Prostaglandin E1-induced desensitization of prostaglandin-sensitive adenylate cyclase of cultured mammalian cells

Printed in Sweden Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved 0014.48?7/79/0?0??9-07%0?.00/0

Experimental Cell Research 118 (1979) 229-235

PROSTAGLANDJN

El-INDUCED

PROSTAGLANDIN-SENSITIVE CULTURED

DESENSITIZATION ADENYLATE

MAMMALIAN

OF

CYCLASE

OF

CELLS

Studies with Broken and Intact Cell Preparations S. R. AYAD and G. R. .I. BURNS Department of Biochemistry, lJni\lersity of Manchester, Manchester, Ml3 9PL, UK

SUMMARY Prostaglandin El-induced desensitization of prostaglandin-sensitive adenylate cyclase has been investigated in intact and broken-cell preparations of two cell lines, P388F-36and PCM,, P388F-36 cells may be characterized by their low intracellular CAMP concentration after exposure to the hormone, whereas PCM, cells accumulate high concentration of CAMP under similar conditions. Broken-cell preparations from both cell lines exhibit a similar prostaglandin-sensitive adenylate cyclase activity. When the activity is examined after prior exposure of intact cells to hormone, the nature of and extent of desensitization is quite distinct in these two cell lines. In PCM, cells, desensitization proceeds rapidlv to completion with no change in the apparent affinity for prostaglandin E;, whereas in P388F-36, thk maximum extent ofhesensitiza& is only 30;lO%, although a IO-fold decrease in affinity for the hormone is observed. GTP and Gup(NHL effects are identical in control and desensitizei preparations. These results are discussed in relation to the regtilation of adenylate cyclase in the two cell lines.

The selective decrease of hormone responsiveness following exposure to hormones has been demonstrated in a variety of cells [l-4]. This phenomenon of desensitization appears to be quite specific since exposure to a particular hormone does not lead to loss of sensitivity to other hormones. Recent studies have indicated that both binding of hormone to receptors and functional coupling of the hormone-receptor complex with adenylate cyclase are required to produce the desensitized state, since hormone analogues which bind to receptors but do not lead to activation of adenylate cyclase are without effect, and moreover, can protect the system from desensitization in the presence of hormone

[51. I6 - 7XIXO3

Various explanations have been proposed to account for this phenomenon. Increased cyclic nucleotide phosphodiesterase seems not to be involved in hormone specific desensitization [6-8], although the activity of this enzyme often increases following incubation of cells with hormone [8-91. Several recent reports however, have provided strong evidence for a decrease in high affinity hormone binding sites following prior incubation with hormone [4-6]. It has been shown that a somatic cell hybrid, PCM1, produced by fusion of Chinese hamster fibroblasts and a mouse lymphoma P388F-36, produced much higher concentrations of CAMP in response to incubation with PGE, than either parental cell line [2]. Moreover, the magnitude of the E.\/’(‘e/lKl,.\I lx , /Y7Y)

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Figs 1, 2. Abscissa: incubation time (mm); ordinate: CAMP (pmoleslmg protein). Fig. I. Time course of CAMP production in PCM, broken-cell preparations. O-0, Assayed with 3 FM PGE,; O-O, basal act. Points shown are the means of duplicate assays.

response in each cell line, shows a qualitative inverse correlation with the cyclic nucleotide phosphodiesterase activity. Intact P388F-36 cells exhibit an extremely poor response to PGEi, whereas brokencell preparations exhibit a highly responsive hormonally stimulated adenylate cyclase activity. Recent reports have indicated that broken-cell preparations from refractory cells maintain their refractoriness [6, 71. In order to compare desensitization in the responsive PCM, cell line and the non-responsive P388F-36 cell lines, we have therefore investigated the phenomenon using a combination of intact and broken-cell preparations. MATERIALS

AND METHODS

Cell culture P388F-36, a mouse lymphatic luekaemia cell line, and PCM,, a somatic cell hybrid produced by fusion of P388F-36 and CH 23 Chinese hamster fibroblasts [IO] were grown in Eagle’s Minimal Essential Medium supplemented with 10% newborn calf serum at 37°C in an atmosphere of 5 % CO* and 95 % air. The origins and hormonal responsiveness of these cell lines have been described previously [2, IO].

