Metabolism of [14C] arachidonic acid in the isolated rabbit spleen

PROSTAGLANDINS

METABOLISM OF [14C] ARACHIDONIC ACID IN THE ISOLATED RABBIT SPLEEN Toshihiro Hidaka, Patrick Y-K. Wong I and Kafait U. Malik 2 Department of Pharmacology, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163, USA ABSTRACT Infusion of [14C] arachidonic acid (AA) into the isolated, Tyrode perfused rabbit spleen resulted in the release of a substance into the venous effluent with the musculotropic activity and chromatographic properties of Drostaglandin (PG)E 2. Smaller amounts of radioactive materials with the chromatographi5 properties of PGF2s , 6-keto-PGFl~ , and PGD 2 were also released. The radiolabeled material released in largest amounts from the spleen was identified as PGE 2 on the basis of: i) Co-chromatography with PGE 2 in three solvent systems, 2) Conversion of the radioactive material and of authentic [jH] PGE 2 to similar products by treatment with sodium borohydride and with potassium hydroxide, and 3) Stability of the musculotropie activity in Tyrode solution at 37°C. Release of the major and minor radioactive products was inhibited by pretreatment of the spleen with either indomethaein or 5,8,11,14-eicosatetraynoic acid. INTRODUCTION Arachidonic acid, a 20-carbon unsaturated fatty acid which is released from tissue phospholipids by a wide variety of stimuli, is converted by mierosomal cyclo-oxygenase to an endoperoxide precursor of thromboxane (Tx)Az and PGI2, PGE2, PGF2e, and PGD2(1). There are reports that rat and guinea-pig spleen homogenates convert a considerable amount of AA into TxB2, the stable hydrolysis product of TxA 2 (2,3). Similarly, infusion of AA into the cat spleen or mechanical

A part of this work was presented at the fall meeting of the American Society for Pharmacology and Experimental Therapeutics, August, 1978, in Houston, Texas and the Fourth International Prostaglandin Conference in Washington, D.C., May, 1979. This work was supported by the National Heart, Lung and Blood Institute, National Institute of Health Grant 19134 and the American Heart Association Grant 77-803. i Recipient of Young Investigator Award, National Heart, Lung and Blood Institute, National Institute of Health. 2 Recipient of USPHS Research Career Development Award, National Heart, Lung and Blood Institute, National Institute of Health (5 KO4 HLO0142).

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PROSTAGLANDINS

stimulation of rabbit spleen slices released a short-lived substance, presumably TxA2, that caused contraction Bf rabbit aorta (4,5). Both TxA 2 and TxB 2 produce vasoconstriction and the latter substances potentiates vascular responses to adrenergic stimuli in the rat spleen (6,7). These observations suggest that the metabolism of AA to TxA 2 could have important implications in the regulation of vascular tone and the events at the adrenergic neuroeffector Junction in the spleen. However, the demonstration that A A p r o d u c e d vasodilation and reduced adrenergically-induced vasoconstriction in the isolated rabbit and rat spleen and that these effects of A A w e r e blocked by indomethacin suggests formation of products other than TxA 2 in this organ (7,8). Our recent findings that PGE2(7,8) and PGI 2 (unpublished work) mimics the inhibitory effects of A A o n the vascular tone and on the vascular responses to adrenergic stimuli in the isolated perfused rabbit and rat spleen indicated that AA could produce its action through conversion to PGE 2 and/or PGI 2. To test this view, we have investigated the incorporation and metabolism of [14C]AA in the isolated rabbit spleen perfused with Tyrode solution. The biological activity of the material(s) generated from AA on passage through the spleen was examined using a number of assay tissues, continuously superfused with the venous effluent. METHODS

