inhibited [3H]DHA binding by 50%: Kaapp is the agonist activation constant for the adenylate cyclase system and is equal to the agonist concentration yielding 50% maximal activation. Kaapp was calculated from the Hofstee plot of the doseactivation curve. supplied by the Ciba-Geigy Laboratories and prostaglandin by John Pike Upjohn.

RESULTS
Characteristics of P-Adrenergic-sensitive Adenylate Cyclase a n d P-Adrenergic Receptors during Desensitization-C6 glioma cells were exposed for 3 h to M isoproterenol. Characteristics of the P-adrenergic adenylate cyclase system were observed in particulate fractions. As reported by others, maximal adenylate cyclase stimulation diminished after desensitization (by 60%, Fig. L4). Furthermore, we observed an increase in the apparent activation constant (KAapp) of isoproterenol from 3.5 X M to 1.3 X M. The Hofstee plots of the dose-response curves given in Fig. 1B (Fig. 1D).
The Hill Coefficients of the isoproterenol displacement were identical (n = 0.98 and 0.96 for control and desensitized systems, respectively (Fig. 2).
For several systems, the Hill coefficients of isoproterenol displacement curves are less than one in the absence of GTP and equal to one in its presence ( 7 ) . The difference between these results and ours is probably due to the fact that we measured binding under adenylate cyclase conditions, especially in the presence of nonpurified ATP and of an ATPregenerating system. Furthermore, our membrane preparation was probably contaminated by GTP and GDP. In fact, under our experimental conditions, GTP does not increase stimulation by isoproterenol and only has a very slight effect on isoproterenol displacement curves (less than a factor 2).
In all experiments, basal, NaF, and Gpp(NH)p-sensitive adenylate cyclase were unchanged. In addition, in each experiment, we verified that alprenolol M) did not significantly reduce basal adenylate cyclase activity. Fig. 3 shows that the shift in the KAapp of isoproterenol for stimulating adenylate cyclase increased with the intensity of desensitization. Thus, KAapp was 3.8 X M in the control, whereas it was equal to 7.7 X IO-' M and 1.1 X M after lh h and 3 h of desensitization, respectively.
An important point was to check that adenylate cyclase activities were in fact measured a t equilibrium and that there was no desensitization a t high isoproterenol concentrations in uitro. T o verify these points we conducted an experiment comparing the dose-activation curves obtained in control and desensitized systems by measuring the adenylate cyclase activities, either between 0 to 5 min or between 8 to 13 min, in order to determine any differences as a function of time, This method is more accurate that the one that consists of measuring cyclic AMP accumulation throughout the entire period. _" , theoretical dose-activation curve of the desensitized system calculated from the equation where ET is the maximal theoretical adenylate cyclase activity of  The results of this experiment showed that basal activity was strictly linear. Maximal activities were slightly higher when measured between 8 to 13 min than between 0 to 5 min (less than 10% difference from linearity during the entire period). However, the important point is that the KAapp values in control and desensitized system were identical when measured either between 0 to 5 min or 8 to 13 min (data not shown).

Influence of the Degree of Desensitization on the Kaapp of PGEl for Adenylate Cyclase System-To ascertain whether
the shift in the &app which occws during desensitization concomitantly with the decrease in the maximal adenylate cyclase stimulation by an agonist is found in other systems, we studied the desensitization of the prostaglandin-sensitive adenylate cyclase system in neuroblastoma cells. Fig. 4 shows that when neuroblastoma cells were incubated for 6 h with increasing concentrations of PGE1, the maximal stimulation of adenylate cyclase by PGEl diminished and the Kaapp gradually shifted to the right. cyclase stimulation in the desensitized system (Fig. 6A). The intensity of adenylate cyclase desensitization did not rise when cells were incubated for 5 instead of 3 h at 4°C. At this temperature, there was no significant reduction in the number of /3-adrenergic receptors (~1 0 % ) following treatment of cells with M isoproterenol for 3 h (Fig. 6B) or 5 h (data not shown).