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Fig. 2. Time course of CAMP production in P388F-36

broken-cell preparations. O-O, Assayed with 9 /.LM PGE,; O-O, basal act. Points shown are the means of duplicate assays.

Preparation

of cell homogenates

In the present experiments, cell were seeded at 1-2~ IO5 cells in 200 ml glass medical flats. When PCM, cells were confluent the monolayer was scraped off the glass with a rubber policeman. For both cell types, cells were then collected by centrifugation at 4°C (150 g, 5 min) and washed twice in buffer by resuspension and centrifugation and finally resuspended in 1 ml of the buffer (50 mM Tris-HCI, pH 7.9, containing 0.25 M sucrose, 5 mM Mg Cl,, 2 mM P-mercaptoethanol and 1 mM ethyleneglycol-his @-amino-ethyl ether) N,N’-tetraacetic acid). Cells were then homogenized as described previously [ 1I] and the homogenate diluted in buffer (IO ml) and centrifuged at 4°C (40000 g, 15 min). The supernatant was discarded and the pellet resuspended in buffer by gentle homogenization. The process was repeated twice and the pellet was finally resuspended in approx. 1 ml of adenylate cyclase assay buffer.

Assay of adenylate cyclase Adenylate cyclase reactions were initiated by the addition of 50-100 pg of protein to a final vol of 200 ~1

Desensitization of prostaglandin-sensitive adenylate cyclase

231

20

10

Fig. S. Abscissa: % desensitization; ordinate: time of pre-incubation with 30 PM PGE, (hours). O-O, PCM,; O-O, P388F-36. Time course of desensitization. Results are the means of duplicate assays of duplicate pre-incubated bottles. Percentage desensitization is defined as: 1-[(PGE,-stimulated act, of desensitized preparation -basal act)/(PGE,-stimulated act, of control preparation - basal act)] x 100. containing 50 mM Tris/HCI, pH 7.4 at 30°C; 2 mM ATP, bovine serum albumin, 0.1 mglml, 0.1 mM EGTA, I mM I-methyl-3-isobutyl xanthine, 5 mM MgCI,, 2 mM /3-mercaptoethanol and an ATP-regenerating system (20 U/ml phosphocreatine kinase and IO mM creatine phosphate). Prostaglandin E,, GTP and guanylylimidophosphate (Boehringer) were included in the assay as appropriate. Incubations were generally carried out for IO min at 30°C although in some experiments the incubation time was varied up to 2 h. Reactions were terminated by the addition of 200 ~1 of 0.2 M HCI. After heating at 90°C for 60 min to hydrolyse the remaining ATP, 100 ~1 of 2 M TrisHCI, pH 7.4, was added and the CAMP content was determined bv a competitive protein binding assay [ 121.Endogenbus CAMP in the preparations was determined in samples of homogenate to which 0.2 M HCI was added before ATP, and these values were subtracted from the total CAMP measured after incubation at 30°C.

Densensitization in broken-cell preparations Broken-cell preparations were prepared and incubated with ATP and PGE, as described above. At varying intervals of time, the incubation mixtures were diluted with ice-cold buffer and centrifuged at 4°C (40000 g, I5 min). The supernatant was discarded and the pellet resuspended in buffer by gentle homogenization. The process was repeated twice and the pellet was finally resuspended in cyclase assay buffer and assayed as described above.