Perfusion of the Spleen. Experiments were performed on albino rabbits of both sexes, weighing 2-3 kg. Rabbits were anesthetized with sodium pentobarbital (30 mg/kg, i.v.). The abdomen was opened by a midline incision, and the spleen was exposed. The splenic artery was separated from its surrounding tissue. Vascular connections of the spleen with the stomach and greater omentum were disconnected by placing ligatures. A small stainless steel tip cannula was inserted into the splenic artery and flushed with oxygenated Tyrode solution. The spleen was isolated and perfused at a constant rate of 5 ml/min using a Harvard peristaltic pump (model 1210) as described (7). Tyrode solution of the following millimolar composition was used: NaCI 137, KCI 2.7, CaCI 2 1.8, MgCI 2 I.I, NaHCO 3 12, NaH2PO 4 0.42 and D(+) Glucose 5.6. The perfusion fluid was maintained at a temperature of 37°C and aerated with a 95% 02 + 5% CO 2 mixture. The efflux of biologically active products (primarily PGs) from the rabbit spleen during the infusion of AA, was detected in the perfusate by continuously superfusing rat stomach strip (RSS), rat colon (RC), chick rectum (CR) and spirally cut strips of bovine coronary artery (BCA) according to the procedure described (9,10).

Eztraction, Separation by Thin-layer Chromatography and Analysis of Prostaglandins and Other Lipids Released in the Venous Effluent During the Infusion of [14~AA in the Isolated Rabbit Spleen. To label the phospholipid pool in the spleen, 5 ~Ci of [14C]AA was infused over a period of 20 minutes as described (11,12). The venous

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PROSTAGLANDINS

effluent was collected during the infusion of [14C]AA. The venous effluent (I00 ml) was acidified to pH 3.0 with formic acid and extracted 3 times with an equal volume of ethyl acetate. The solvent was combined and evaporated under vacuum. The extract was dissolved in chloroform/methanol (4:1, v/v) and applied to thin-layer silica gel chromatographic plates in parallel with PG standards. The plates were developed in two different solvent systems: PG(1), organic phase of iso-octane/ethyl acetate/acetlc acid/water (25:55:10:50, v/v), and PG(II), ethyl acetate/acetic acid (99:1, v/v). When the solvent system PG(1) was used, the plates were developed twice. The Rf values of 6-keto-PGFl~ , PGF2=, TxB2, PGE2, PGD2, PGA2, and PGB 2 and AA were approximately 0.19, 0.29, 0.43, 0.47, 0.64, 0.76, and 0.94 in solvent system PG(1) and 0.13, 0.18, 0.46, 0.32, 0.63, 0.74, and 0.86 in solvent system PG(II), respectively. TxB2, on the plates that were developed in solvent system PG(1), migrated close to PGE 2 but these compounds were well separated on plates developed in solvent system PG(II). On the other hand, 6-keto-PGFl= migrated close to PGF2~ on TLC plates developed in solvent system PG(II), but well resolved from it on plates developed in solvent system PG(1). The radiolabeled products on TLC plates were detected with a Packard radiochromatogram scanner and quantitated by liquid scintillation counting in Bioflour, after cutting each zone carefully according to the migration of standards and the radiolabeled material(s). The larger amount of the radiolabeled material that was detected on TLC plates in the zone corresponding to PGE 2 standard [solvent system PG(II)] was ~luted with methanol and dried by a stream of nitrogen. The extract was dissolved in absolute methanol at 0°C and subjected to sodium borohydride treatment or alkaline treatment with potassium hydroxide as follows:

8odium Borohydride Treatment. An aliquot of this solution containing 38 x 103 dpm in cold 2 ml absolute methanol was put into a test tube and an equivalent amount of [3H]PGE2 was dissolved in 2 ml of absolute methanol at 0°C in another test tube. Ten mg of sodium borohydride was added in both test tubes and the reaction was allowed to proceed for 20 min at 0°C and then for 60 min at room temperature. Thereafter, 3 ml of distilled water was added to the reaction mixture, and the reaction products were extracted 3 times with 5 ml of ethyl acetate, after adjusting to pH 3.5 with formic acid. The combined organic layers were washed with 3 ml of water, dried under a nitrogen gas stream and separated by TLC in solvent system PG(1). Potassium Hydroxide Treatment. A solution of the extract containing 25 x 103 dpm and an equivalent amount of [3H]PGE2 in 2 ml of absolute methanol were put into two separate test tubes and dried under a N 2 gas stream. These samples were then treated with 2 nLl of 1/15 N KOH (pH 13) in methanol at 37°C for 60 min. The reaction product was extracted and chromatographed as above. Evaluation of the Radiolabeled Material Detected in Zone Corresponding to PGE 2 by a Double Isotope Method. The identity of the major radiolabeled