Effects of Cycloheximide on the Degree of Desensitization-When Cs glioma cells were incubated with cycloheximide (50 pg/ml) for 1 h before desensitization, the latter was not reduced. There was a 69% drop in the p-adrenergic stimulation of the adenylate cyclase and 51% loss of P-adrenergic receptors (Fig. 7, A and I?). This loss in adenylate cyclase Time Course for /3-Adrenergic-sensitiue Adenylate Cyclase and for P-Adrenergic Receptor Losses-Time course for the reduction of maximally stimulated /?-adrenergic-sensitive adenylate cyclase and of p-adrenergic receptor density were not identical (Fig. 5A). In this experiment, adenylate cyclase desensitization was maximal after l h, whereas the loss of padrenergic receptors still continued after 6 h. Further evidence that the rate of adenylate cyclase desensitizaton was greater than the velocity of /?-adrenergic receptor reduction is given in Fig. 5B which is the mean of three experiments.
Intensity of Desensitization at ~O C -C S glioma cells were incubated with isoproterenol (lop5 M) for 3 h at 4OC. As shown in Fig. 6, the degree of desensitization of the adenylate cyclase system was less than that obtained at 37°C. There was only a 32% reduction of the maximal adenylate cyclase stimulation, compared to 57% at 37°C (Table I). Note that in this case too, there was an increase in the Kaapp for isoproterenol adenylate stimulation was the same whether or not cycloheximide was present throughout washing (data not shown). However, when C6 glioma cells were incubated for 15 h with cycloheximide before desensitization started, there was no significant decrease in the number of p-adrenergic receptors (Fig. 7C), whereas desensitization of adenylate cyclase, although reduced, still persisted (38%, Fig. 7 0 ) . Mean results of four experiments were as follows: 1) loss of P-adrenergic  receptors: 44.0 k 3.6% for the control and 6 k 3% after cycloheximide pretreatment and 2) loss of adenylate cyclase stimulation: 67 f 2% in the control and 29 f 8% after cycloheximide pretreatment. We checked that the CAMP production system was still working after this long term treatment with cycloheximide by measuring intracellular cAMP by radioimmunoassay. In control cells, cAMP rose from 4.9 to 860 pmol/mg of protein when M isoproterenol was present for 10 min, and in cycloheximide-treated cells, CAMP rose from 3.2 to 539 pmol/mg of protein. Stimulations by NaF and Gpp(NH)p were not modified by pretreatment with cycloheximide.
In another experiment, we used 20 p g / d of puromycin instead of cycloheximide for 15 h of pretreatment. We did not observe any change in the number of /I-adrenergic receptors during desensitization, whereas in the corresponding control experiment, there was a 41% reduction. Although there was also a reduction in adenylate cyclase desensitization after puromycin treatment, the results were difficult to interpret, since there was also a 72% decrease in basal adenylate cyclase activity in puromycin-treated cells.
Coupling Function between Receptor Occupancy and Adenylate Cyclase Stimulation-We previously found that in Cs glioma cells, the coupling function between receptor occupancy (RH) and adenylate cyclase stimulation ( E ) was not linear (50% of adenylate cyclase stimulation was obtained with less than 50% of receptor occupancy). In the present report, this function was analyzed in more detail. An Eadie plot of this function is shown in Fig. 8A. The receptor occupancy [RH] was normalized to maximal occupancy RT). A straight line was obtained indicating that adenylate Lyclase activation can be described as a Michaelis-Menten function of receptor occupancy. Thus, with E * = adenylate cyclase minus basal activity, ET is the maximal theoretical adenylate cyclase activity (RH -P m), whose value is shown on this line intercept with abscissa (1.22 nmol of cAMP/5 min/mg). Fig. 8A shows that this value is 120% of the maximal experimental adenylate cyclase activity. The slope of this line allows calculation of K (apparent affinity of R H for the enzyme) expressed relatively to [RT] concentration (RT = maximal concentration of hormone receptor complex measured in binding experiments). In this case, K = 0.24 X [RT]. Note that these reactions occur within the membrane, whose volume is unknown. Thus, it is impossible to ascertain the absolute values of [RT] and K .
