Eur. J. Biochem. 143, 243-250 (1984) ’( FEBS 1984

Regulation by adenosine of the vasopressin-sensitive adenylate cyclase in pig-kidney cells (LLC-PK,,) grown in defined media

Christian ROY Centre de Pharmacologie-Endocrinologie (Centre National de la Recherche Scientifique - Institut National de la Recherche Medicale), Montpellier

(Received March 23/May 29, 1984) - EJB 840308

LLC-PK,, cells, a kidney-derived cell line, had sustained growth in a defined medium. When compared to the parent cell line growing with 10 % fetal bovine serum, LLC-PK,, cells had about 100-times fewer vasopressin receptors. Upon modifications of the cell culture medium, the vasopressin response of the adenylate cyclase could be increased by more than 10-fold with a parallel increase in vasopressin receptor number. Using cells with high or low receptor densities, the stimulatory and inhibitory effects of N6-~-2-phenylisopropyl-adenosine on the modulation of the adenylate cyclase responsiveness to vasopressin were investigated. When high concentrations of GTP were added, low concentrations of phenylisopropyladenosine inhibited the enzyme, while higher concentrations were found to be stimulatory. The adenylate cyclase activity stimulated by vasopressin could only be inhibited by phenylisopropyladen- osine under these conditions in membranes with high receptor density; only the increase in enzyme activity due to high GTP concentration was inhibitable. The analysis of the dependency of the adenylate cyclase activity as a function of the vasopressin concentration showed that, besides reducing the maximum velocity of the system for vasopressin, the addition of phenylisopropyladenosine generated an heterogeneity in the adenylate cyclase response to vasopressin (as judged by a curvilinear Eadie plot). A high-affinity component in the adenylate cyclase response appeared when phenylisopropyladenosine was added.

The growth of the cells in a medium containing adenosine deaminase gave results identical to those obtained for control cells. However, growing the cells with both phenylisopropyladenosine and adenosine deaminase abolished the inhibitory effects of the former on the adenylate cyclase and greatly reduced its stimulatory action. Under these conditions, the vasopressin response of the adenylate cyclase was not further regulated by phenylisopropyladen- osine. These results indicate a role of adenosine on vasopressin response, especially at low physiological concentrations of the hormone where a high-affinity component of the hormonal response could be demonstrated.

Adenosine appears to be an important regulator of ade- nylate cyclase activity in many tissues. The enzyme activity can be lowered upon occupancy of the A, receptors or enhanced when A, sites are occupied [I - 61. These two types of receptor coexist in the same tissue structure [2, 6, 71. The apparent affinity of the A, receptor in every tissue tested has always been found higher than that of the A, receptor in the same tissue [S - 1 I]. The ability to eliminate endogenous adenosine from membrane preparations allows the determination of the both A, and A, receptor effects on the adenylate cyclase activity [4, 101. This is achieved by using N6-~-2-phenylisopropyl- adenosine (PIA) as an adenosine substitute, taking advantage of its metabolic stability in the presence of adenosine de aminase.

LLC-PK,, cells are a kidney-derived cell line growing in a defined medium with no hormone added. A previous study showed that LLC-PK,, cells had a very low receptor density when compared to the parent cell line continuously grown in the presence of 10 %fetal bovine serum. By making appropriate addition to the cell culture medium, the amount of vasopressin receptor per cell could be increased [12]. This increase was

Abbreviations. PJNaCl = 137mM NaC1,0.9mM CaCl,, 2.7 mM KCI, 0.9 m M KH,PO,, 24mM Na,HPO, and 0.5 mM MgCI, ; PIA, Nh-~-2-phenylisopropyl-adenosine; [Lys8]vasopressin, vasopressin with lysine as the residue at position 8.

correlated with a proportional enhancement of the adenylate cyclase responsiveness to vasopressin.

At variance with numerous other studies, I have in- vestigated the effects of PIA on both basal and hormonally stimulated adenylate cyclase activities. Besides the well re- ported effects of adenosine on several other systems, PIA behaved differently on hormonally stimulated adenylate cy- clase. Taking advantage of our ability to modulate the extent of the hormonal response upon modification of the cell growth medium [12], the modulation of the [Lys8]vasopressin response by PIA was compared under situations where the cells had either low or high [Lys8]vasopressin receptor densities. In addition, the effects of a chronic exposure of the cells to adenosine deaminase and PIA or adenosine deaminase alone were tested on the PIA response of the adenylate cyclase. The desensitization of both A, and A, receptors were studied on the modulation of the [Lys8]vasopressin response.