product that comigrated with PGE 2 standard was

MAY 1980 VOL. 19 NO. 5

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PROSTAGLANDINS

further examined by a double isotope method. An aliquot of the lipid extract of the venous effluent, collected during infusion of [14C]AA into the spleen, was mixed with an appropriate amount of [3H]PGE 2 and applied to silica gel TLC plates. The platesDwere then developed in either the PG(I) or PG(lI) solvent systems. The PGE 2 zone from these p l a t e s w a s successively eluted and rechromatographed on thin-layer silica gel plates using different solvent s y s t e m s i n the following order: PG(I), iso-octane/ethyl acetate/acetic acid/water (25:55:10: 50, v/v) - PG(II), ethylacetate/water (99:1, v/v) and PG(III), chloroform/methanol/acetic acid (90:10:5, v/v) or in the order: PG(II)PG(I)-PG(III). The radioactive products on each TLC plate were detected using a radiochromatograph scanner and the zone corresponding to the major radioactive peak on each plate [i.e., TxB 2 + PGE 2 zone in the solvent system PG(I); PGE 9 zone in solvent system PG(II)] was eluted wlth methanol. The radloactlvlty correspondlng to [ C] and [ H] in PGE 2 zone was separately counted. The ratio of [14C]/[3H] in the PGE 2 zone of each TLC plate was calculated and compared by taking the ratio on the TLC plate as I00 percent. The radioactivity in zones other than PGE 2 was also measured on the second of the successive thin-layer developments. •

•

~

•

•

•

14

3

•

Biological Activity of Material(s) Eluted from Various Zones of TLC Plates. The venous effluent from the perfused spleen, before and during the infusion of [14C]AA and in the absence and presence of indomethacin, was collected and acidic lipids were extracted. A onetenth aliquot of the total extract was ehromatographed on silica gel plastic plates using the PC (I) and PG(II) solvent systems• The plates were divided into several zones according to the migration of radiolabeled products and PGs and TxB 2 standards. The products were eluted with methanol, dried and reconstituted into 500 ~i of saline and divided into two portions. One portion was used to measure the radioactivity and second portion was assayed for musclotropic activity using the isolated strip of the fundus of rat stomach, superfused with Krebs solution (3 ml/min, 37oc) containing indomethacin, 2 ~g/ml. Authentic FGE 2 and PGF2e were used as the standard and the contraction of the stomach strip was measured with a Harvard muscle transducer and recorded on a physiograph.

Materials [14C] arachidonic acid in toluene: methanol, 9:1 (specific activity 55 mCi/mmol) and [3H]PGE 2 in ethanol/water (7:3)(specific activity, 130 Ci/mmol) were obtained from New England Nuclear Co., and their purity was examined by thin layer chromatography before use. Five ~Ci of [14C]AA was dissolved in 2 ml of sodium carbonate solution (I mg/ml). Toluene in the solution was then evaporated with a stream of nitrogen gas for 15 min, and Tyrode solution was added to bring the final volume of the mixture to 4 ml (pH 9.0). Prostaglandin standards were gifts from Upjohn Co. Arachidonic acid (AA), indomethacin and 5,8,11,14-eicosa tetraynoic acid (ETYA) were purchased from Sigma Chemical Co. Preeoated plastic sheets (Polygram SIL G,

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MAY 1980 VOL. 19 NO. 5

PROSTAGLANDINS

0.25 nml, 20 x 20 mm) and precoa~ed glass plates (silica gel F 254, 0.5 ram, 20 x 20 mm) were obtained from Brinkman Instruments, Inc. The scintillation cocktail, Biofluor, was purchased from New England Nuclear Co. All other chemicals were of analytical grade. RESULTS

