PROSTAGLANDINS

DESENSITIZATION OF PROSTAGLANDIN-ACTIVATED PLATELET ADENYLATE CYCLASE

Barry Cooper, Andrew I. Schafer, David Puchalsky, and Robert I. Handin

Division of Hematology, Department of Medicine, Peter Bent Brig- ham Hospital, West Roxbury Veterans Administration Hospital and Department of Medicine, Harvard Medical Sdhool, Boston, MA.

ABSTRACT

Prostaglandin D2 (PGD2) is one of several prostaglandins that can inhibit platelet aggregation and activate adenylate cyclase. Platelets were exposed to varying concentrations of PGD2, washed, and the adenylate cyclase response to prostaglan- dins, epinephrine, and sodium fluoride determined. platelets with 5 x 10-5

Incubating M PGD2 for 2 hr resulted in a 45% decrease

in PGD2 activation of adenylate cyclase and a 25% decrease in stimulation by PGEl. Fluoride activation (‘l-fold) epinephrine inhibition (30%) and basal enzyme activity were unchanged by expo- sure of the platelets to PGD2. Desensitization was concentration dependent, with loss of enzyme activity first noted when platelets were incubated with 10e7 M PGD2. Enzyme sensitivity could be par- tially restored when desensitized platelets were washed free of PGD2 and incubated in buffer for 2 hr; complete resensitization required incubation for 24 hr in plasma. Regulation of prosta- glandin sensitive platelet adenylate cyclase could be of impor- tance in mediating the response of platelets to aggregating agents.

INTRODUCTION

Prolonged exposure of cells to hormones may result in dimi- nished physiologic responsiveness, a condition which is referred to as tolerance or desensitization. The molecular basis for this phenomena has been well characterized for a number of polypeptide hormones and for the catecholamines and is generally felt to be a result of a ligand-induced change in the state of the cellular receptors (l-5). There are fewer studies demonstrating desensiti- zation of cells to the effects of prostaglandins (6-9). The plate- let is a potentially interesting cell for such studies as the oxi- dation products of arachidonic acid, including certain prostaglan- dins, regulate platelet responses to aggregating agents (10). Several prostaglandins including PGEl, PG12, and PGD2 inhibit ag- gregation and stimulate adenylate cyclase activity (11-15). PGEl is not produced by platelets and probably has no physiologic role in platelet metabolism, PG12, the most potent platelet inhibitor, is synthesized by endothelial cells and may affect platelet-vessel wall interactions. PGD2 is also of interest as sufficient quanti-

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PROSTAGLANDINS

ties (17 nM) are produced by platelets and released into plasma during aggregation to potentially affect platelet function and stimulate adeny- late cyclase (15). We have studied the effects of exposing platelets to PGD2 on subsequent adenylate cyclase activity and can demonstrate ligand-specific desensitization following prolonged incubation.

METHODS

Platelet rich plasma (PRP) was prepared from 50 ml of venous blood anticoagulated with 13.5% ACD (NIH Formula A) by centrifu- gation at 160g for 10 min. The PRP was adjusted to pH 6.5 with additional ACD and a platelet pellet was prepared by centrifuga- tion at 1500g for 10 min at 25'C. The pellet was resuspended in buffer containing 8 mM Na2HP04, 2 mM NaH2P04, 10 mM EDTA, 5 mM KCl, 135 mM NaCl, pH 7.2, in the same volume as the original PRP. Equal volumes of the platelets were then incubated at room temperature in the presence of prostaglandin (PGD2) or buffer for 2 hr. The platelets were then washed three times in buffer, and the supernatants decanted. The platelet pellet was then frozen and thawed in a dry ice-acetone bath, and suspended in 1 ml of ice-cold Trig-saline (15 mM Tris-HCl, 138 mM NaCl, pH 7.6).

Enzyme activity of the platelet lysates was measured imme- diately after thawing by a ~9 dification (16) of the method of Salomon et al (17) using [a PI-ATP as substrate and directly measurin

!? the [32P]-cyciic AMP product. Assay mixtures contained

5.0 X 10 dpm [32P]-ATP (New England Nuclear, lo-30 Ci/mmol); O.lmM ATP (Sigma); 25 mM Tris-HCl (pH 7.4); 5 mM MgC12; 2 mM cyclic AMP (Sigma), 0.1% bovine albumin (Pentex); 10 mM theophyl- line; 1 mM EGTA; and an ATP-regenerating solution consisting of 20 mM creatine phosphate and 1 mg/ml creatine kinase (Sigma). Reactions were initiated by the addition of 20 ul of platelet suspension and incubated for 10 min at 37'C at a final volume of 50 ul. Enzyme activity was linear for at least 20 min at protein concentrations up to 4 mgfml. Data was expressed as nmol cyclic AMP/mg protein/l0 min or as the ratio of stimulated to basal acti- vity. Determination of protein was by the method of Lowry et al

(18). Prostaglandins were a gift from Dr. John Pike (UpJohn Co., Kalamazoo, Mich).