MATERIALS AND METHODS

Cell culture

Pig kidney cells (LLC-PK,,) derived from the LLC-PK, cell line (ATCC CL 101) were kindly given by Dr J.S. Handler (NIH, Bethesda, MD, USA). The cells were grown in Coon’s modification of Ham’s F12 medium [13]. Selenium con-

244

centration was 10 nM. Penicillin and streptomycin concen- trations were 40 IU/ml and 40 pg/ml respectively. The cells were split at a ratio of 1 : 3 every two weeks as already described [12]. The medium was changed every two days and the day preceding the experiments. The whole set of experiments described in this paper was carried out in passages 6-21 upon thawing a new batch of cells. Cells could be induced for an increase in vasopressin receptor number by altering the composition of the cell growth medium. The supplemented medium was added 3 - 7 days before the adenylate cyclase experiments (the com- position of this supplemented medium will be described in a subsequent paper). When the cells were treated with adenosine deaminase (1 Ujml) and/or PIA (30 pM), the treatment started 30 h before thc adenylate cyclase assay, the medium being changed one more time, 7 h before the enzyme preparation.

Preparation of particulute fractions

Each 65-cm2 petri dish was rinsed three times with ice- cold PJNaCl without Ca2+ and Mg2+. The cells were scraped off with a rubber policeman in 3ml of the homogenization buffer ( 5 mM Tris/HCl p H 7.4, 1 m M EDTA). The cells were then disrupted by means of a tight Dounce homogenizer (15 strokes). One petri dish (14-16mg protein, 3ml) was diluted to 15 ml and centrifuged at 10000 x g for 10 min. The pellet was resuspended by vortexing in 2 ml of the homogeni- zation buffer, diluted to 15ml and again centrifuged at 10000 x g for 10 min. The final pellet was then resuspended in such a

volume that the amount of enzyme during the adenylate cyclase assay was 0.3-0.5 mg protein/ml. (Each petri dish yielded 1.8 -2.0mg protein in the second 10000 x g pellet.) For all the growth conditions tested (supplemented versus control media, treatment with adenosine deaminase and/or PIA), two, three or four distinct petri dishes treated under the same experimental conditions were pooled in order to minimize possible variations from one dish to another. Enzymes were always used fresh for the adenylate cyclase assays.

Adenylate cycluse assay

The incubation medium (40 - 100 pl) contained 50 mM Tris/HCl pH 7.4, 5 mM MgCl,, 10 pM CAMP, 10 pM papav- erine, 8 U adenosine deaminase/ml, 300 pM [LX-~'P]ATP (0.1 - 0.25 Cijmmol 10000 cpm cyclic [3H]AMP, 0.5 mg/ml creatine kinase, 2.5 mg/ml sodium phosphocreatine and 115 mM NaCl. Incubation time was usually 5 min; however, when hormonal effects were studied as a function of the hormone concen- tration, the incubation lasted 20min in order to ensure the equilibrium of the interaction between the hormone and the receptor [141. The adenylate cyclase reaction was stopped as already described [I 51 and cyclic [32P]AMP purified according to [16]. When the effects of adenosine were tested, 5pM coformycin was added to the incubation medium to block adenosine deaminase.

Hormonul binding assay

The [3H]vasopressin binding assay was performed as al- ready described using a millipore filtration technique [14]. Total binding was corrected for non-specific binding measured with 2 pM unlabeled [Lysx]vasopressin.

Chenziculs

Nh-~-2-Phenylisopropyl-adenosine (PIA), adenosine de- aminase, creatine kinase and phosphocreatine were from

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Fig. 1. PIA regulatory eifccts on basal and hornzone-stimulated ade- nylate cyclase activities. The cells used were grown in the minimum medium for 11 days and then switched to the supplemented medium 3 days before the adenylate cyclase experiment. The adenylate cyclase assay lasted 5 min; the activity was measured under basal conditions (striped bars), with 10 mM [Lys*]vasopressin (LVP, dotted bars), or with 1 pM [Lys*]vasopressin (white bars). The concentrations of GTP and PIA used were 0 , l pM and 10 pM and of PIA 0,0.3 pM and 30 pM, respectively

Boehringer (Mannheim, FRG), ATP, cyclic AMP and pa- paverine were from Sigma (St Louis, MO, USA). Radiochemi- cals were purchased from NEN (Boston, MA, USA). [Lys']Vasopressin was from Bachem (Buddendorf, Switzer- land). [3H][Lys']Vasopressin was tritiated and purified as already described [I 71.