Release of PGE2-1ike Material Into the Venous Effluent During the Infusion of AA Into the Isolated Spleen of Rabbit Application of PGE2, I0 ng, directly into the fluid superfusing the assay tissues, caused a marked contraction of RSS, CR, RC and BCA; whereas PGI 2 in similar doses relaxed BCA and contracted all other assay organs (Fig. I). PGF2~ , 30 ng, caused a marked contraction of RSS, CR and RC and a small contraction of BCA. Prostaglandin D 2 and TxB 2 had 1/200th and 1/1000th the musculotropic activity of PGE 2 on the assay organs, respectively (not shown in Fig. i). During infusion of AA into the spleen, a material was released into the venous effluent which caused contraction of RS, CR, RC and BCA. Allowing the effluent from the spleen obtained during AA infusion to stand at 37°C or room temperature for 30 minutes, did not diminish the biological activity. Infusion of indomethacin, 1 ~g/ml, into the spleen abolished the output of the material that caused contraction of the assay organs but did not alter the response of the tissues to direct application of prostaglandins E2, F2~ , or 12 (Fig. I).

Radiolabeled Products Released Into the Venous Effluent During [14C]~ Infusion in the Absence and Presence of Indomethacin and ETYA Into the Isolated Spleen of Rabbit The venous effluent obtained ~uring an infusion of [14C]AA into the spleen contained 8.1% of the initially infused [14C]AA radioactivity. The largest amount of radioactivity extracted from the venous effluent was detected in the zone corresponding to PGE 2 standard in two solvent systems (Fig. 2 AI and BI). In addition, smaller amounts of radioactivity were detected in areas corresponding to PL, 6-keto-PGFl=, PGF2~ , PGD2, and PGA 2 standards. Very little radioactivity was detected in the TxB 2 zone. The average amount of radioactivity in various zones of the TLC plates, from 4 isolated spleens perfused with [14C]AA is shown in Fig. 3. The data presented in this figure was obtained from the TLC plates developed with solvent system PG(1) except for the amounts of PGE 2 and TxB2, which were quantitated from the TLC plates developed in the solvent system PG(1), because of poor resolution of these two products in the former solvent system. The venous effluent collected from the spleen 90 minutes after the termination of [14C]AA infusion-when the spleen still retained very high levels of radioactivity derived from incorporation of [14C]AA into the phospholipid poolsdid not contain an appreciable amount of the radiolabeled products. Infusion of either indomethacin. I ~g/ml, or ETYA, i0 ~g/ml, which neither altered the uptake of [14C]AA into the spleen, nor the release of radioactivity into the perfusate, inhibited conversion of [14C]AA into radiolabeled products corresponding to PGE2, PGF2= , PGD 2 and 6keto-PGF2= standards (Figs. 2 A2, B2, A3, B3 and 3).

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PROSTAGLANDINS

15om

16 n~n

I---INDO M E THAC I N - - % u g/ml--( IS)-~ PGE2 PGI2 PGF2w lOng lOng 30ng

AA l~Jg/ml

(DIR) (DIR)(DIR)

(IS)

SP (DIR)

AA PGI 2 1.ug/ml lOng (IS)

(DIR)

PGE 2 lOng

PGF2q 3Ong

(DIR)

(DIR)

FIGURE i: Efflux of prostaglandin (PG) E-like material into the venous effluent of isolated rabbit spleen during infusion of arachidonic acid (AA). The venous effluent was continuously superfused over the isolated rat stomach fundus strip (RSS), chick rectum (CR), rat colon (RC) and bovine coronary artery (BCA). AA, 1 ~g/ml, contained in Tyrode solution was infused into the spleen (IS). Prostaglandins E 2, F2~ and 12 were given as bolus injections directly (DIR) into the fluid superfusing the assay organs. The venous effluent collected from the spleen during infusion of AA was allowed to stand at 37°C for 30 minutes and superfused directly over the assay organs at a rate of 5 ml/min (SP). Indomethacin, 1 ~g/ml, contained in Tyrode solution was infused into the spleen.

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PROSTAGLANDINS

T

A,

900

f

cDm

1

SQ., TxB 2

PL)

O O OO O O 6kFld F2d

e2

O

D2 A2B2 A A

T

,To cpm

)RG.