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Fig. 4. Abscissa: adenylate cyclase act. (pmoles cAMP/mg proteinlmin); ordinute: PGE, cont. (PM). O---O, Control assayed without GTP; O---O, intact cells pre-incubated for 40 min with 3 FM PGE,; n---n, intact cells pre-incubated for 4 h with 3 PM PGE,; O-O, control assayed with 50 PM-GTP; m-m, intact cells pre-incubated for 40 min with 3 PM PGE, assayed with 50 PM GTP; A-A, intact cells pre-incubated for 4 h with 3 FM PGE,. assayed with 50 PM GTP. PGE, dose-response curves for control and desensitized broken-cell preparations of PCM,.

Pre-incubation and refractory broken-cell preparations At confluencv or limiting cell density, PGE, was added to cell cultures at an appropriate concentration. Cells were then incubated at 37°C for varying intervals of time, and homogenised and assayed as described above.

Protein determinations All protein determinations were performed using the method of Lowry et al. [l3], with bovine serum albumin as standard.

RESULTS Time-course of CAMP accumulation by broken-cell preparations Figs 1 and 2 show the time course of CAMP accumulation by broken-ceil preparations of PCM, and P388F-36. It can be observed that the basal activity of PCM, adenylate ‘cl/’(‘c/IKC\/ I8, /Y7Y)

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Fig. 5. Abscissa: adenylate cyclase act. (pmoles cAMP/mg protein/min); ordinate: PGE, cont. (PM). O-O, Control; O-O, intact cells pre-incubated for 4 h with 3 PM PGE,. PGE, dose-response curves for control and desensitized broken-cell preparations of P388F-36.

cyclase is linear up to 3 h, whereas the hormonally stimulated activity decreases after 40-60 min. In the case of P388F-36 cells, both basal and PGE,-stimulated activities decrease after an initial linear phase, although the hormonally-stimulated enzyme activity begins to decrease earlier than the basal activity (30 min compared with 100 min). Since an ATP-regulating system was included in the incubation mixture, it is unlikely that the decreases in enzyme activity are due to a decrease in substrate concentration. The stability of the regenerating system is demonstrated by the maintenance of linearity for PCM,, basal activity over the whole time course of the experiments. In PCM, the decrease in hormonally-stimulated activity seems therefore to be clearly associated with hormone-specific desensitization. In P388F-36 however, the decrease in basal activity makes such a conclusion less certain. The lag of 30-60 min before the onset of the decrease in hormonally stimulated adenylate cyclase may be compared with the results of Roy et al. [14], where a lag of about 20 min was ob-

served before the onset of desensitization of vasopressin-sensitive adenylate cyclase of pig-kidney membranes. In some experiments, the crude particulate preparations were incubated as described above and then washed by repeated centrifugation and resuspension as described above. However, significant technical problems complicated attempts to study desensitization by this means, since extensive losses (>90%) in both basal and hormonally stimulated activity were observed with preparations pre-incubated with or without PGE,. A similar problem has recently been reported with frog erythrocyte membranes [ 151. Pre-incubation in intact cells and assay of broken-cell preparations

Due to the problems described above, further studies were carried out by pre-incubation of intact cells with hormone, and subsequent assay of particulate preparations from these cells. Time-course of desensitization

Fig. 3 shows the time-course of desensitization in PCM, and P388F-36 cells,. It can be observed that desensitization in PCM, cells proceeds rapidly to completion without a detectable lag. The time course is very similar to that observed when both pre-incubation and assay of responsiveness are carried out in intact cells [2]. In P388F-36, the poor hormone-responsiveness of the intact cells prevents the direct examination of desensitization. However, as seen in fig. 3, broken-cell preparations from cells pre-incubated with PGE, do show a decreased hormone responsiveness compared with control preparations. It can be seen that this desensitization is neither as rapid nor as extensive as that observed in PCMl cells, approaching only

Desensitization

of prostaglandin-sensitive

30-40% desensitization after 4 h pre-incubation. Experiments in which the pre-incubation time was extended to 18 h indicated that no significant further increase in the extent of desensitization could be observed, although a decreased basal activity was noted after these extended incubation times, which complicated the interpretation of the data (not shown).