RESULTS

The effects of two different doses of PIA were compared both on basal and hormone-stimulated adenylate cyclase activities from cells which had been grown in supplemented medium (Fig. 1). In the absence of GTP, 30 pM PIA increased the enzyme activity both under basal conditions or when using an inframaximum dose of hormone. In contrast, 30pM PIA reduced the adenylate cyclase activity when measured with a maximum dose of hormone. The addition of 1 p M GTP enhanced the stimulatory effect of 30 pM PIA on basal enzyme activity. GTP potentiated the stimulatory effect of the two doses of [Lyss]vasopressin which were used. However, in both cases, PIA tended to be inhibitory. The inhibition was more pronounced as the [LysK]vasopressin concentration used in- creased. Finally, when adding a high dose of GTP (10 pM) to the assay medium, as little as 0.3pM PIA inhibited the hormonal response, irrespective of the hormone concentration used. However, the intensity of the observed inhibition was larger for the higher hormonal concentrations used. Under the same experimental conditions (10pM GTP) PIA exerted a biphasic effect on basal adenylate cyclase activity. A low dose of PIA (0.3pM) inhibited the activity while 30pM PIA was stimulatory. To summarize, the results in Fig. 1 showed that: (a) irrespective of the GTP concentration used, 30pM PIA inhibited the maximally hormone-stimulated adenylate cyclase activity; (b) the inhibitory effects of lower doses of PIA were potentiated by the addition of GTP; (c) the extent of the observed inhibition depended on the hormonal dose used; (d) finally, the effects of PIA were biphasic when measured on

245

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Fig. 2. Basal and [LysX]vasopressin-stirnulated adenylate cyclase activities as a function of the PIA Concentration. Effects on cells with low or high [Lys8]vasopressin receptor densities. The adenylate cyclase activity was measured as a function of the PIA concentration with (closed symbols) or without (open symbols) GTP (10 pM). When added, [Lyss]vasopressin concentration was 1 pM (A, A). (A) Cells grown in Coon’s modification of Ham F12 medium; (B) cells grown in supplemented medium (note the differences in ordinate scales). In the absence of any addition, the basal adenylate cyclase activiries for both enzymes were identical

basal adenylate cyclase activity, in the presence of 10 pM GTP (inhibitory then stimulatory).

The specificity for enzyme inhibition and activation by adenosine analogs was in a good agreement with the results described in the literature [3, 8-10]. For the A, receptors (stimulatory) the order of the effectiveness of the different analogs was the following: 5’-N-ethylcarboxamide adenosine 2 PIA > 2-chloroadenosine = adenosine > 5’-deoxyaden- osine + 1 ,Nh-etheno-adenosine. The following order of po- tency was observed for the A, (inhibitory) receptors: PIA > 5’- N-ethylcarboxamide adenosine > 2-chloroadenosine > aden- osine > 3’-deoxyadenosine +- 5’-deoxyadenosine + 1,N6- etheno-adenosine. In addition the inhibitory effects of PIA could be antagonized by the classical antagonists of adenosine A, receptors (3-isobutyl-I -methylxanthine > theophylline > caffeine) (results not shown).

The effects of PIA appeared to differ qualitatively and quantitatively when measured either on basal or on hormone- stimulated adenylate cyclase activities. We took advantage of our ability to enhance the vasopressin response by supplement- ing the growth medium with different hormones and vitamins. The dependency of the enzyme activity as a function of the PIA concentration was carried out on enzyme preparations orig- inating from cells grown either in the minimum medium (Fig.2A) or in the supplemented medium (Fig.2B). Under both growth conditions, basal adenylate cyclase activities behaved similarly and had the same specific activities. In the absence of added GTP, PIA stimulated the basal enzyme activity. However, in the presence of 20 pM GTP, low doses of PIA inhibited the enzyme activity while higher doses of PIA were stimulatory. Whatever the intensity of the vasopressin response we were able to achieve in the absence of GTP, PIA enhanced the enzyme activity over that measured with vaso- pressin alone. However, when GTP was added, low doses of PIA reduced the [Lys8]vasopressin-stimulated enzyme activity. Except for the cells growing in supplemented medium, this inhibition was followed by a stimulation as the PIA con- centration increased. This stimulation can mainly be accounted for by the observed effect of PIA on basal enzyme activity; these effects on basal enzyme activity were additive to those

