The effect of aspirin on prostaglandin synthetase

THE

EFFECT

OF

ASPIRIN ON SYNTHETASE

PROSTAGLANDIN

G. J. ROTn Division of Hematology, Departmentof Medicine, University of Connecticut School of Medicine, Farmington, Connecticut 06032, U.S.A.

INTRODUCTION

Aspirin (acetylsalicylic acid) is familiar to all as a "household analgesic". The drug was first isolated in the late 1800's and since then, has maintained its place in medicine cabinets around the world. 2~ Within the past decade, several investigators have demonstrated that aspirin influepces prostaglandin (PG) synthesis by inhibiting the initial enzyme of the biosynthetic pathway. ~'4'~'~'2° In this review, the mechanism by which aspirin works against this enzyme will be discussed, and some areas worthy of further investigation will be pointed out. ASPIRIN-MEDIATED ACETYLATION OF A PLATELET PARTICULATE PROTEIN

Aspirin consists of an acetyl group esterified to the hydroxyl group of salicylic acid (Fig. 1). Such a phenolic ester can be expected to acetylate any number of materials of biologic interest. One might also expect that aspirin could exert its pharmacologic effect by acetylation, the transfer of the acetyl group to an unknown acceptor. Our work in this area began with an attempt to correlate the acetylating property of aspirin with the drug's effect on platelets. 12 Aspirin is known to be an inhibitor of platelet function, and this antiplatelet effect occurs under specific conditions; namely, within minutes--20 min --at low concentrations of aspirin--20 to 50 p1~.21 Our work with aspirin and platelets was based on the assumption that aspirin acts on platelet function by acetylating a single platelet protein. If this assumption is correct, such a reaction must occur under appropriate conditions, for example 20 to 50/~M aspirin in 20 rain. For the first experiments, we used washed normal human platelets, aspirin of high specific activity (200 Ci/mol) carrying a tritium label in the acetyl portion of the molecule, and sodium dodecyl sulfate (SDS) gel electrophoresis to permit separation of radiolabeled, acetylated proteins. Platelets were incubated with [acetyl-aH]aspirin to permit ASPIRIN AS AN ACETYLATING AGENT SALICYLIC ACID

ACETYL SALICYLIC ACID (Aspirin)

COOH

0 OH

•

0

O-ACETYL RESIDUE

~-CH~

?

'"

6H I R group of protein

FIG. 1. Aspirin is a phenolic ester which readily transfers its acetyl group to suitable acceptors such as amino or hydroxyl groups of proteins. [Acetyl-aH]aspirin transfers the I'aH] label to acetylated proteins. 461

,162

G.J. Roth

I

Z

8~o

0

~

;~

~ ~ ~ ~

4

~,

8

,~ ~m~,oa _. . . . i, % 1~.,t0~ Pt~

¢

~

~

~ ~ •

.~_

~

/ ~

~ ~

12 • 16 20 24 C~L SLICE NUMBER

~ ~ ~ ~ ~~~

2~

~

~

32

FIG. 2. Acetylationof platelet protein by 30/~ [acetyl)H]aspirin for 20 rain at 3T'C. Following sonication, solubleand particulate fractions wereprepared and analyzed for radioactivity by SDS gel electrophorcsisas described)2 (Reproducedby permission o4"the J. Clin. lnve~t.).

