Inhibitors of Cyclic Nucleotide Phosphodiesterases Inhibit Protein Carboxyl Methylation in Intact Blood Platelets* detergent chloride of platelets

The cycle of protein-carboxyl methylation and de- methylation was studied in intact blood platelets. Platelets rapidly incorporated ~-[rnethyZ-~H]methio- nine and after a delay of about 20 min, they evolved [3H]methanol. This evolution, and the amount of [3H] methanol liberated by treatment with base, was inhibited in a dose-dependent fashion by the cyclic nucleo- tide phosphodiesterase inhibitors 3-isobutyl-1-meth-ylxanthine, papaverine, dipyridamole, and RA233 (2,6-bis(diethanolamino)-4-piperidinopyrimido[5,4-~] pyrimidine). Each of these compounds increased the incorporation of [3H]methionine into platelets. The effects of RA233 were studied in more detail. Inhibition of [3H]methanol production was not potentiated by stimulators of the adenylate cyclase or the guanylate cyclase. The majority of the base-labile radioactivity was trichloroacetic acid precipitable. Thin layer chro- matography of extracts of platelets incubated with L-[36S]methionine showed that RA233 did not induce a cellular accumulation of [35S]S-adenosylhomocysteine, and that it actually increased the amount of cellular [36S]S-aden~~ylmethionine. Discontinuous polyacryl- amide gel electrophoresis at acid pH using the cationic


Inhibitors of Cyclic Nucleotide Phosphodiesterases Inhibit Protein Carboxyl Methylation in Intact Blood
The cycle of protein-carboxyl methylation and demethylation was studied in intact blood platelets.

Platelets rapidly incorporated ~-[rnethyZ-~H]methionine and after a delay of about 20 min, they evolved [3H]methanol. This evolution, and the amount of [3H] methanol liberated by treatment with base, was inhibited in a dose-dependent fashion by the cyclic nucleotide phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine, papaverine, dipyridamole, and RA233 (2,6-bis(diethanolamino)-4-piperidinopyrimido[5,4-~] pyrimidine). Each of these compounds increased the incorporation of [3H]methionine into platelets. The effects of RA233 were studied in more detail. Inhibition of [3H]methanol production was not potentiated by stimulators of the adenylate cyclase or the guanylate cyclase. The majority of the base-labile radioactivity was trichloroacetic acid precipitable. Thin layer chromatography of extracts of platelets incubated with L-[36S]methionine showed that RA233 did not induce a cellular accumulation of [35S]S-adenosylhomocysteine, and that it actually increased the amount of cellular
[36S]S-aden~~ylmethionine. Discontinuous polyacrylamide gel electrophoresis at acid pH using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride of platelets incubated with [3H]methionine showed incorporation of radioactivity into more than 30 protein bands, including one which co-migrates with calmodulin. The incorporation into the majority of these bands was inhibited by RA233 in a dosedependent fashion. It is suggested that caution should be used in ascribing the pharmacological effects of known phosphodiesterase inhibitors to increases in cyclic nucleotides, because some of these effects could be due to inhibition of protein carboxyl methylation.
The methyl esterification of free carboxyl groups of proteins occurs in all of the eukaryocytic cells that have been examined (l), and a substantial body of evidence suggests that this reaction may be involved in the regulation of such cellular processes as exocytotic secretion and chemotaxis (2, 3). The investigation of such involvement is complicated by the finding that several combinations of reagents used to inhibit transmethylation reactions also induced an accumulation of cyclic AMP within the target cell, in part by the inhibition of 3',5'-cyclic nucleotide phosphodiesterases (4). In this paper, we report that several compounds which are known to inhibit cyclic nucleotide phosphodiesterases also inhibit protein-carboxyl methyl esterification in intact platelets, apparently by * This work was supported by a grant-in-aid from the American Heart Association. 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. a mechanism not involving the accumulation of cyclic nucleotides.