seen in the presence of hormone. As far as the enzyme response to [Lyss]vasopressin from cells grown in the supplemented medium was concerned, only an inhibition was seen in the presence of GTP. When the PIA concentration was increased in the presence of vasopressin and GTP, the maximum drop in enzyme activity was obtained for PIA concentrations close to those eliciting the lowest enzyme activities under conditions where the PIA effects were biphasic (no added GTP).

The results presented in Fig. 2 were analysed in another way in Fig.3. The increase in enzyme activity due to GTP was plotted as a function of the PIA concentration, both for basal and [Lys8]vasopressin-stimulated adenylate cyclase activities. Increasing the PIA concentration tended to abolish the effects of GTP. Therefore, the inhibitory effects of PIA can only be observed when GTP was added to the assay medium. The stimulatory effect of PIA did not require any addition of GTP and the addition of GTP did not prevent or increase its stimulatory action. Similar results were obtained whatever the hormonal response detected on the enzymes prepared from cells grown under different conditions.

Considering the differential effects measured on both the basal adenylate cyclase activity and on the activity measured in the presence of vasopressin, the effects of PIA and/or GTP were tested on the [3H][Lyss]vasopressin binding (see Table 1). From the Scatchard analysis of hormonal binding data, no significant effect of PIA or GTP could be detected on the maximum binding capacity of the membranes nor on the affinity of the vasopressin receptor.

The dependency of enzyme activity was therefore studied as a function of the GTP concentration as shown in Fig. 4. Basal enzyme activity, with or without PIA, was stimulated upon the addition of GTP. In the presence of PIA, the threshold dose for GTP activation was lowered and a decline in enzyme activity occurred only at high GTP concentrations. At low GTP concentrations, no PIA effect was seen on hormone-stimulated enzyme activity. However, as GTP increased, the activity in the presence of 0.3 pM PIA declined, exhibiting a biphasic depen- dency as a function of the GTP concentration; such an observation could also be made at higher PIA concentrations (30pM). With no PIA added, the dependency of

246

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Table 1. Luck of effects of PIA undjor G T P on the maximum binding capacity and affinity vasopressin for its receptor Results are given k SD

Additions Kd

PIA GTP

PM nM fmol/mg protein

0 0 5.6k2.3 253 11 30 0 6.2 k 1.4 269 k 20 0 10 8.9 2 3.1 320 20

30 10 l . l k1 .5 2692 9 0.3 0 5.62 1.1 246k 8 0.3 10 10 k3.8 322 k 16

[Lys8]vasopressin-stimulated adenylate cyclase activity reach- ed a plateau as a function of the GTP concentration. In Fig.4B the increase in adenylate cyclase activity due to [Lys8]vasopressin as a function of the GTP concentration was plotted. The higher the PIA concentration, the lower was the hormonal effect. The concentrations of GTP yielding the maximum response to hormone were similar at both PIA concentrations tested. As PIA concentration increased the absolute decrease in enzyme activity was enhanced when compared to control conditions (no PIA added).

Therefore, the effects of PIA (0,0.3 pM and 30 pM) with or without 10pM GTP were studied as a function of the [Lys8]vasopressin concentration in the assay medium (Fig. 5). For such a study, cells grown in supplemented medium were used. The results obtained on basal adenylate cyclase activity were similar to those described in Fig. 1 and 2. In the absence of GTP, the addition of 0.3 pM PIA did not make any important difference to the dependency of the enzyme activity as a function of the [Lyss]vasopressin concentration (Fig. 5). However, when using 30 pM PIA, the inhibitory effect of PIA developed but only at high [Lys8]vasopressin concentrations.