any protein acetylation to proceed. The platelets were first sonicated and centrifuged. The proteins of the soluble and particulate fractions were then treated with SDS and subjected to SDS polyacrylamide gel electrophoresis. Using the technique of gel slicing, solubilization, and scintillation counting, the acetylated proteins in either the soluble or the particulate fractions could be separated, detected, and quantitated by their tritium label. The results of a typical experiment are shown in Fig. 2. Platelets were incubated with 30 ~4 [:acetyl-~H]aspirin for 20 rain at 37°C, sonicated and centrifuged to give supernatant (soluble) and particulate (microsomal) fractions. Each fraction was applied to a separate SDS gel and subjected to electrophoresis. Each gel was then analyzed for the presence of acetylated protein as tritium, and a chromatogram was drawn plotting the amount of [~H]acetate in various gel slices. The particulate fraction contained a single [acetyl-~H]protein, molecular weight (MW) 70,000, while the soluble fraction conrained two [acetyl-~Hlproteins of MW 225,000 and 55,000. When identical experiments were performed with [~H]aspirin which carried the [~H]label in the ring portion of the molecule, no [~H]labeling of platelet protein was observed. The result indicates the [~H'llabeling of platelet protein by [acetyl-~H'laspirin does reflect an acetylation reaction. The next experiment was performed to test the concentration dependence of the acetylation of each protein (Fig. 3). Platelets were incubated with 3, 10, 30, I00, and 300 VM [acetyl-~H]aspirin for 20 rain at 37°C, and then soluble and particulate fractions were analyzed for the amount of [~H'lacetate in each protein. As shown in Fig. 3, the extent of labeling of the 70,000 MW particulate protein reached a maximal extent with approximately 50 VM aspirin in 20 min, the same conditions required for the antiplatelet effect of the drug. The acetylation of the soluble proteins did not proceed under the appropriate conditions, and these acetylation reactions were not investigated further. The "permanence" of the aspirin effect might be inferred from the above data which shows that aspirin covalently modifies the 70,000 MW protein. However, an additional experiment was performed to demonstrate this point. The experiment was based on the following assumption. If aspirin-mediated acetylation is permanent, then platelets exposed at one time point to a sufficient amount of aspirin will have all of their acetylation sites filled. If the same platelets are treated a second time with aspirin, no further acetylation will occur. The results of the actual experiment are given in Fig. 4. Three volunteers ingested gram doses of nonradioactive aspirin to provide the initial "treatment" in vivo. At intervals of 1, 3, 6, and 13 days, platelets were prepared from the blood of these subjects. The platelets were incubated with [acetyi-~H'laspirin in vitro and analyzed for the extent of [~H]acetate incorporation into the particulate protein. This con-

The effect of aspirin on prostaglandin synthetic

~

i

:

~

~.....~ 5 5 ~ M W ~ ' ~

: 851~XJMW ~o*e,e P~rh
~--o ~

I

~

,

~

I

~5~MW S ~

P,~ ~,~,,~ ~ ~t~

~

~

~

463

I

.~-/

.;...-

I

~

~

•

~

%

~ ~ • [AEETYL'3~ "SP~RIN CO4~CENTRAT~ON luMI

F ~ . 3. C o ~ n t r a t i o n ~ ~ i~iv~ p ~ t d ~ proton ~tylatmn. W ~ h ~ p l a t d ~ ~e i~bat~ ~th [~yl-~H]~n (3, 10, ~ , I ~ ~ d ~ ~ ) for ~ rain at ~7~C. ~ i u ~ e a ~ p~iculate fra~m~ (2 x 10t ~ a t e l ~ ~ ~ l ) w~e ~ y ~ for [~H] in l a ~ l ~ p r o t o n . (Repr~ by ~ n ~ t ~ J. ~in. inv~t.[

stitutes the second "treatment". The time course of increasing uptake of [SH]acetate in the protein of interest parallels the appearance of new nonacetylated platelets from the marrow over a 10-13 day period, approximately the same time as platelet lifespan in humans. In other words, the first treatment of platelets with aspirin gave a "permanent" effect on the platelets present, and the appearance of "acetylatable" platelets over the next 13 days reflects the appearance of new platelets from the bone marrow. 200

Z

Z 0- -

120

I--

<~

~ ~-

,<

gO

=~

< ~

/

~0

• Svb~t | o ~,b~e<~ 2 "- $ ~ 3

/

_~

-I

1 0

~,~*

~

1 ~

~

~

t 3

t 6

0AYS

P~ST

A~

~3 INGESTION

*~

F~. 4. Inhibition of ia v~Iro l~rti~late proton ~cetyl~tion by ~pinn in~tion. ~atelets ~e ~ nt ~ t ~ (I, 3, 6 ~ 13) from s u b ~ who ~ i n ~ t ~ 3 ~ ~ ~ n . Plate~ts ~ r e t r ~ t ~ in ¢lgro ~ t h [ ~ t y ~ S H ] ~ h n to five ~ i ~ l ~tylat~n ~ t~ p~t~late protein. ~ o r e ~pi~n t r i t e r , t ~ s u b ~ " p l a t e ~ (2 ~ tO*) i ~ r ~ r a t ~ l ~ m u~ the ~ ~iti~. (R~r~ by ~ m ~ n ~ t ~ J. ~in. Inv~t.).