MATERIALS AND METHODS
Human blood platelets were prepared from normal donors after they had ingested 700 mg of aspirin. The blood was anticoagulated with '/E volume acid-citrate-dextrose (1.25 g/lOO ml citric acid, 3.8 g/ 100 ml trisodium citrate, and 2 g/lOO ml glucose), and centrifuged to prepare platelet-rich plasma. About 10 ml of this platelet-rich plasma (containing 2-3 X lo9 platelets) was cooled to 15 "C and centrifuged a t 2000 rpm for 10 min. The platelet pellet was gently resuspended in 10-15 mi of HEPES' buffer (140 mM NaCl, 5 mM KCl, 15 mM HEPES, 1 mg/ml glucose, pH 7.3), and centrifuged again. The platelets were then resuspended in the required volume of the same buffer and used immediately.
The incorporation of radioactivity into the platelets was determined by centrifuging them through silicone oil (5) in microsedimentation tubes (Sarsted No. 702). The pellet was harvested by centrifugation and solubilized by sonication in Scintiverse liquid scintillation mixture (Fisher Scientific) and its radioactivity and that of the supernatant were determined.
The formation of radioactive methanol was determined as follows. Platelets were incubated with [methyl-3H]methionine, and ['*C]methanol was added as an internal recovery standard. A sample was taken to determine the 3H/14C ratio, and at the appropriate times, 50 p1 of the incubation mixture was withdrawn and added to 50 pl of methanol containing 2% (v/v) acetic acid. After mixing and centrifugation, 50 pl of the supernatant was pipetted onto a twist of filter paper lodged in the neck of a scintillation counting vial containing 10 ml of Scintiverse, and the vial was immediately capped. Care was taken to ensure that the filter paper did not come into contact with the scintillation mixture; such contact was easily discovered by removing the filter paper and examining it under ultraviolet light. The vial was allowed to stand for at least 2 h and then the 3H/14C ratio of the material that had evaporated into the scint.illation mixture was determined. Preliminary experiments established that volatile 3H production ceases in the methanol/acetic acid mixture (which can conveniently be stored overnight a t -10 "C), that the filter paper is rapidly dried by the desiccating nature of Scintiverse (under actual experimental conditions, the half-time for ["C]methanol to transfer from the filter paper to the mixture is about 10 min), and that after drying none of the 3H retained on the filter paper becomes volatile. Fractional distillation of the volatile 3H released from platelets established its identity as methanol (Fig. 1).
Separation of methionine metabolites was carried out by thin layer chromatography. Samples of the platelet suspension were diluted with ice-cold EDTA/saline (140 mM NaC1, 10 mM Na-EDTA, pH 7.0) and centrifuged. The supernatant was discarded and the pellet was dispersed in 100 pl of 0.1 M acetic acid under nitrogen and the tube was briefly immersed in a boiling water bath and then cooled on ice and centrifuged. 