RESULTS

Desensitization of prostaglandin activated adenylate cyclase: Fig. 1 illustrates that incubation of platelets with increasing concentrations of PGD2 for 2 hr results in a dose-related decrease in PGD2-sensitive adenylate cyclase activity. Platelets incubated in buffer alone for 2 hr prior to freeze-thaw lysis had 14-fold activation at 10m6 M PGD2. Platelets incubated with 10e7 M PGD2 for 2 hr prior to washing and lysis had only lo-fold activation

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by 1O-6 M PGD2. Incubation of the platelets with 10-6 M and lo-% PGD2 resulted in a further decrease in enzyme activity with 8.5-fold and 7.5-fold activation by 10-o M PGD2 respectively.

0 IOQ lO-6

POOL PREUUCUBATION

IO-S

(Ml

Fig. 1 Effect of Prior Incubation of Platelets With Various Concentrations of PGD2 on Subsequent PGD2-Activated Adenylate Cyclase Activity.

Fig. 2 describes the results of experiments in which plate- lets were incubated in either buffer or 5 x 10e5 M PGD2, washed free of prostaglandin, followed by measurement of adenylate cyclase stimulation by PGD2 and PGEl. Fig. 2A indicates that the platelets preincubated with PGD2 had markedly decreased PGD2-sensitive enzyme stimulation at all doses of prostaglandin studied, with maximal stimulation of only lo-fold compared to 17.5-fold enhance- ment in control platelets (p < 0.001). Incubating platelets in PGD2 also resulted in a diminished sensitivity to PGEl, with only Ll-fold stimulation by 10e5M PGEl compared to 28-fold activation

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4-

/

i

I

10-7 10-6 lo-5 PGD2 (Ml

I I I 10-7 0-6 o-5

PGE, (M)

Fig. 2. Desensitization of Prostaglandin-Activated Aden late Gyclase. Platelets were incubated for 2 hr with 5 x lo- s M PGD2

(0 - 0) or with no addition (0 - O), washed free of prostaglan- din, and adenylate cyclase activity measured in the presence of increasing concentrations of PGD2 (A) or PGEl (B). Results are mean 2; SF,M for 8 experiments.

in platelets incubated in buffer (p < 0.01). This 25% decrease in PGEl stimulated enzyme activity was less than the 45% loss of PGD2 activated adenylate cyclase under the same incubation condi- tions. Incubation of platelets with PGD2 did not alter basal adenylate cyclase activity [90.+ 22 (SEM) pmole/mg protein/lOmin] compared to incubation in buffer alone [72 + 16 (SEM) pmole/mg protein110 min]. In addition, as indicated in Table 1, fluoride activated enzyme activity and epinephrine inhibition of basal levels were comparable in platelets incubated in buffer or in the presence of PGD2. These data indicate that the desensitization of platelet adenylate cyclase induced by exposure to PGD2 was specific for prostaglandins with the maximal effect noted on the

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PGD2-sensitive enzyme. We also performed two experiments incubating platelets with lo-5 M PGD2 for 2 hr in plasma rather than buffer. Comparable results were found with an average decrease of 51% in PGD2-sensitive enzyme activity, 26% in PGEl-stimulated activity, and no change in fluoride activated enzyme.

TABLE 1 EFFECT OF INCUBATION OF PLATELETS WITH PGD2 ON CATECHOLA- MINE AND FLUORIDE SENSITIVE ADENYLATE CYCLASE

Addition

Fluoride (10s2M)

Adenylate Cyclase Activity (Relative to Basal) Control Prostaglandin Incubationb

7.7 + 1.2a 6.6 + 1.4

Epinephrine (10m4M) 0.73 + .02 0.68 + .09

aMean + 1 SD for n=8 experiments b5 x 10-5 M PGD2

Fig. 3 illustrates the time course of desensitization. Platelets were incubated in buffer alone or in buffer containing 10B5 M PGD2 for varying periods of time. No desensitization was noted at 5 min but after 30 min of incubation with PGD2, 30% inhibition of PGD2-stimulated enzyme activity was noted. After 2 hr, enzyme activity was decreased over 40%.

Resensitization of prostaglandin-activated adenylate cy- clase: Fig. 4 illustrates that platelets desensitized to prosta- glandins partially recovered when washed free of PGD2 and then reincubated in buffer for 2 hr prior to lysis and measurement of adenylate cyclase activity. Under these conditions about 50% of the sensitivity to PGD2 was restored (Fig. 4A) with complete recovery of the PGEl-sensitive enzyme response (Fig. 4B). When the desensitized platelets were incubated in plasma for 24 hr in the absence of prostaglandin, complete recovery of enzyme response to PGD2 was noted (Fig. 5). Basal enzyme levels, fluoride activa- ted, and epinephrine-inhibited enzyme activity were unchanged either in control or prostaglandin desensitized platelets following this 24 hr incubation in plasma.