•

SOL)F

0 0 0OX"20 0

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E2

0

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A3

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T

ORG

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B2

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SOL.

0 0 00e20Tx 0 6kFld F2d

E2

0

D2 A2B 2 A~

°;"'Co ooo,,P[ F2d E2 TxB2 D2 A2B2

FIGURE 2: Chromatograms of the radiolabeled products extracted from the venous effluent that was collected during infusion of [14C]AA into the spleen in the absence (AI and BI) and presence of indomethacin (A2 and B2) and ETYA (A3 and B3). Products from the extract were separated by TLC in two different solvent systems: iso-octane/ethyl acetate/acetic acid/water (25:55:10:50, v/v)(Al, A2 and A3) and ethyl acetate/acetic acid (99:1, v/v)(Bl, B2 and B3). PL = phospholipid; 6KFIe = 6-keto-prostaglandin FI~; E2, F2e , D 2, A 2 and B 2 = prostaglandins E2, F2e , D2, A 2 and B2; TxB 2 = thromboxane; AA = arachidonic acid; ORG = origin and Sol. F = solvent front.

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PROSTAGLANDINS

m no inhibitor ( n : 4) ,:.::~.'-::-:~:~,with indomethacin 1.ug/ml ( n : 2 ) I---I with ETYA l O . u § / m l ( n = 4 ) x

6

ItL

origin

6kFl~

F2ot

E2

TxB2

L D2

A2B2

FIGURE 3: Amount of radiolabeled products extracted from the venous effluent during infusion of [14C]AA into the spleen of rabbit in the absence and presence of indomethacin and ETYA. The products were separated by TLC and the amount of radioactivity in zones corresponding to the origin and areas of PGs and TxB 2 was determined by liquid scintillation counting. All the data were obtained from the TLC plates developed in iso-octane/ethyl acetate/acetic acid/water (25:55:10:50, v/v) except for the amounts of radioactivity corresponding to PGE 2 and TxB~ which were quantitated from TLC plates developed in ethyl acetate/acetic acid (99:1, v/v) because of poor separation of these two products in the former solvent system, n = denotes the number of spleens perfused. For abbreviations see Figure 6.

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PROSTAGLANDINS Effect of Sodium Borohydride and Potassium Hydroxide on the Radiolabeled Material R~leased from the Spleen During Infusion of [14C]AA and Detected in the Zone Corresponding to PGE2 The radlolabeled material that was released from the spleen during infusion of [14C]AAand comigrated with PGE 2 when treated with sodium borohydride was converted to more polar products having the chromatographic mobilities of PGF2e , and PGFpR on TLC plates developed in solvent system PG(1) (Fig. 4). Authentic~3H] PGE 2 treated with sodium borohydride was converted into products having a similar chromatographic profile. Treatment with potassium hydroxide (pH 13.0) dehydrated both authentic [3H] PGE 2 and the radiolabeled product formed from [14C]AA, to a less polar material having the chromatographic mobility of PGB 2 (Fig. 4).

Double Isotope Analysis of the Radlolabeled Material Released From the Spleen During Infusion of [14C]AA and Detected in the Zone Corresponding to PGE~ When the major radlolabeled product formed from [14C]AA in the spleen was eluted from the PGE 2 + TxB 2 zone of the TLC plates developed in system PG(1), and rechromatographed with solvent system PG(II) (Step II), 65-70% of [14C] radioactivity was detected in the PGE 2 zone; whereas 10-15% and 5-10% of [14C] radioactivity was localized in PGF2~ and TxB 2 zones, respectively. On the other hand, when the radiolabeled material eluted from the PGE 2 zone of the TLC plates developed in solvent system PG(II) was rechromatographed in solvent system PG(1) (Step II), 80-82% of the [14C] radioactivity was detected in the PGE 2 zone and 7-10% in the 6-keto-PGFl=zone. When the radiolabeled material recovered from the TLC plates in both series of experiments (Step II) was eluted and rechromatographed using chloroform/methanol/ acetic acid as the solvent system (Step III), all of the radioactivity appeared as a s i n g l e peak in the PGE 2 zone. Cochromatography of the major [14C] product formed "-^~ ..... [14C ]AA and the authentic [3H] PGE 2 in differentsolvent systems in successive steps resulted in a similar ratio of [14C]/[3H] after Step II (Table I). 35% of the radioactivity detected in the PGE 2 + TxB 2 zone on the TLC plates developed in solvent system PG(1) and at least 20% of the total radioactivity in the PGE 2 zone on the TLC plate developed in solvent system PG(II), was due to the presence of radioactive products other than PGE 2. Even allowing for this, PGE 2 was the major radiolabeled product released into the perfusate during infusion of [14C]AA into the spleen.