adenylate cyclase

233

of GTP in assays of control or desensitized preparations of P388F-36 in the present study was also without effect, although guanylyl imidophosphate increased activity to the same extent in both control and desensitized preparations (data not shown). DISCUSSION

Although the phenomenon of desensitization is readily observed in most intact cells, detailed investigations of the mechanism and nature of the process are difficult due In order to further investigate the differ- to the inability to manipulate experimental ences in desensitization between PCM, and conditions sufficiently. Several studies have thus been directed P388F-36 cells, the dose-response of control and refractory cell preparations was ex- towards investigations of the phenomenon in broken-cell or purified plasma memamined (figs 4, 5). Fig. 4 shows the dose-response curves brane preparations [5, 14, 171. However, for control and refractory PCM, prepara- incubation of frog erythrocyte membranes tions. It can be seen that desensitization is followed by washing procedures led to 90 % manifested by a decrease in the maximum losses in PGE,-sensitive adenylate cyclase activity of adenylate cyclase with no change [15] and similar technical problems were in the apparent dissociation constant for encountered in the present study. AccordPGE, (1.6?0.2~10-’ M). In view of some ingly we have adopted two alternatives for reports that GTP can reverse desensitiza- investigating the phenomenon, namely foltion [5] dose-responses were also examined lowing the time course of CAMP production in the presence of GTP. It can be seen that by broken-cell preparations, and the dethe response to GTP is essentially un- velopment of refractoriness in intact cells changed in desensitized preparations and followed by assay of broken-cell preparaalso that GTP is unable to reverse desensi- tions. Several reports have previously demonstrated that such preparations retain their tization under these conditions. In contrast to PCM, cells, P388F-36 cells inability to respond to hormone [6, 71. showed only a modest decrease in the maxWhen the time course of CAMP producimum adenylate cyclase activity although a tion by broken-cell preparations of PCM, pronounced increase in the apparent dis- was investigated, PGE,-stimulated activity sociation constant (Kd) was observed. Fig. was linear for 40-60 min, after which the 5 is representative of the results obtained hormone-stimulated activity rapidly deand similar decreases in affinity were ob- clines so that after 3 h, the rate of CAMP served in three separate experiments (con- production approached the basal rate which trol &=2.8*1x 10es M; desensitized Kd was constant over the whole of the 3 h 2.1*0.6x lo-’ M). Neither basal nor PGE,- period. The lag before the onset of desenstimulated adenylate cyclase of P388F-36 sitization is similar to that observed for the desensitization of vasopressin-sensitive cells are responsive to GTP [16]. Inclusion

PGEl dose-response of particulate fractions prepared from control and refractory cells

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Ayad and Burns

adenylate cyclase of pig-kidney membranes

1141. Although both basal and PGE,-stimulated adenylate cyclase activities of P388F36 broken-cell preparations were initially linear, both showed a pronounced subsequent decline, although the onset of this decrease occurred earlier for the hormonally stimulated system than for the basal (30 min vis-a-vis 80 min). The basis for this decrease in basal activity is not known, but this observation makes interpretation of the desensitization difficult in this cell line. When intact PCM, cells were pre-incubated with PGE, the time course of desensitization as measured in broken-cell preparations was similar to that observed when intact cells were assayed after pre-incubation [2]. No detectable lag was observed, in contrast to the studies in broken-cell preparations. However, a pronounced lag of l-2 h before the onset of desensitization was much less extensive than for PCM, cells even after protracted pre-incubation times. This lag and the smaller extent of desensitization may reflect the low intracellular concentrations of CAMP which accumulate in P388F-36 cells during incubation with the hormone in contrast to the high intracellular concentrations observed in PCM, cells after similar incubations [2]. If this is the explanation of the differences, it would suggest that CAMP itself may play an important role in the desensitization process. It is pertinent to note that high ATP concentrations are required to elicit desensitization in certain membrane preparations [14, 171, although in frog erythrocytes, GTP, ATP and several other nucleotide have been shown to reverse the desensitization. There thus appears to be quite distinct differences in the mechanism of desensitization in difference cell types. In the present studies, no reversal of desensitiza-