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Fig. 4. PIA-induced inhibition of the adenylate cyclase response to (Lysa]vasopressin as a function of the G T P concentration. (A) The basal adenylate cyclase activity was measured under control conditions ( x ) or with 30pM PIA added (0). The enzyme activity was also measured in the presence of 1 pM [Lys8]vasopressin with the following PIA concentrations: 0 (o), 0.3 pM (A----A) and 30pM (0). (B) The increase in activity due to [Lys*]vasopressin was plotted as a function of the GTP concentration at a PIA concentration of (0) 0,0.3 pM and (0) 30PM

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Fig. 5. Dependency of the adenylate cyclase activity as a function of the [Ly~~jvasopress in concentration: effects of PIA and GTP. The ade- nylate cyclase activity was measured as a function of the [Lys*]vasopressin concentration at three different PIA concentrations: 0 (o), 0.3 pM (.---+) and 30 pM (A). (A) No GTP was added; (B) l n IIM GTP WRP nrecpnt in the RCPRV The riirvm rpnrecent the inrrencr

in enzyme activity due to the hormone. Basal adenylate cyclase activity is represented by the bars on the graphs

When GTP was added to the assay medium, 0.3 pM or 30 pbf PIA had similar effects on the absolute increase in enzyme activity due to vasopressin (see also Fig. 2). The effects observed on basal adenylate cyclase activity were comparable to those described in Fig. 1.

Differences in the dependency of the enzyme activity as a function of the vasopressin concentration were identified, as shown in Fig. 6. The increases in enzyme activity due to the hormone (results from Fig. 5 ) were plotted according to Eadie. When no PIA was added, with or without GTP, the enzyme had a Michaelian behaviour pattern (monophasic plot). However, the Eadie plot of the results revealed a heterogeneity in the enzyme activity when either high PIA concentrations were used in the absence of GTP, or for both PIA concentrations in the presence of GTP. This corresponded in both cases to conditions where the inhibition of the [Lys8]vasopressin response could be demonstrated at high concentration of the hormone. Curve fitting of the data obtained for the non-linear plots gave values for the apparent affinities of the hormone of 10pM/I and 0.45nM/1. In both cases, the ratio of the velocity of high- affinity to low-affinity forms of the enzyme was equal to 0.45.

Taking advantage of our ability to grow cells in defined media, we grew cells in media containing 1 IU/ml adenosine deaminase with or without 30 pM PIA. (Control experiments showed that cells grown with or without adenosine deaminase behaved similarly with respect to the parameters studied; therefore cells grown with this enzyme were used as control cells for comparing the effects observed on cells grown with PIA and the enzyme.) The data presented in Table 2 show the effects of these different growth conditions on basal and hormonally stimulated adenylate cyclase activities with or without GTP (10 pM) and/or PIA (30 pM). As mentioned above, the results obtained either under control conditions or with cells grown in the presence of adenosine deaminase were similar. However, due to the addition of PIA and this enzyme to the cell growth medium, the following observations can be made : (a) basal adenylate cyclase activity measured with or without GTP was not altered upon intact cell treatments; (b) basal enzyme activity from cells grown with both adenosine deaminase and PIA lost most of its sensitivity to the stimulatory action of PIA

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Table 2. Effects of growing cells with PIA on the modulation of the vasopressin response by PIA ADA = adenosine deaminase ~- ~

Enzyme activity GTP PIA CAMP production with additions to cell growth medium

ADA + PIA none ADA

- Basal -

+ + Increase in activity due to 1 pM [Lys8]vasopressin -

-

+ +

pmol in 5 min/mg protein

53 58 195 233 131 118 292 286 412 446 339 315 514 526 278 259

52 64

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-10 -9 -8 -7 -10 -9 -8 -7 log ( ILy~~ lVosopress in concn/M)

Fig. I . Adenylate cyclase response as a function of (Lys8/vasopressin concentration in cells treated with adenosine deaminasee, with or without PIA. The cells were grown as indicated under Materials and Methods with (A) adenosine deaminase alone or (B) with adenosine deaminase and 30 pM PIA. The increase in adenylate cyclase activity due to [Lys*]vasopressin was plotted as a function of the hormone concentration using different adenylate cyclase assay conditions: (0----4) no GTP, no PIA; (O---+) no GTP, 30pM PIA; (A-A) 10pM GTP, no PIA; (A----A) 10pM GTP, 30 pM PIA. The curves represent the increase in adenylate cyclase activity due to [Lysx]vasopressin. The bars represent the basal adenylate cyclase activity (no hormone added) using the same combinations of GTP and PIA

with and without GTP. The addition of adenosine deaminase to the cell growth medium did not alter the response to vasopressin, GTP and PIA, alone or in combination. However when the cells were grown in the presence of both adenosine deaminase and PIA, the inhibitory effects of PIA on the vasopressin response were greatly reduced, even when GTP was added. Such results were suggestive of a desensitization process of both stimulatory (as measured on basal adenylate cyclase activity) and inhibitory effects of PIA (as measured on the hormonal response) upon a prior exposure of cells to PIA.