The interaction of H with the receptor may be described as a Michaelis-Menten function of [ H ] since the Hill coefficient for agonist binding is very close to 1 (see Fig. 2). and the maximal experimental stimulation is:

From Equations 1 and 2, it follows that
Evolution of Coupling between P-Adrenergic Receptor Occupancy and Adenylate Cyclase Activation during Desensitization-As shown above adenylate cyclase activation can be described as a Michaelis-Menten function of receptor occupancy (Equation 1). Consequently, the adenylate cyclase activities in control and desensitized systems are: The observation that basal, NaF, and Gpp(NH)p-stimulated adenylate cyclase activities do not change suggests that ET is the same in control and desensitized systems. Furthermore, the Eadie plot of the function relating adenylate cyclase stimulation to receptor occupancy for the desensitized system (Fig. 8A) shows that ET is equal to 1.20 nmol of cAMP/5 min/ mg which is identical with the ET for the control (1.22 nmol of cAMP/5 min/mg).
The values of ET in control and desensitized systems were computer-derived from least square regression analysis. The accuracy of ET determination was however less in desensitized than in control systems, since EZaX was far from ET. In two other experiments in which all the parameters for binding and adenylate cyclase were measured on the same enzyme, the ET values in the desensitized system were 93 and 87% of the control. In all desensitization experiments with CS glioma cells, straight lines were in fact obtained (Fig. 9, A , B, 0. Similar plotting of the dose-activation curves for prostaglandin-sen-sitive adenylate cyclase in neuroblastoma cell membranes also gave straight lines (Fig. 9D).
One advantage of this method is that it allows the agonist dissociation constant KD for the receptor implicated in adenylate cyclase stimulation to be determined from adenylate cyclase experiments. As shown in Fig. 9, A, B, C, this KO is very close to that determined by binding experiments. The mean KO value determined from cyclase experiments is 1.4 f 0.2 X M (n = 6) compared to 1.9 & 0.5 X M (n = 5) from binding measurements. Within a single experiment, the above method of plotting adenylate cyclase activity resulted in very close KO values whether membranes were prepared after cells had been incubated for different periods at the same agonist concentration (Fig. 9B) or for the same period at different agonist concentrations (Fig. 9D).
The experiment shown in Fig. 1, in which all binding and adenylate cyclase parameters were measured on the same membranes, indicated that, in membranes from desensitized cells the number of P-adrenergic receptors determined by binding was 48% of the control (Fig. 1 0 . If desensitization only involved a loss of /?-adrenergic receptors without any change in the coupling, the maximal adenylate cyclase stimulation would, according to the coupling function shown in w w-  Fig. 8B, amount to 66% ET, i.e. 82% of the maximal stimulation of the control. This value is obviously far higher than the one observed experimentally after desensitization (41% of the control).
Thus, it is reasonable to suppose that some change in coupling occurs. Since, in the experiment presented in Figs theoretical isoproterenol dose activation curve can be computed from Equation 1 for the desensitized system. This curve fits the experimental one reasonably well (Fig. lA). The significance of K in terms of molecular events and its possible modification during desensitization will be discussed later.

DISCUSSION
A careful comparison between the respective losses in hormonal responsiveness and hormonal receptors during desensituation should give interesting indications on the relations between the two. Several authorsshave already compared the loss of maximal hormonal responses with that of binding sites (2-5). However, direct comparison between the percentage of receptors accessible to a ligand and the percentage of maximal hormonal stimulation after desensitization can lead to erroneous cpnclusions as regard receptor uncoupling during desensitization, unless the relationship between receptor occupancy and adenylate cyclase stimulation is linear, which is generally not the case (8-12). This is why we carefully analyzed this relationship in the system under study, Le. CS glioma cells.