464

G.J. Roth

These initial studies indicated that aspirin acetylates a single membrane-bound protein in human platelets under the same conditions that the deug inhibits platelet function. We conclude that the aspirin effect on human platelet function is due to this specific acetylation reaction. 12 E V I D E N C E T H A T T.HE A C E T Y L A T E D P R O T E I N PROSTAGLANDIN SYNTHETASE

IS

Since several investigators had shown the inhibition of prostaglandin synthetase by next set out to determine if the acetylated protein was identical to the prostaglandin-forming enzyme. Several lines of evidence were developed. 11.1s First, we looked for the presence of an aspirin-acetylated protein in the tissue most commonly used for prostagiandin synthetase studies--seminal vesicles of sheep and beef. An aspirinacetylated protein was found in the membrane fraction of both sheep and beef seminal vesicles although slightly higher aspirin concentrations (100 #M) and longer incubations (30-60 min) were required to give maximal acetylation. Is The MW of the acetylated protein in these tissues was identical to that found in human platelets. All later experiments were performed using sheep seminal vesicles. Second, we compared the time course of aspirin-mediated protein acetylation of the 70,000 MW protein to the time course of aspirin inhibition of the activity of the prostaglandin-forming enzyme. As shown in Fig. 5, aspirin (100 #~) was added at time 0 to a suspension of acetone-pentane powder of sheep vesicular gland. The mixture was tested at intervals for enzyme activity using a malonaldehyde assay and for the extent of protein acetylation using SDS gel electrophoresis. The time course of enzyme inactivation and protein acetylation is essentially identical. Third, we assessed the ability of various substrates and inhibitors of prostaglandin synthetase to inhibit aspirin-mediated acetylation. The assumption was aspirin, 't'17 w e

100

• -0

•

50

20

10

2

•

ENZY~~CTIVITY (~ OF CONTROL)

{~

PROTEIN~CETYLATION (Z RE~t~,ININGUNACETYLATED)

I

I

.b

'

go

TIME (Min.) Fro. 5. Time course of aspirin-mediated enzyme inhibition and protein acetylation. Acetone/ pentane powder of ram seminal vesicles was treated with 100 pM [acetyl-SHlaspirin at 37°C. At intervals, aliquots were removed from enzyme assay by malonaldehyde formation (e) and for protein acetylation by $D$ gel el~rophoresis IO). Result~ were expressed as % of original activity(60 nmol malonaldehyde/mg/min) or as % remaining unacetylated (compared to maximal acetylation of 1250 cpm/prep).

The effect of aspirin on prostaglandin synthetase TAn~

465

1. Inhibition of Aspirin-Mediated Acetylation by Fatty Acids and lndomethacin

Inhibitor

Inhibitor Conc. (pM)

CPM

50

294 < 20

> 95

10 100 10 100 10 1130

243 96 291 181 110 25

17 43 1 38 63 92

Control 5,8,11,14-Eicosatetraenoic (arachidonate 20:4) 9,12,15-Octadecatrienoic (linolenic 19: 3)

11,14,17-Eicosatrienoic (20:3) Indomethacin

% Inhibition

Ram vesicular gland microsomes were incubated with 100pw [acetyi-3H]aspirin for 20 min at 37°C with or without inhibitor and analyzed for the content of 13HI m the 70,000 MW microsomai protein by SDS gel electrophoresis. Controls were performed with no inhibitor added.

made that aspirin interacts with an active-site region of the enzyme and that various enzyme substrates such as arachidonic acid or inhibitors such as indomethacin would compete with aspirin for this same site(s). As a result, these substances would interfere with aspirin-mediated aeetylation of the 70,000 MW membrane-bound protein. As noted in Table 1, the various substrates and analogues did inhibit acetylation as did indomethacin. Lastly, we did "co-purification" experiments to determine if enzyme purification procedures gave preparations with a proportion increase in their specific content of enzyme activity and in protein susceptible to aspirin-mediated acetylation. 11 As noted in Fig. 6, a variety of prostaglandin synthetase preparations of increasing enzyme activity, measured by oxygen electrode assay (/~mol O2/min/mg), were incubated with [acetyl-aH]aspirin (100 pM, 60 min, 37°C) to give maximal acetylation and the content of the [acetyl-aH]protein was then measured, expressed as cpm/mg. Specific enzyme activity correlated well with the specific content of [acetyl-aH]protein. All the data noted above are consistent with the hypothesis that the aspirin-acetylated, membrane-bound protein of sheep Vesicular gland is identical to the prostaglandinforming enzyme in this tissue.~S