It was therefore of interest to check whether or not the low- affinity or the high-affinity components of the adenylate cyclase response to [Lys*]vasopressin were altered, as shown in Fig.7. When cells were grown in the presence of adenosine deaminase, the results obtained were in a good agreement with all those described above (see Fig. 5 and 6). However, growing cells with both PIA and adenosine deaminase induced a different pattern. The addition of GTP and/or PIA in the adenylate cyclase assay medium no longer altered the shape of

the dependency of the enzyme activity as a function of the concentration of [Lys*]vasopressin. All four curves were sim- ilar in shape, differing slightly by their maximum velocity. They all exhibited the same apparent affinity for the hormone. In no instances was there an indication of an enzyme heterogeneity (as shown in Fig. 6).

Results (not shown) indicated that with respect to the experiments described above, the enzyme partially desensitized to [Lys*]vasopressin behaved similarly to the control one.

DISCUSSION

As for many other adenylate cyclase systems, the existence of both A, (inhibitory) and A, (stimulatory) receptor sites for adenosine [I - 61 has been demonstrated in LLC-PK,,> cells. The requirements for demonstrating the inhibitory effects varied from one tissue to another. In this cell line, the addition of GTP was a necessary prerequisite for demonstrating such an

249

inhibition. In fact, PIA was found to inhibit the increment in enzyme activity due to GTP both on basal and on maximally stimulated adenylate cyclase activities. Therefore PIA ap- peared to act as a regulator of GTP action. Due to the addition of PIA, an heterogeneity in the adenylate cyclase response to vasopressin could be demonstrated. This allowed the de- monstration of a potentiation of the hormonal response at very low and physiological vasopressin concentrations. Such poten- tiating effects of adenosine on the hormonal responses were described for other hormone-sensitive adenylate cyclase sys- tems but in intact cells [18, 191. It is tempting to speculate that adenosine might act as a double regulator of hormonal action by enhancing low hormonal effects and blunting higher ones. Such effects might, however, not be a general feature of all adenylate cyclase systems.

Preliminary experiments (not shown) using the LLC-PK, cells (i.e. the parent cell line of the cells used in this study which grow in the presence of serum) showed that: (a) the vasopressin stimulation of the adenylate cyclase was biphasic; (b) the inhibitory effects of vasopressin developed only at high GTP concentrations; (c) the relative magnitude of the inhibitory effect increased as the vasopressin concentration was en- hanced; (d) these cells were not responsive to PIA. Therefore this cell line was apparently able to regulate the vasopressin action by bypassing the adenosine pathway. Such a loss of the adenosine regulation in these cells might be a consequence of growing the cells in the presence of fetal bovine serum (the addition of serum had been shown to modify some biochemical properties of various cell lines [12, 20-221). This further strengthened the validity of using, whenever possible, chemi- cally defined media to study hormonal regulations. In these experiments such a possibility helped greatly in differentiating between the PIA action on basal adenylate cyclase activity from those measured on hormonally stimulated enzyme. Such an analysis was rendered possible by our ability to modulate the vasopressin response upon changing the cell culture conditions and the fact that basal adenylate cyclase activity had similar characteristics for both induced or non-induced cells.

Manipulation of the cell growth medium composition, which increased the number of vasopressin binding sites, allowed the measurement of [3H][Lyss]vasopressin binding. In none of the experimental conditions tested was an effect on hormonal binding observed. Therefore the following dis- cussion will be focused on steps beyond the hormone-receptor interaction. Finally, the ability to use defined media allowed modifications of the cell growth medium under carefully controlled conditions, such as the possibility of adding aden- osine deaminase and/or PIA to the cell growth medium.