We showed that a Michaelis-Menten type of function relates ,&adrenergic receptor occupancy to adenylate cyclase stimulation (see Fig. 8 It is difficult to demonstrate that ET represents the total enzyme pool activity in our system. The fact that ET, like basal, Gpp(NH)p, and NaF-stimulated activities, does not change significantly during desensitization is a good argument for this assumption. It has been suggested that the activity measured in the presence of Gpp(NH)p plus isoproterenol reflects total enzyme activity (18). However, in our system, Gpp(NH)p reduced the maximal stimulation produced by isoproterenol (10). We calculated that, in turkey erythrocytes (12) K = 3 [RT] (see above). Thus, according to Equation 4, ET/E,,, should be equal to 4. In fact, in this system, the activity measured in the presence of Gpp(NH)p plus isoproterenol was reported to be 5 times higher than in the presence of the natural effector GTP (18). Considering that these data are taken from two different laboratories, this concordance is rather good, In pig kidney plasma membranes, on the other hand, the coupling function is not a Michaelis-Menten function, because the adenylate cyclase stimulation is not a Michaelis-Menten function of the concentration of the agonist (vasopressin). This is probably due to a partial irreversibility of the adenylate cyclase stimulation (8,19).
A coupling model for the C6 glioma cell system would have to take into account the following observations: 1) the binding for the agonist is noncooperative (Fig. 2); 2) activation of E is a Michaelis-Menten function of RH; and 3) [ET] does not change when the number of receptors is reduced. The literature gives at least one model which fits these facts, z.e. the collision coupling proposed by Levitzki and colleagues (20) which is set out as follows The rate constant for enzyme activation is Having thus defined the relationship between adenylate cyclase activation and receptor occupancy in the membranes from control cells, we were able to compare the loss of adenylate cyclase response to the loss of P-adrenergic receptors during desensitization.

KO
When Cs glioma cells were incubated with M isoproterenol for 3 h, the adenylate cyclase system became desensitized and the following observations were made: 1) the number of P-adrenergic receptors accessible to r3H]DHA decreased (45.4 f 2.7%, n = 10). The affinity of these receptors for both the antagonist C3H]DHA and the agonist isoproterenol did not change ( Table I) Nonlinearity of coupling in a system could be very useful in regulating its responsiveness. For instance, for a small loss in active receptors, the resulting shift in apparent affinity should considerably reduce the physiological response to low hormonal concentrations which are probably also the physiological ones. In several hormonal systems the physiological response in fact shows reduced hormonal apparent affinity rather than a reduced maximal response after desensitization (23-25). Su et al. (2) suggested that desensitization proceeds in two steps, one being the modification of the coupling between receptor and adenylate cyclase and the other the loss of padrenergic receptors (2). The present work gives three different types of finding in support of this idea.
The fist results from a comparison of the time courses for the decline in /3-receptor number and in maximal adenylate cyclase, respectively, during desensitization. As shown in Fig.  5, hormonal responsiveness diminishes faster than the number of P-adrenergic receptors. These results are very similar to those reported by S u et al. (2). However, if as established above, the coupling function is not linear, we cannot expect the time courses for the loss of binding and the loss of adenylate cyclase responsiveness to be superimposable. For example, in our CS glioma cell system, adenylate cyclase activity should diminish more slowly than the number of binding sites when desensitization is slight; conversely, receptor number should dwindle faster than adenylate cyclase activity when desensitization is greater (see Fig. 8). This is a complex situation and obviously other arguments should be found to substantiate the idea that a change in coupling precedes the loss in receptors.
The second type of finding in support of two steps desensitization arises from experimental situations in which the loss of binding sites can be suppressed but the loss of hormonal responsiveness persists. We obtained two such situations, one of them by long term treatment of cells with protein synthesis inhibitors before desensitization, and the other by performing desensitization at 4°C. The effects of protein synthesis inhibitors on desensitization have been tested in many systems (for review see Ref. 14), including CS glioma cells (26) with contradictory results. We do not know of any study in which such inhibition was reported to affect the loss of specific receptor sites during desensitization. In our system, no reduction of desensitization was observed when cycloheximide was added 1 h before isoproterenol (Fig. 7, A and B ) . On the contrary, when CS glioma cells were preincubated for 15 h with cycloheximide before exposure to isoproterenol, P-adrenergic receptor loss was inhibited almost completely, whereas desensitization of P-adrenergic-sensitive adenylate cyclase was only reduced (Fig. 7, C and D). This could indicate that a protein having a slow turnover is involved in the loss of receptors.