5 pl of the supernatant was spotted on the preabsorbent zone of the thin layer plate, together with chromatography standards (about 50 nmol each of S-adenosylmethionine, methionine, methionine sulfoxide, methionine sulfone, S-adenosylhomocysteine, homocysteine, and 5'-deoxy-5'-methylthioadenosine). The plate (250 p, silica GF Uniplate, channeled; Analtech, Newark, DE) was developed in an equilibrated tank with butanol/acetic acid/water The abbreviations used are: HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid 16-BAC, benzyldimethyl-n-hexadecylammonium chloride.
(6:2:4). The standards were located by examining the plate under 254 nm ultraviolet light, and by the ninhydrin reaction. The radioactivity was located by autoradiography and was measured by scraping the absorbent into a counting vial containing 1 ml of 10 mM HC1, and then adding 10 ml of scintillation mixture. The ninhydrin reaction caused a loss of radioactivity detected in the methionine spot, and so was not used to locate the radioactive spots. Recovery of counts was not significantly different from 100%.
Polyacrylamide gel electrophoresis was carried out a t acid pH with the cationic detergent 16-BAC, using slight modifications of our published method (6). The incubation of platelets with 13H]methionine was terminated by the addition of an equal volume of a solution containing 16-BAC (7%, w/v), urea (5 M), glycerol (lo%, v/v), acetic acid (50 mM), dithiothreitol (0.1%, v/v), EDTA (2 mM) made to volume with the electrode buffer. The sample (generally 100 pl) was applied to one of 15 wells cast in the stacking gel of a 32 cm X 14 cm X 1.5 mm Protean slab gel (Bio-Rad Laboratories, Richmond, CA). The composition of the stacking gel was: acrylamide (4%, w/v), N, N'-methylenebisacrylamide (0.35%), urea (lo%, w/v), potassium phosphate (125 mM, pH 4.0), ferrous sulfate (4.3 pM), and ascorbic acid (0.43 mM). After bubbling with nitrogen gas, hydrogen peroxide was added to a final concentration of 0.45 mM. The running gel contained acrylamide (7.5%, w/v), N,N'-methylenebisacrylamide (0.26%, w/v), urea (15% w/v), ferrous sulfate (7.7 p~) , and ascorbic acid (0.43 mM) in potassium phosphate buffer (150 mM), pH 2.0. After bubbling with nitrogen gas, polymerization was initiated by adding hydrogen peroxide 0.34 mM. The electrode buffer was glycine (150 mM) and phosphoric acid (27.5 mM). 16-BAC (0.1%, w/v) was added to the upper electrode buffer. Electrophoresis was carried out overnight a t a constant current of 50 mA. The gel was fixed in two changes of methanol/acetic acid/water (4:1:5) stained with Coomassie blue, destained, hydrated with two changes of distilled water, and impregnated with 2 M sodium salicylate containing 100 mM acetic acid and 1% (v/v) glycerol. It was then dried onto filter paper without applied heating using a rotary vacuum pump protected with a trap and exposed to Kodak x-ray film a t -80 "C for 1 week.
The isotopes used were purchased from New England Nuclear: 16-BAC was purchased from Gallard-Schlesinger, Carle Place, NY. Prostaglandin E, was purchased from Upjohn Co., Kalamazoo, MI, and prostacyclin was a gift from the same company.