In the following discussion the different molecular species are tentatively referred to, for brevity, as: R, and R,, re- spectively, for the stimulatory and inhibitory adenosine re- ceptors; N, and N,, respectively, for the stimulatory and inhibitory regulatory components of the adenylate cyclase, C. RH represents the vasopressin-receptor complex. Since we did not measure the binding of adenosine (or its analogues) we must assume that, in the absence of GTP, either the affinity of R, for adenosine was lower than that of Ri for adenosine or that R, cannot be negatively coupled to C. In the presence of low doses of GTP, the apparent affinity of R, was enhanced as well as its intrinsic activity. The biphasic action of GTP observed suggests that the affinity of N, for GTP was higher than that of N,. Such data may be interpretated in two ways: either N, and N, compete for an interaction with C, or R, and R, compete for N, which in turn activates C, R, having a lower intrinsic activity than R,. This latter hypothesis does not hold since even basal

adenylate cyclase activity could be inhibited, unless N, and N, interact with C without having been coupled to any receptor.

When the membranes were prepared from low-density or high-density vasopressin receptor cells, in the abscence of added GTP, the actions of vasopressin and PIA were additive, suggesting eventually two distinct mechanisms of activation, or that C was present in a sufficient amount to allow the expression of both types of receptors (RH and %).

The differences in the pattern of the dose dependencies as a function of either GTP or PIA suggested that R, had a better affinity than R, for PIA and that RH had a higher affinity for N, than for Ni. Therefore, the differences in the potentiating effects of GTP might simply reflect the facts that the affinity of RHN, for GTP was higher than that of RHN,. In the case of PIA, the differences in affinity might be too low to elicit such a potentiation. Such a conclusion might also lead to the hy- pothesis that both vasopressin and adenosine binding could induce, not only the activation of adenylate cyclase via N,, but also a (simultaneous) inhibition via N, of C. The level ofenzyme activity would be the result of a balance between activation and inhibition.

Taking into account that both RH and R, might not have the same intrinsic efficiency either for inhibition or stimulation, the inhibition of vasopressin action could be explained on the basis of a competition between RHN, and RN, for C and/or that RH has an affinity for N, different from that of R, for N,.

Therefore, if we now make the assumption that there is only one single adenosine receptor, we can account for the results described. The existence of adenosine analogues which are selectively either stimulatory or inhibitory may only reflect a difference in their absolute intrinsic activity. Such observations might be, depending on the hormonal system considered, a consequence of the integrity of the coupling step after homoge- neization as well as possible alterations in receptor binding characteristics following cell disruption. In addition in most systems, there was no indication of an heterogeneity in adenosine binding, suggesting the existence of a single class of adenosine receptors [23 - 261. Along with this hypothesis, the results show that both stimulatory and inhibitory effects of PIA disappeared upon intact cell exposure to PIA. Such an observation, albeit not formal, might be taken as evidence for the desensitization of the same receptor.

The distinction between A, and A, receptors was made on the basis of their respective ability to inhibit or stimulate the adenylate cyclase activity upon occupancy by adenosine ana- logues. This discrimination upon the choice of analogues still implies that the physiologically relevant ligand is adenosine. It might be suggested that, as for [Lys8]vasopressin in the LLC- PK, cells or for isoproterenol in fat cells [27], each ligand which is able to be coupled to the adenylate cyclase per se is able to interact with N, and/or N, via its receptor. Each ligand may have a different ratio K, for NJK, for N,. In addition, inhibitory ligands may be those which have lost all their stimulatory properties (the K,,, of R, for N, being much larger then the K , of R, for N,) while the reverse holds for stimulatory ligands. Such a conclusion could be supported by the fact that, in a given system, only a single homogenous population of binding sites was detected [23 - 261. This is a logical conclusion if the interaction of the receptor with either N, or N, was hampered (or if such a ligand had antagonistic properties, yet the definition of an antagonist has to be carefully checked since it may involve the antagonism of either a stimulation or an inhibition) [4].

In addition to the above discussion, the existence of the desensitization of an inhibitory receptor coupled to an in-

hibition of the adenylate cyclase has apparently not been demonstrated. Since both activating and inhibitory effects of PIA could be desensitized upon a prior exposure of cells to this compound, such an observation further supports the existence of a single class of receptors mediating both effects. In addition, all the data presented have are consistent with the data obtained by Ui et al. [27] on the reversal of the adenylate cyclase inhibition elicited by an activating ligand in the presence of GTP and pertussis toxin.