Bioassay of the Products ElutedFrom Various Zones of TLC Plates When the products released into the venous effluent of rabbit spleen before the infusion of [14C]AA were separated by TLC and analyzed using the RSS, a small amount of musculotropic activity (equivalent to an average of 1.0 ng of PGE 2) was found in the zone corresponding to PGE 2. The biological activity in the eluates prepared from other zones of the TLC plates was below the limit of sensitivity of the bioassay (< 0.5 ng). When the venous effluent

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813

PROSTAGLANDINS

o o o L~ '

~

% I ~'~

3000

~ ~ L I

3

~m

~ 6kr-~ F ~

~,~, o o o ; % o o / ' ~ ~A,,

E2

3000

Im

SOI-F D2 ~

t

SC ~F~

E2 O2 A2~

A^

~

FIGURE 4: Radiochromatograms of the product formed by treatment with sodium borohydride (NaBH4) and potassium hydroxide (KOH) of the major radiolabeled material, isolated from the venous effluent d~ring infusion of [14C]AA into the spleen (A), and authentic [ H]PGE9 (B). The products were separated by TLC using iso-octane/ ethyl acetate/acetic acid/water (25:55:10:50, v/v) as the solvent system. A1 and B1 = no treatment; A2 and B2 = NABH 4 treatment; A3 and B3 = KOH treatment. F2B = prostaglandin F2B. For the rest of the abbreviations see Figure 6.

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PROSTAGLANDINS

obtained during infusion of [14C]AA into the s p l e e n w a s similarly analyzed, the highest amount of the material with musculotropic activity was found in the zone corresponding to PGE 2 + TxB 2 [solvent system PG(1)] and PGE 2 [solvent system PG(II)] (equivalent to 32.2 ng and 32.4 ng of PGE2~ respectively). The eluate from the PGF2s zone [solvent system PG(1)] also showed musculotropic activity (equivalent to 4.0 ng of PGE 2 or 13.8 ng of PGF2e). These estimates obtained by bioassay were very close to the amounts calculated from the radioactivity, assuming no dilution with endogenous substrate. The amounts of materials in the PGE 2 + TxBp zone [solvent system PG(1)] and PGE 2 zone [solvent system PG(1) T and PGE 2 zone [solvent system PG(II)] calculated fro m the radioactivity as PGE 2 were 39.9 ng and 41.3 ng, respectively, and the content of the material in PGF2s zone [solvent system PG(1)] obtained from the radioactivity was 17.9 ng. In experiments, where [14C]AAwas infused into the spleen in the presence of indomethacin, eluates from various zones of TLC plates, including the PGE 2 and PGF2~ zones, showed no musculotropic activity. TABLE I Double Isotope Analysis of the Major Radiolabeled Product in PGE 2 Zone of TLC Plates

Experiment

Solvent

Rf value

System

II

[14C]

[3H]

[14C]/[3H]

Recovery

Counts/Minute

%

(I) PG(1) (2) PG(II) (3) PG(III)

0.46 0.33 0.64

2557 1245 737

15949 11969 7060

0.160 0.104 0.104

I00 65 65

(i) PG(1) (2) PG(II) (3) PG(III)