DISCUSSION

These studies demonstrate desensitization of prostaglandin- activated adenylate cyclase when intact platelets are exposed to PGD2 for 2 hr. This enzyme desensitization is prostaglandin spe- cific and dependent on the concentration of PGD2 in the incuba- tion buffer. Desensitization of cells to prostaglandins have been demonstrated in other cell systems. Remold-O'Donnell (6) incuba- ted guinea pig macrophage cells with PGE2 for 2 hr and found that PGE2-activated adenylate cyclase decreased 80% with no change in

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__ e. I 1 I I I I I I I I 1

30 60 90’ 120

TIME (MINUTES)

Fig. 3. Time Course of Desensitization of Prostaglandin-Activated Adenylate Cyclase. Platelets were incubated with 10e5 M PGD% or with buffer alone far varying periods of time, washed free of prostaglandin, and adenylate cyclase activity measured in the presence of 10-5 M PGD2. Results are mean of two determinations. Activity is shown as the % inhibition of control enzyme activity incubated in buffer.

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24

16

PGD2 (M) PGE, (IU)

Fig. 4. Resensitization of Prostaglandin Activated Platelet Adenylate Gyclase. Platelets were desensitized by incubation with lo-5M PGD2 for 2 hr as described in Fig. 2. Platelets were then washed free of prostaglandin and incubated in buffer for 2 hr at 25O. The platelets were then centrifuged at 1500g for 10 min, and the pellet freeze-thawed prior to measuring PGD2-(A) and PGEl-(B) stimulated adenylate cyclase activity. Enzyme activity is shown for control platelets (0 - O), desensitized platelets (0 -- O), and resensitized platelets (A --- A). Each point re- presents the mean of duplicate determinations from a representa- tive experiment.

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PGD2 MA)

Fig. 5. Resensitization of PGD2-Activated Adenylate Cyclase by Incubation in Plasma. Platelets were treated as in Fig. 3 with the exception that the washed, desensitized platelets were incuba- ted for 24 hr at 25' in platelet poor plasma prepared by centrifu- ging ACD-anticoagulated blood at 1500g for 10 min. Following this incubation, the plasma was adjusted to pH 6.5 with ACD, centrifuged at 1500g for 10 min, and the platelet pellet washed once in buffer prior to freeze-thawing and measuring adenylate cyclase activity. Enzyme activity is shown for control platelets (g - 0), desensi- tized platelets (0 --- O), and platelets resensitized by incubation in plasma (A --- A). Each point represents the mean of duplicate determinations from a representative experiment.

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catecholamine or fluoride sensitive enzyme. Similar studies have also been done with human astrocytoma cells (8) and with synovio- cytes (7). Lefkowitz et al (9) demonstrated that incubation of frog erythrocytes with PGEl for 2 hr diminished the subsequent adenylate cyclase response to PGEl and decreased the amount of [3H]PGEl binding to the desensitized cells. In previous studies cultured cells became resensitized to the effects of prostaglandins 6-12 hr after the prostaglandin was removed from the incubation media (7,8). In our experiments we noted partial resensitization after 2 hr of incubation and total resensitization when platelets were washed free of prostaglandin and incubated in plasma for 24 hr.

These studies on human platelets are the first reported examples of prostaglandin-induced desensitization and resensitiza- tion of adenylate cyclase in a non-nucleated cell, and suggest that both processes may not require protein synthesis. Su et al (8) reported that the blockade of protein synthesis with cyclo- heximide did not alter the rate or degree of prostaglandin desen- sitization and subsequent resensitization in the astrocytoma cell. However, other investigators found that protein synthesis was necessary for catecholamine-induced adenylate cyclase desensitiza- tion or resensitization (19,20). While these data appear contra- dictory, it is possible that agonist-induced desensitization of this enzyme may not involve a common mechanism in all cell sys- tems.

It has been well established that platelet adenylate cyclase activity may influence platelet aggregation. Aggregating agents inhibit adenylate cyclase while compounds which inhibit aggregation, including the prostaglandins, stimulate the enzyme. The ability of the enzyme to respond to a given mediator may also be influenced by prior exposure to the compound. For example, recent studies from our laboratory have shown that desensitization of the platelet alpha receptor by exposure to epinephrine results in platelets which no longer respond to epinephrine as an aggrega- ting agent (21). The studies reported here extend these observa- tions and raise the possibility that chronic exposure to certain prostaglandins like PGD2 might alter subsequent responses to aggregating stimuli. These studies also demonstrate that the blood platelet is a useful and convenient model for studying the molecular basis of ligand-induced desensitization and resensitiza- tion.

ACKNOWLEDGEMENTS

The study was supported by Grant No. 13-505-778 from the American Heart Association Massachusetts Affiliation Inc., funds from the Medical Research Service of the Veterans Administration, NIH Grant HL 17513, and NIH Training Grant 5T32HL07142-03. Dr. Handin is a recipient of NIH RCDA #KOlHL00236.

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Received 11/6/78 - Approved 2/21/79

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