We wish to thank Michele Benoit and Angie Turner for typewrit- ing assistance as well as M. N. Balestre for technical assistance. This word was supported by the Centre National de la Recherche Scientifique and by the Institut National de la Santt et de la Recherche Medicale.

REFERENCES

1. Jakobs, K. H., Saur, W. & Johnson, R. A. (1979) Biochim.

2. Haslam, R. J. & Lynham, J. A. (1972) Life Sci. 11, 1143-1154. 3. Londos, C. & Wolff, J. (1977) Proc. Natl Acad. Sci. USA 74,

4. Londos, C., Cooper, D. M. F. & Wolff, J. (1980) Proc. Nut/ Acad.

5 . Birnbaummer, L., Nakahara, T. & Yang, P. C. (1974) J . Biol.

6. Lad, P. M., Nielsen, T. B., Londos, C., Preston, M. S. & Rodbell,

7. Steer, M. L. & Wood, A. (1979) J. Biol. Chem. 254,10791 - 10797. 8. Premont, J., Perez, M., Blanc, G., Tassin, J. P., Thierry, A. M.,

Herve, D. & Bockaert, J. (1979) Mol. Pharmacol. 16,790- 804.

Biophys. Acta 583, 409 - 421.

5482 - 5486.

Sci. U S A 77, 2551 - 2554.

Chem. 249, 7857- 7866.

M. (1980) J. Bid. Chem. 255, 10841 - 10846.

9. Premont, J., Perez, M. & Bockaert, J. (1977) Mol. Pharrnacol. 13,

10. Cooper, D. M. F., Londos, C. & Rodbell, M. (1980) Mol.

11. Van Calker, D., Muller, M. & Hamprecht, B. (1979) J . Neurochem.

12. Roy, C., Preston, A. & Handler, J. S. (1980) Proc. Natl Acad. Sci.

13. Coon, M. G. & Weiss, M. C. (1969) Proc. Natl Acad. Sci. USA 62,

14. Bockaert, J., Roy, C., Rajerison, R. & Jard, S. (1973) J . Biol.

15. Roy, C. (1979) Biochim. Biophys. Acta 587, 433-445. 16. Salomon, Y., Londos, C. & Rodbell, M. (1976) Anal. Biochem. 58,

17. Pradelles, P., Morgat, J. L., Fromageot, P., Camier, M., Bonne, D., Cohen, P., Bockaert, J. & Jard, S. (1972) FEBS Lett. 26,

662 - 670.

Pharmacol. 18, 598 - 601.

33, 999-1005.

USA 77, 5979- 5983.

852- 859.

Chern. 248, 5922-5931.

541 -548.

189 -202. 18. Wolff, J. & Cook, G. H. (1977) J . Biol. Chem. 252, 687-693. 19. Hall, A. K., Preston, S. L. & Behrman, H. R. (1981) J . Biol. Chem.

20. Heldin, C. H., Wasteson, A. & Westermark, B. (1980) Proc. Nut1

21. Murakami, M., Masui, H., Sato, G. & Raschke, W. C. (1981)

22. Fukase, M., Birge, S. J., Jr, Rifas, L., Avioli, L. V. & Chase, L. R.

23. Murphy, K. M. M. & Snyder, S. M. (1982) Mol. Pharmacol. 22,

24. Williams, M. & Risley, E. A. (1980) Proc. Natl Acad. Sci. USA 77,

25. Trost, T. & Schwabe, U. (1981) Mol. Pharmacol. 19, 228-235. 26. Goodman, R. R., Cooper, M. J., Gavish, M. & Snyder, S. H.

27. Murayama, T. & Ui, M. (1983) J . Biol. Chem. 258, 3319-3326.

256, 10390-10398.

Acad. Sci. USA 77, 6611 -6615.

Anal. Biochern. 114, 422 - 428.

(1 982) Endocrinology 110, 1073 - 1075.

250 - 257.

6892- 6896.

(1982) Mol. Pharmacol. 21, 329- 335.

C. Roy, Centre de Pharmacologie-Endocrinologie du Centre National de la Recherche Scientifique, Rue de la Cardonille, Boite postale 5055, F-34033 Montpellier-Cedex, HCrault, France