0.31 0.47 0.64

1440 948 671

13591 11099 7763

0.106 0.085 0.086

i00 80 81

DISCUSSION Our studies on the metabolism of AA in the isolated spleen perfused with Tyrode solution leads to the following conclusion. AA is incorporated into the phospholipld pool in the spleen and a part of it is converted into various cyclo-oxygenase p r o d u c t s ; t h e major product resembling PGE 2 in its biological and chromatographic characteristics. When [14C]AAwas infused into the spleen, it was taken

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PROSTAGLANDINS

up by the tissue and only a small portion (8% .of the ~otal [14C] radioactivity) was recovered in the perfusate. The major part of the radioactivity in the spleen was incorporated into phospholipids, primarily phosphatidylcholine (unpublished observations). Studies on the chromatographic mobility of the products released into the venous effluent, during the infusion of [14C]AA into the spleen, indicated that this fatty acid was metabolized principally to PGE 2 and, to a lesser extent, to 6-keto-PGFl= , PGF2= and PGD 2. This conclusion is in agreement with our finding that the infusion of AA into the spleen released a stable material having musculotropic activity that closely resembled PGE 2. Preferential metabolism of AA in rabbit spleen to PGE 2 contrasts to metabolism of AA in other organs of this species. For example, in the isolated rabbit heart, the major product of AA metabolism was 6-keto-PGFl= and only a small amount of PGE 2 and PGF2¢ were observed (13). In the kidney, the principal products released during the infusion of A A w e r e 6-keto-PGFl= and PGE 2 (14). These observations indicate that the activity of enzymes participating in the formation of various products of AA, viz, PGI 2 synthetase, PGE 2 isomerase and PGF2e reductase may vary in different organs of rabbit. TxB2, the stable derivative of T x ~ , which was identified as one of the principal products of AA metabolism in rat and guineapig spleen homogenates (2,3), was formed in very small amounts in the rabbit spleen during the infusion of [14C]AA. Inhibition of the production from [14C]AAof radiolabeled products having the TLC characteristics of PGE2, 6-keto-PGF _, PGF2~ and TxB 2 by indomethacin (15), or ETYA (16), indicates that these products are derived by the cyclo-oxygenase pathway in the rabbit spleen. The radiolabeled products that were released in the venous effluent were probably not derived indirectly from [14 C]AA incorporated into phospholipids, but directly from [~4C]AA infused into the spleen because no appreciable amount of the products were detected in the venous effluent collected 90 minutes after the termination of infusion of [14C]AA, even though the spleen retained high levels of incorporated [14C]AA. This view was supported by our observation that the specific activity of the released PGE2, calculated from the ratio of musculotropic activity to radioactivity, was that of the infused substrate. Analysis by TLC in different solvent systems and double isotope analysis showed that the principle radiolabeled product formed in larger amounts from [14C]AAclosely resembled PGE 2. Sodium borohydride and potassium hydroxide converted both the radiolabeled product of [14C]AA and authentic [3H]PGE 2 to derivatives having similar chromatographic mobillties. Moreover, bioassay of the products of [14C]AA eluted from various areas of TLC plates showed musculotropic activity malnly-in the PGE 2 zone that was absent in experiments performed in the presence of indomethaein. Thus, PGE2, is most likely the product contributing to the biological activity of the material in the venous effluent released during infusion of AA into the rabbit spleen. Whether the metabolism of exogenous AA infused in the isolated perfused rabbit spleen occurs primarily in the smooth muscle cells or blood vessels supplying the spleen is not known. Since all the bio-

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chemical pathways for the transformation of PG endoperoxide to PGs including PGI2, PGE2, PGF2~, and PGD 2 are potentially available in the intact vessel wall, except for the pathways involved in TxA 2 production (17), it is possible that at least a part of the product formed from [14C]AA in the isolated perfused rabbit spleen could result from its metabolism in the splenic vessels. Supporting this view are our preliminary results that in the isolated perfused extracapsular vessels of the spleen, [14C]AA was converted mainly to a product with the TLC characteristics of PGE 2 and to a minor extent to 6-ke~o-PGFl= (unpublished work). The results of this study taken together with the release of PGE 2 by sympathetic nerve stimulation and norepinephrine (18) and the inhibitory effect of PGE 2 and its precursor AA (7,8) on the vascular responses to adrenergic stimuli strongly suggest that PGE 2 may function as a modulator of adrenergically-induced vasoconstriction in the isolated rabbit spleen. ACKNOWLEDGEMENTS We wish to thank David Ash and Nguyen K.L. Khanh for the technical assistance and Susan Mangum for typing this manuscript. We also thank Dr. L. Cagen for his helpful suggestions and Dr. John J. Pike of Upjohn Co. for supplying prostaglandins and thromboxane B 2. REFERENCES