COR~'LATIqN Of OXYGENASEENZYM~CONTENT WITH [ACE'~t'L-3H] P]~OTEIN CON~I~i"

i:[ 7

o

•

/m

~o

~

,~

~

~

m

CP~/MG x ~0"3

FIG, 6. Comp~ison of the specific c~ymc activity (retool Oz/~n/mg) with the s ~ f i c [~H]a~tyl-protcin content (cpm/mg) in v ~ o u s parti~iy purifi~ e ~ y m e prep~ations. (Rcprodu~d by ~rmission of Prostaglandins). J.P.L.T. 20~ I-4

P*

~66

G . J . Roth

STOICHIOMETRY

OF

PROSTAGLANDIN OF

ASPIRIN-MEDIATED SYNTHETASE

THE

ACETYLATION

AND

ACETYLATED

OF

IDENTIFICATION

RESIDUE

Subsequent work has been done using highly purified prostaglandin synthetase from sheep vesicular gland. 13'~4 Enzyme purification was done with established methods which include the isolatiqn of microsomes, solubilization in Tween 20, DEAE cellulose chromatography, preparative isoelectric focusing, and gel filtration. 6'9'1a Nearly homogeneous preparations of the enzyme were obtained in both aspirin-acetylated and active forms. The final material was purified about 18-fold from microsomes and gave a single band on SDS gel electrophoresis (MW 70,000), and a unique amino terminal sequence (ala-asp-pro-gly-aia-pro-ala-pro-val-asn-pro-met-gly-) using automated Edman degradation. 1'~ To test the stoichiometry of aspirin-mediated acetylation, the purified [acetyl-3H]enzyme was assayed for its amino acid and [3H]acetate content. Assuming an approximate molecular weight of 70,000, each mole of enzyme contained one mole of acetate. A similar result was obtained with purified active enzyme which was acetylated with [acetyl-3H]aspirin and then assayed for [3H]acetate and amino acid content. Finally, aminoterminal sequence analysis of the aspirin-acetylated enzyme demonstrated the presence of one mole of amino-terminal amino acid (alanine) per mole of acetate} 4 The results indicate that prostaglandin synthetase consists of a single polypeptide chain of approximate MW 70,000 and that aspirin transfers one acetate per enzyme molecule during the acetylating and inactivating event. A series of experiments were performed to identify the specific amino acid acetylated by aspirin. ~3 The starting material for this work was purified [acetyl-3H]enzyme. The enzyme was fragmented sequentially with cyanogen bromide, trypsin, and pronase. After each proteolytic treatment, the peptide containing the [3H]acetate was isolated by gel filtration alone or gel filtration plus ion exchange chromatography on Dowex 50 or Dowex 1. The behavior of the three successive [acetyl-3H]peptides on Sephadex G-25 is shown in Fig. 7. The final material isolated after pronase digestion was assumed to be a single acetylated amino acid. It was identified as N-acetyl-L-serine by several means. First, the ['3H1 material co-chromatographed with authentic N-acetyl-serine on both Sephadex G-10 and Dowex 1. Second, following acid hydrolysis, the material was determined to contain mainly serine by amino acid analysis. Third, the ['3HI material cocrystallized from ethyl acetate with authentic N-acetyl-L-serine. The acetylated serine was initially assumed to be an NH2-terminal residue since this would be the most direct explanation for the presence of a free ~-amino group which could accept the acetate moiety. 13 This hypothesis was tested directly because it predicts

VT

x

m~

1

~

/\ ~0~-~ •

~ . ~%

•

~

~ ~

~

~

~

~ ~~ 5

I0

~

~ '.

~~

.....

20

." ~" ~"

.~.~. . .~. . . . . FRACTIONS

,,

~ ~

~

~ . . . .

~0

2~

....

'

(I ~L)

Fro. ~. ~el 61tr~tion of suc~sive [~H]~txl-~ptid~ p r ~ u ~ bX proteolxtic c l ~ v ~ e of [ ~ t~l-S~]p~os¢~din synt~et~e. 7 x 10 s cpm of C N B r di~est ~ , 4.5 x l0 ~ cpm of trypsin diB~t ~ , oc 4.4 x ~0 ~ cpm of p c o n ~ di~es¢ •O. ~. ~ -, were applied ¢o ~ I x 48 cm ~ l u m n of ~ p h ~ d ~ O - ~ ~ d elu~¢e fractions (I ml) wece ~nalTzed for [~H] in Chree sep~te a~tim~ts.