Although, like cycloheximide, puromycin eliminates the loss of binding sites, it is not impossible that this drug exerts some kind of action not mediated by protein synthesis inhibition. In any case, cycloheximide treatment was able to dissociate desensitization of the P-adrenergic adenylate cyclase from the loss of P-adrenergic receptors. Such a dissociation was also shown when desensitization was conducted at 4°C (Fig. 6). It is tempting to propose that the loss of binding sites is the result of internalization as suggested recently by Chuang et al. (27) for the P-adrenergic receptor of frog erythrocytes. Note that internalization of epidermal growth factor is suppressed at 4°C (28,29).
In another CS glioma cell line (C62B), Terasaki et al. reported different characteristics for desensitization than those described here (30). The main difference is that these authors did not see any change in the number of receptors after desensitization. This is probably due to a difference in the cell lines used in these two studies. Furthermore, a decrease in the number of ,&adrenergic receptors after desensitization was found recently in CS glioma cells by Mallorga et al. (31) with cells which originated from the same source as those used in our experiments (Benda,32,33).
The third type of finding in support of two-step desensitization was obtained from the analysis of the different parameters for P-adrenergic-sensitive adenylate cyclase system during desensitization, as described under "Results." The double reciprocal plots of agonist concentrations in control and desensitized systems always produced straight lines both in the ,&adrenergic-and prostaglandin-sensitive adenylate cyclase systems (Fig. 9). The result of this analysis is interesting for three reasons: 1) it allows determination of the affinity of the agonist for the receptor (KO) involved in adenylate cyclase stimulation without binding experiments. Note that, in CS glioma cells, the KD determined by this method (1.4 f 0.2 X M, n = 6) is very close to that determined by binding experiments (1.9 f 0.5 X M, n = 5). This confiis that [3H]DHA really labels the ,&adrenergic receptors coupled to adenylate cyclase. Furthermore, the low affinity of PGEI for its receptors calculated by this method (KO = 2.6 f 0.6 X 10"j M, n = 3) explains why it is impossible to measure PGE, binding under adenylate cyclase assay conditions. 2) Double reciprocal plot analysis indicates that the Michaelis-Menten nature of the function relating binding occupancy to adenylate cyclase stimulation does not change during desensitization; otherwise no straight lines would be obtained for these plots.

3) Such analysis enables calculation of the value Q which is equal to K / K ' x [RT']/[RT].
When CS glioma cells are desensitized for 3 h with M isoproterenol, the value of Q is 0.13 (Fig. 9A), which is different from the ratio of total receptor concentration in the desensitized system over the receptor concentration in the control system, as determined in binding experiments (0.48, Fig. 1). A change in the coupling function therefore occurs during desensitization. A 3.7-fold increase in the constant K is occurring (K' = 3.7 K ) . Since K = kOfl/k1 (a/l + a ) , the increase observed during desensitization might result from changes in three parameters. 1) k,ff might be modified. Although this is unlikely, such a change would explain heterologous desensitization as described in astrocytoma cells ( 5 ) .
2) k,, might diminish either because of a reduction in k1 or in a (ratio between active and inactive agonist receptor complexes).
Note that these changes in either kl or a have completely different meanings in terms of molecular events. When kl changes, all the efficient receptor.agonist complexes (RH") are capable of activating the adenylate cyclase but with a lower apparent affinity. In the case of a change in a, the ratio between the active and inactive form of the receptor agonist complexes is reduced during desensitization. It is of interest to note that the Gpp(NH)p-induced decrease in agonist a f f iity for &receptors seems to be reduced by desensitization (34, 35). A report by Kent et al. (22) shows that when agonist binding was measured in Tris buffer on control membranes of turkey and frog erythrocytes, ,&adrenergic receptors were found in both low and high affinity states. The ability of an agonist to activate adenylate cyclase correlated with the amount of high affinity state receptors formed in the presence of the agonist. During desensitization there was impairment of high affinity state formation (22).
Although further experiments are needed to discriminate between the proposed hypotheses explaining the decrease of K during desensitization, our results clearly show a modification in the coupling between P-adrenergic receptors and adenylate cyclase during desensitization and also that experimental conditions can be found for dissociating this change in coupling from the loss of P-adrenergic receptors.