RESULTS
In preliminary experiments, we confirmed that washed platelets rapidly concentrate radioactivity when they are incubated with [methyl-'HH]methionine, and this radioactivity subsequently appears in S-adenosylmethionine, protein, and a volatile product with a distillation profile identical with that of authentic [14C]methanol (Fig. 1).
The generation of methanol by platelets labeled with ['HI methionine and then freeze-thawed was a function of the pH of the solution in which they are thawed (Fig. 2). At pH below 5, the rate of production of ['Hlmethanol by hydrolysis was negligible, but at high pH values, a rapid evolution of volatile radioactivity occurred. At pH 10, this amounted to about 10% of the platelet-associated radioactivity and was complete in about 2 min. In a series of experiments, we established that the rate of ["Hlmethanol production at pH 7.4 studied in freeze-thawed platelets was unaffected by the addition of EDTA, Ca", Mg2+, tosylarginine methyl ester, diisopropyl fluorophosphonate, N-ethylmaleimide, or formaldehyde, suggesting that under these circumstances the majority of the hydrolysis of methyl esters was not enzymatic.
Methanol production in intact platelets was studied by sampling platelets during their incubation with ['Hlmethanol and estimating the radioactivity in the volatile fraction. After a delay of 10-20 min, ['Hlmethanol production accelerated and reached a stable rate in about 1 h. This stable rate was  Washed platelets were incubated with 30 pCi of 13H]methionine for 90 min, and then sedimented. They were resuspended in 140 mM NaCI, 10 mM Na-EDTA, pH 7.0, and frozen in 100-pl aliquots containing about 4000 cpm of [I4C]methanol and about 10' platelets. They were thawed by the addition of 300 p1 of buffer having the indicated pH, and the mixture was incubated at 37 "C. At the times indicated thereafter, the volatile 3H was determined as described under "Materials and Methods" and is expressed as a fraction of the 3H in the platelets. The buffers used were 100 mM citric acid, pH 4 with NaOH, 100 mM citric acid, pH 5 with NaOH, 100 mM NaH2P04, pH 6 with NaOH, 100 mM NaH2P04, pH 7 with Tris base; 100 mM Tris base, pH 8 with H3P04, 100 mM NaH2P04, pH 9 with NaOH, and 100 mM H3B03, pH 10 with NaOH.
unaffected by agents which induce platelet aggregation and exocytotic secretion (thrombin and the calcium ionophore, A23187) (data not shown). Compound RA233, a phosphodiesterase inhibitor structurally similar to dipyridamole (Fig. 3), inhibited the rate of production of volatile 'H without extending the delay (Fig. 4). This effect was potentiated neither by stimulating the adenylate cyclase with prostacyclin nor by stimulating the guanylate cyclase with sodium nitroprusside (Fig. 4).
This effect of RA233 was not due to inhibition of the uptake of radioactivity by platelet; indeed, the addition of RA233 increased this accumulation by about 250% in a dose-related fashion (Fig. 5 , top). This figure also shows that RA233 inhibited the incorporation of 'H into base-labile methyl esters, revealed by the increase in volatile 3H after basecatalyzed hydrolysis. The majority of these methyl esters were precipitated by trichloroacetic acid, and RA233 reduced both the total incorporation of 3H into trichloroacetic acid precipitate and the fraction made volatile by base treatment (Fig.  6). In these figures, the [3H]methanol production is expressed as a percentage of the 3H originally added to the platelets. The inhibitory effect of RA233 would have appeared much greater had we expressed the [3H]methanol as a percentage of 'H taken up by the platelets.
In order to determine if RA233 inhibited the synthesis within platelets of the methyl donor, S-adenosylmethionine, we incubated platelets with [35S]methionine and with or without RA233 and washed them. 5 p1 of the lysates of these platelets were chromatographed on a thin layer system which separated S-adenosylmethionine from methionine and its oxidation products (Fig. 7). The radioactivity in the spots was determined by liquid scintillation counting. In the control  MeOH and incubated with the indicated concentration of RA233 for 60 min. The volatile 'H/"C ratio before and after the platelet suspension was incubated at 37 "C with 125 mM Na2C03 was determined. Additional samples were centrifuged and the percentage of the total 3H that was found in the platelet pellet was determined. In samples from which platelets were omitted but which were otherwise processed in parallel, 0.26% of the 3H was volatile, and this was not affected by the addition of RA233 or Na2C03. This figure was subtracted from the data, which were then reexpressed as the percentage of total 3H. samples (total applied, -5.0 x lo5 cpm), about 6% was found as S-adenosylmethionine plus 5'-deoxy-5'-methylthioadenosine and 16% as S-adenosylhomocysteine. In the RA233 samples (total, 1.46 X IO6 cpm), these figures were 7 and 7.5%, respectively. We confirmed the identity of the radioactivity migrating with S-adenosylmethionine by subjecting the extracts to acid hydrolysis at 100 "C for 10 min before chromatography. This is known to hydrolyze S-adenosylmethionine to 5'-deoxy-5'-methylthioadenosine (7). As can be seen in Fig. 7, this treatment substantially reduced the radioactivity found with S-adenosylmethionine with a corresponding increase in the radioactivity recovered with 5'-deoxy-5'-methylthioadenosine. Fig. 8 shows a fluorograph of a polyacrylamide gel electrophoretic separation of platelet proteins extracted with the cationic detergent 16-BAC. This method (6) preserves baselabile carboxymethyl esters by maintaining an acidic pH (pH < 4.0) throughout the whole procedure. Examination of the figure shows that the inclusion of RA233 decreases the radioactivity associated with most of the platelet proteins, and that this was more obvious in some of the lower molecular weight proteins. One such protein co-migrated with authentic calmodulin. Table I shows that compounds commonly used as phosphodiesterase inhibitors have the same effect as RA233. Papaverine, dipyridamole, and isobutylmethylxanthine increased the incorporation of radioactive methionine into platelets, and inhibited both the production of methanol and base-labile methyl esters expressed in terms of the 3H originally added. In this regard, papaverine was about 3 times as potent as