The effectof aspirin on prostaglandin synthetase

467

that the NH2-terminus of the acetylated enzyme would be a "blocked" serine and, therefore, inaccessible to conventional sequence analysis. This proved not to be the case since the NH2-terrninus of both the active and acetylated enzyme is alanine, x3,t8 Therefore, the acetylated residue is, in fact, an internal O-acetyl-serine, and the presence of N-acetyl-serine in the original identification ~3 arose from an O--, N acyl shift following enzyme fragmentation. 7 The presence of th~ internal O-acetyl serine has been confirmed by structural study of the [acetyl-3H]tryptic peptide prepared from the Iacetyl-3H]en zyme and by the observation that the acetate to protein bond in the intact enzyme is cleaved by alkaline 2M hydroxylamine (Roth, G. J., unpublished observations). DIRECTIONS FOR ADDITIONAL WORK Questions remain regarding how aspirin affects prostaglandin synthetase and how the drug should be used as an antiplatelet agent. In discu~ssing the mechanism of aspirin's effect on the enzyme itself, some aspects of the enzyme should be emphasized. A number of investigators have purified the enzyme to near homogeneity and have characterized it to some extent. 6'9'18'19 All workers agree on certain points: first, that the enzyme is labile, membrane-bound, active in non-ionic detergent with a subunit MW of 70,000 by SDS gel electrophoresis; second, that the enzyme has both oxygenase and peroxidase activity; third, that the enzyme rapidly becomes inactive as it catalyses the formation of P G G to P G H ; and finally, that the purified enzyme requires exogenous heine and a cofactor such as phenol or tryptophan for full activity. Some areas of disagreement remain: for example, the non-berne iron content of the enzyme and the number of subunits present in the enzyme. ~'6'9'18'~9 The most important question has to do with the heine-protein nature of the enzyme. There is no doubt that the enzyme does contain heine during the early stages of purification, for instance, following ion exchange chromatography but before isoelectric focussing. However, the majority of the heine is apparently lost during further purification so that, in working with highly purified enzyme, exogenous heine must be added to restore activity. My question relates to whether or not the enzyme suffers some irreversible denaturation during the initial loss of the heine moiety such that a native holo-enzyme can no longer be formed by addition of exogenous heine. This question is raised because the reconstituted heine-enzyme has the following unusual properties: (1) The absorption spectrum of the reconstituted enzyme has a relatively low peak in the gamma region, x9 (2) The heine group is lost relatively easily during gel filtration. 6 (3) The heine moiety can be provided in either free form or as a second hemoprotein such as hemoglobin, x° (4) The reconstituted heine enzyme is not sensitive to cyanide or carbon monoxide. ~° (5) The addition of free heine group to the enzyme results in rapid enzyme inactivation. ~° For these reasons, one must consider the possibility that prostaglandin synthetase is still not available for study in a native undenatured state with an intact heme-binding region. The site of aspirin mediated acetylation may relate in some way to this heme-binding region. For example, acetylation of the serine residue by aspirin may result in a change in heme-binding and subsequent loss of activity. Therefore, further work is required to define how aspirin alters enzyme function. An effect on heme-binding is one possibility. The second question concerning aspirin as an antiplatelet agent deals with the sensitivity of the platelet and endothelial cell enzymes to aspirin. Conflicting information is available. All investigators agree that platelet prostaglandin synthetase is exquisitely sensitive to aspirin. In vitro, the platelet enzyme is completely acetylated by 50/ZM ASA in 20 min. More than 90% of platelet enzyme activity is lost following the ingestion of a single 325 mg tablet of aspirin. 2'~2 The controversy deals with the sensitivity of the endothelial enzyme to aspirin. Jaffe and Weksler have shown that the enzyme in cultured human endothelial cells from umbilical veins are quite sensitive to aspirin, 90% inhibition after 1 hr using 6.2/~M aspirin, a Conversely, Burch et al., presented data to show that the enzyme in aortic microsomes was only 1/250th as sensitive to aspirin as was the platelet enzyme, a Perhaps the main difference between these two studies is the state of the