tion was observed when GTP was included in the assays. A most unusual finding was the apparent decrease in affinity for PGE,, in P388F-36 cells after pre-incubation with the hormone (fig. 5). In PCM, as in other studies of the phenomenon, only the maximum extent of activation was affected, the apparent Kd remaining unchanged. Previous studies of the adenylate cyclase system of P388F-36 cells have indicated that whilst broken cells respond well to PGE,, intact cells have a very poor response [2]. The presence of specific high affinity PGE, receptors has also been demonstrated in plasma membranes from this cell line [ 111. These cells have a high phosphodiesterase activity [2, 181 which could account for the poor response of the cells, but the possibility that the hormone receptors were in some way inaccessible or unable to form a functional complex with the hormone in intact cells could not be discounted. However, the observed changes in the response to PGE, of broken-cell preparations from intact cells pre-incubated with the hormone, strongly suggest that intact cells are able to bind hormone to adenylate cyclase-associated receptors. The low degree of decrease in the maximum response and the change in apparent affinity of the receptors, in contrast to PCM1, may indicate, however, that binding does not lead to the same changes in the receptor-enzyme complex which accompany binding in normal responsive cells. Lefkowitz et al. [15] have reported that despite the extensive losses in PGE,-sensitive adenylate cyclase described above, no such losses are observed when the binding of the hormone to prostaglandin E, receptors is studied. If binding to receptors of PCM, and P388F-36 cells is similarly unaffected by pre-incubation, such studies may allow further progress to be made in

Desensitization

of prostaglandin-sensitive

our understanding of the desensitization phenomenon in these cell lines. The authors wish to express their thanks to Professor G. R. Barker for providing the facilities for this work and to Miss J. Worthington for secretarial assistance. Financial support from the Cancer Research Campaign and the Medical Research Council is gratefully acknowledged.

REFERENCES I. 2. 3. 4.

Franklin, T J & Foster, S J, Nature 246 (1973) 146. Ayad, S R & Foster, S J, Cell 3 (1974) 135. Remold-O’Donnell, E, J biol them 249 (1974) 3615. Mukherjee, C, Caron, M G & Lefkowitz, R J, Proc natl acad sci US 72 (1975) 1945. 5. Mukherjee, C & Lefkowitz, R J, Proc natl acad sci US 73 (1976) 1494. 6. Shear, M, Insel, PA, Melmon, K L & Coffino, P, J biol them 251 (1976) 7572. 7. Franklin, T J & Twose, P A, FEBS lett 66 (1976) 225.

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8. Newcombe, D S, Ciosek, C P, Sshikawa, Y &

Fahey, J V, Proc natl acad sci US 72 (1975) 3124. 9. Browning, E T, Brostrom, C 0 & Groppi, V E, Mol pharmacol I2 (1976) 32. IO. Ayad, S R & Delinassios, J G, Biochem genet I2 (1974) 147. Il. Ayad, S R &Burns, G R J, Exp cell res 108(1977) 12. Brown, B L, Albano, J D M, Elkins, R P & Sgherizi, A M, Biochem j 121 (1971) 561. 13. Lowrv. 0 H. Rosebrouah. N J. Farr. A L & Randall, R J, J biol chemi93 (l95J) 265.’ 14. Roy, C, Guillow, G & Jard, S, Biochem biophys res commun 72 (1976) 1265. 15. Letkowitz, R J, Mullikin, D, Wood, C L, Gore, T B & Mukherjee, C, J biol them 252 (1977) 5295. 16. Ayad, S R & Foster, S J, Exp cell res 109 (1977) 87. 17. Bockaert, J, Hunzicker-Dunn, M & Birnbaumer, L, J biol them 251 (1976) 2653. 18. Ayad, S R & Wright, J F, Biochem genet 15 (1977) 1001. Keceived June 19, 1978 Accepted August 24, 1978

Exp Cell Res II8 ( 1979)