RA233.
Theophylline significantly increased the rate of production of methanol.

DISCUSSION
Blood platelets, like nucleated cells (l), actively incorporate methionine, from which they synthesize S-adenosylmethionine and the methyl donor for a variety of reactions including the methyl esterification of free carboxyl groups of proteins (8). This esterification nullifies the negative charge and hydrophilic nature of the carboxyl groups and can be reasonably predicted to have a substantial effect on the properties and function of proteins. The esters are unstable and hydrolyze spontaneously a t neutral or alkaline pH, with the formation of methanol.
In the course of our investigation of the involvement of protein methylation in the regulation of platelet function, we examined the effects of a series of compounds known to inhibit platelet aggregation in vitro. These included dipyridamole (Persantine), which is used clinically as a coronary vasodilator and an inhibitor of platelet aggregation, and papaverine, a vasodilator and a spasmolytic. We found it convenient to do most of our studies with compound RA233, an analogue of dipyridamole which is more soluble than are the others in physiological salt solutions. Each is known to inhibit the cyclic AMP phosphodiesterases of platelets (9).
The addition of RA233 to platelets inhibited their production of methanol in a dose-related fashion and this inhibition was paralleled by a reduction in the base-labile methylation (detected either in whole platelets or in their proteins precip-  itated with trichloroacetic acid), and by a reduction in the amount of radioactivity in individual protein bands resolved on 16-BAC polyacrylamide gels, a procedure we have recently described for detecting base-labile protein methylation (6). There can thus be no doubt that RA233 inhibits protein methylation. Dipyridamole was about equiactive with RA233, and papaverine, which bears no structural relation to these two compounds, was about 3 times as potent.

Isobutylmethylxanthine also inhibited protein methylation
We investigated the mechanism of this inhibition. It was not due simply to the inhibition of the uptake of the isotopically labeled methionine; indeed, each of these compounds substantially increased the incorporation of the methionine. It was not due to the accumulation within the platelets of cyclic nucleotides, since neither prostaglandin E, (a stimulator of the adenylate cyclase) nor sodium nitroprusside (a stimulator of the guanylate cyclase) (10) potentiated the effect of the compounds. It was not due to an acceleration in the rate of hydrolysis of methyl esters, because this would have caused an increase rather than a decrease in the rate of ['HI methanol production, and it was not due to the accumulation of S-adenosylhomocysteine which might be brought about by the inhibition of S-adenosylhomocysteine hydrolase. It would appear therefore that these drugs act (directly or indirectly) Our demonstration that phosphodiesterase inhibitors inhibit protein methylation is of particular significance when compared with the finding that a variety of maneuvers used to inhibit the methylation of proteins and other substrates (often by causing the accumulation of S-adenosylhomocysteine or one of its analogues) greatly enhance the accumulation of cyclic AMP in intact cells, in part by inhibiting its hydrolysis (4,11). It is not unreasonable, therefore, to raise the possibility that the activities of cyclic nucleotide phosphodiesterases in the intact cell might be influenced by protein methylation. Calmodulin is known to be both an excellent substrate for protein methylation (12), and to regulate the activity of cyclic nucleotide phosphodiesterases (13). We have detected the methylation of a protein in platelets co-migrating with authentic calmodulin, and this methylation is decreased by RA233. The available evidence suggests, however, that carboxymethylation of calmodulin decreases, rather than increases, its ability to potentiate the hydrolysis of cyclic GMP by phosphodiesterases (12).
That the experimental inhibition of methylation reactions is usually accompanied by increases in cellular cyclic AMP has cast a doubt over the ability of such experiments of establish a role for methylation reactions in the regulation of cellular processes (4). Our results admit to uncertainty in the opposite direction: it is possible that some of the effects of compounds traditionally attributed to their ability to inhibit cyclic nucleotide phosphodiesterases may in fact be due to their ability to inhibit protein methylation.

TABLE I
Effect of drugs on [3H]methionine metabolism Washed platelets (3 X 10s/ml) were incubated with ['Hlmethionine (50 pCi/ml) and the indicated drug a t 37 "C for 90 min. Duplicate samples were centrifuged and the radioactivity in the pellet was determined, and triplicate samples were taken for the determination of volatile radioactivity before and after treatment with 166 mM Na2C03 for 10 min a t 37 "C. The volatile radioactivity found in a parallel incubation to which no platelets were added has been subtracted from the results (0.94% f 0.02%). *, significance a t p < 0.01 by unpaired two-tailed Student's t test. In the absence of added compounds, platelets incorporated 7.5% of the radioactivity and the duplicate determinations of uptake did not differ from each other by more than 1% of total; these results were not analyzed statistically. Note that the volatile radioactivity is expressed as a percentage of the 'H added to the platelets, not as a percentage of intracellular 3H.