468

G.J. Roth

endothelial enzyme, whether it is studied in the intact cell or in a suspension of microsomes. Possibly, the difference in the results will prove to be the denaturation of prostaglandin syntbetase during the preparation of microsomes giving rise to a loss in sensitivity to aspirin. The resolution of this question will have direct bearing on the use of aspirin as an antiplatelet agent for the prevention of vascular disease complications. If the results of Jaffe and Weksler are relevant to the in vivo effects of aspirin, then one would use low doses of aspirin at appropriate intervals to maximize the drug's effect on platelets but to permit the endothelial cell to resynthesize active enzyme between doses. SUMMARY

Aspirin covalently modifies and inactivates prostaglandin synthetase by acetylating a single serine residue of the enzyme. Acetylation selectively inhibits oxygenase activity, the formation of PGG2 from arachidonic acid. The consequence of the aspirin modification is currently unknown but could relate to some disruption in cofactor binding (such as heine) to the enzyme. The relative sensitivity of the platelet and endothelial enzymes to asprin has not been clearly defined. Acknowledgement--This work was supported in part by grant HL-20794 from the National Institutes of Health.

REFERENCES 1. BuRc~ J. W., BAENZIGER,N. L., STANFORD,N. and M~-~Rus, P. W. Proc. Natn Acad. Sci. USA 75, 5181-5184 (1975). 2. BURCH,J. W., STANFORD,N. and M~ERUS, P. W. J. Clin. Invest. 61, 314-319 (1978). 3. FERR~R~ S. H., MONCAD~ S. and VANF~J. R. Nature New Biol. 231, 23?-239 (19?l). 4. H ~ R G , M. and SAMUELS$ON,B. Proc. Natl Acad. Sci. 71, 3400-3404 (1974). 5. HEMLER,M. and LANDS,W. E. M. Lipids 12, 591-595 (1977). 6. HEm.~.~,M., LANDS,W. E. M. and S u r r ~ W. L. J. Biol. Chem. 251, 5575-5579 (1976). 7. IWA~,K. and ANOO,T. Methods Enzymo/. 11, 263-270 (I967). 8. JA~v., E. A. and WE~S~R, B. B. .I. Clin. Invest. 63, 532-535 (1979). 9. MI¥~OTO, T., O~3INO,N., YAu~O~O, S. and HAY~dsm, O. J. Biol. Chem. 251, 2629-2636 (1976). 10. OHK~,S,, OGn~o, N., Y~AMO~O, S. and HAYA~Sm,O. J. Biol. Chem. 7,54, 829-836 (1979). 11. ROM~,L. H., LANDS,W. E. M., ROTH, G. J. and M ~ u s , P.W.'Prostaglandins I1, 23-30 (1976). 12. RO~tH,G. J. and MAJ~RUS, P. W. J. Clin. Invest. 56, 624-632 (1975). 13. ROTH,G. J. and Slo~ C. J. J. Biol. Chem. 2A3, 3782-3784 (1978). 14. RoT~ G. J., Slog, C. J. and OZOLS, J. J. Biol. Chem. 255, 1301-1304 (1980). 15. RffrH, G. J., S'rA~O~D, N. and M ~ u s , P. W. Proc. Nail Acad. Sci. USA 72, 3073-3076 (1975). 16. SMI~, J. B. and WILLts, A. L. Nature New Biol. 231, 235-237 (1971). 17. SMI~, W. L. and LANDS,W. E. M. J. Biol. Chem. 246, 6700-6704 (1971). 18. VAN D~t OUDERg~, F. J., Bu'cr~Nt~, M., NUO~REN, D. H. and VA~ DOap, D. A. Biochim. Biophys. Acta 487, 315-331 (1977). 19. VAN DEit OUDEIUtA,F. J., BUYTENtl~K,M., SLIKKERV~R,F. J. and VAN DotP, D. A. Biochim. Biophys. Acta 572, 29-42 (1979). 20. VAN~,J. R. Nature New Biol. 231, 232-235 (1971). 21. WEIss, H. J., ALE~ORT,L. M. and Koc~wA, S. J. Clin. Invest. 47, 2169-2180 (1968). 22. WOODeUt¥, D. M. In The Pharmacological Basis of Therapeutics, 4th ed., pp. 314-347. (GOOUMA~,L. S. and GILMAN,A., eds.) MacMillan, New York, 1970.