1)

Moncada, A. and J.R. Vane. Arachidonic acid metabolites and the interactions between platelets and blood-vessel walls. N. Engl. J. Med. 300:1142, 1979.

2)

Pace-Asciak , C.R. and G. Rangaraj. Distribution of prostaglandin biosynthetic pathways in several rat tissues. Formation of 6-ketoprostaglandin FI=. Biochim. Biophys. Acta 486:579, 1977.

3)

Hamberg, M. On the formation of thromboxane B 2 and 12L-hydroxy5,8,10,14-eicosatetraenoic acid (12 ho-20:4) in tissues from the guinea-pig. Biochim. Biophys. Acta 431:851, 1976.

4)

Nijkamp, F.P., S. Moncada, H.L. White and J.R. Vane. Diversion of prostaglandin endoperoxide metabolism by selective inhibition of thromboxane A 2 biosynthesis in lung, spleen or platelets. Europ. J. Pharmacol. 44:179, 1977.

5)

Gryglewski, R. and J.R. Vane. The release of prostaglandins and rabbit aorta contracting substance (RCS) from rabbit spleen and its antagonism by antiinflammatorydrugs. Brit. J. Pharmacol. 45:37, 1972.

6)

Svensson, J. and B.B. Fredholm. Vasoconstrictor boxane A 2. Acta Physiol. Scand. 101:366, 1977.

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effect of throm-

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Malik, K.U. and J.C. McGiff. Modulation of adrenergic transmission by prostaglandins (PG) in the isolated splenic vasculature of rabbit and rat. Fed. Proc. 35:297, 1976.

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Vane, J.R. The release and fate of vasoactive hormones in the circulation. Brit. J. Pharmacol. 35:209, 1969.

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Gilmore, N., J.R. Vane and J.H. Wyllie. by the spleen. Nature 218:1135, 1968.

ii)

Isakson, P.C., A. Raz, and P. Needleman. Selective incorporation of 14C-arachidonic acid into the phospholipids of intact tissues and subsequent metabolism to 14C-prostaglandins. Prostaglandins 12:739, 1976.

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Isakson, P.C., A. Raz, S.E. Denny, A. Wyche and P. Needleman. Hormonal stimulation of arachidonate release from isolated perfused organs. Relationship to prostaglandin biosynthesis. Prostaglandins 853:853, 1977.

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Isakson, P.C., A. Raz, S.E. Denny, E. Pure and P. Needleman, A novel prostaglandin is the major product of arachidonic acid metabolism in rabbit heart. Proc. Natl. Acad. Sci. 74:101, 1977.

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Needleman, P., S.D. Bronson, A. Wyche, M. Sivakoff and K.C. Nicholaou. Cardiac and renal prostaglandin 12. Biosynthesis and biological effects in isolated perfused rabbit tissues. J. Clin. Invest. 61:839, 1978.

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Vane, J.R. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature (New Biol) 231:232, 1971.

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Downing, D.T., D.G. Ahern and M. Bachta. Enzyme inhibition by acetylenic compounds. Biochem. Biophys. Res. Commun. 40:218, 1970.

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Bunting, S., R. Gryglewski, S. Moncada and J.R. Vane. Arterial walls generate from prostaglandin endoperoxides a substance which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation. Prostaglandins 12:897, 1976.

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Hidaka, T. and Malik, K.U. Prostaglandin E 2 is the major product of arachidonic acid released by sympathetic nerve stimulation (SNS), administered norepinephrine (NE) and bradykinin (BK) from isolated perfused rabbit spleen. Fed. Proc. 38:751, 1979.

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