Prostaglandins Leukotrienes and Medicine 11: 419-430, 1983

EFFECTS OF BRAOYKININ AND BOVINE SERUM ALBUMIN ON ARACHIDONIC ACID AND PROSTAGLANDIN RELEASE FROM PERFUSED RAT HEART

Jeffrey Paul and J. E. Kinsella*, Institute of Food Science, Cornell University, Ithaca, NY 14853

Abstract The isolated perfused rat heart was used to study if arachidonic

acid (AA) release affected prostaglandin (PG) production. The perfused heart avidly absorbed and esterified exogenous AA, mostly into polar lipids. The administration of bradykinin (BK) after prelabeling with l-Cl'+-AA caused a two-fold increase in the amount of AA released. Bovine serum albumin (BSA) stimulated release 2.5fold. Perfusion with AA sa- turated BSA enhanced the release of l- I%-AA 23-fold indicating the pre- sence of an active deacylation/reacylation system in rat heart.

BK caused a four-fold increase whereas BSA had no effect on the amount of prostaglandins released from perfused heart indicating that BK acted on a specific AA pool available to cyclooxygenase whereas the BSA stimulated a separate pool of AA which apparently was not available to the cyclooxygenase. Introduction

Prostaglandins (PG) and related substances play key roles in modu- lating numerous physiological functions and may be involved in diseases such as atherosclerosis, hypertension, and gastrointestinal ulcers (l-4). Although much research has been devoted to determine the physiological actions of PG's, the mechanisms controlling their biosynthesis remain to be elucidated (3). Experiments performed in the early 1970's supported the hypothesis that substrate (arachidonic acid) availability was the rate limiting step in PG synthesis. Researchers usin bradykinin (BK), a pep- tide known to stimulate the synthesis of PG's (4 4 , and mepacrine, a phospholipase A, inhibitor, showed that the BK effect was rendered inac- tive if the latter was concurrently administered to the perfused lung (5). Furthermore, if phospholipase A2 was infused into perfused lungs it stim- ulated PG release (5) while mepacrine inhibited these actions. These studies suggested that availability of free AA controlled PG synthesis (596).

If substrate availability is the rate limiting step, then there should be an increase in the concentration of AA during or just prior to the pro- duction and release of PG's. Isakson et al. (6,7) and Hsueh et al. (8,9) demonstrated that when BK was administered to the perfused rabbit heart, the release of free AA was not observed unless defatted bovine serum albumin (BSA) was present in the perfusate. These data suggested that the principal action of BK in stimulating PG synthesis was via its ability to activate

419

phospholipase AZ. However, detection of AA was impossible without the con- comitant administration of defatted BSA to bind the released AA. In the absence of this exogenous binding, these authors claimed that a very active reacylation system exists in the heart which quickly reacylates the excess AA released by BK action.

Spector (10) concluded that the FFA/albumin molar ratio controls the amount of FFA released or taken up by tissues. A high FFA/albumin ratio permits mOre FFA to enter cells whereas when defatted albumin is used, the flux of FFA is extracellular as a result of the strong binding affinity of the albumin for FFA.

A number of basic questions remain to be answered concerning the role of substrate availability and prostaglandin synthesis: Is the AA released by tissue in response to stimulants (BK and BSA) derived from the same pools of esterified AA and is the increased AA release asso- ciated with a concomitant increase in PG synthesis?

Because the freshly perfused heart is physiologically responsive (6,8) and facilitates study of fatty acid uptake and release and PG synthesis we studied the effects of BK and BSA on AA release and deter- mined if a relationship existed between the amount of AA released and PG produced in the perfused rat heart.

Materials and Methods

All solvents (analytical grade) were purchased from Mallinckrodt (St. Louis, MO). The l-14C-Palmitic acid (10 pCi/mMole) was obtained from New England Nuclear (Boston, MA) while 5,6,8,9,11,12 3H arachidonic acid (120 pCi/mMole) was obtained from Amersham (Arlington, Heights, IL). Authentic standards and standard mixture of fatty acid methyl esters for gas chromatographic analyses were purchased from Nucheck Prep. (Elysian, MN). Lipids, prostaglandins for thin layer chromatography, bovine serum albumin and bradykinin-lysine were obtained from Sigma (St. Louis, MO).

Heart Perfusion. Sprague Dawley rats weighinq 200-300 g were in- jected with sodium heparin (1 unit/100 g) to prevent clotting 30 min before sacrifice. Diethyl ether was used to anaesthetise the rats. The beating heart was excised and placed in ice-chilled buffer. The aorta was cannulated with an 18 gauge blunt-tipped needle and perfusion was immediately initiated (11). A classical Lanqendorff preparation was used in which the perfusate enters the coronary arteries by retrogade flow through the aorta. A modified Krebs-Heinseleit buffer (11) con- taining half the normal concentration of calcium and magnesium equili- brated with 02:C02 (95:5) at 37"C, pH 7.4 was used. The flow was kept constant at 10 ml/min using a peristaltic pump (Polystatic Pump, Buchler Instruments).

The perfused heart was allowed to stabilize for 15 min before each experiment was begun. The labeled fatty acid (palmitic or arachidonic) was infused by a peristaltic pump (Manostat Cassette Pump, Manostat, New York, NY) at 0.3 ml/min for 15 min to label the cardiac lipid pool. After this labeling period, buffer was pumped through the heart for 25 min to ensure the removal of all nonspecifically absorbed fatty acids (7). At the end of the washout period, 70 ml of perfusate was taken for analysis of PG and FA content. This served as the control.

420

The following treatments were administered to assess the effect of BK and serum albumin on AA and PG release:

(i) Bradykinin (20 pg) was given as a bolus injection proximal to the aortic loop and the perfusate (70 ml) was collected during and after hormone injection.

(ii) Defatted BSA was infused for 15 min at a rate of 0.3 ml/min. Diluted by the perfusate it had a final concentration of 3.3 mg/ml, which is the physiological concentration. Collection of the perfusate (70 ml) was begun 5 min after the onset of the BSA infusion.

(iii) Fraction V BSA (non-defatted) was administered as described in (ii) and 70 ml of perfusate collected.

(iv) Defatted BSA saturated with AA (1:6 BSA:AA molar ratio) was given as described in (ii) and 70 ml of perfusate collected.

Imnediately following each experiment the heart was frozen in liquid N, and stored at -70°F for subsequent lipid analysis.

Extraction and Separation of Heart Lipids. Cardiac lipids were ex- tracted using 20 volumes of chloroform/methanol 2:l (12). The washed chloroform layer was evaporated to dryness and the total lipids were separated into the major lipid classes by thin layer chromatography (TLC). Authentic lipid classes were cochromatographed to facilitate identification. The solvents used were chloroform:methanol:acetic acid: water (30:15:4:1) for the separation of phospholipid classes, and hexane: diethyl ether:acetic acid (80:20:1) for the separation of neutral lipid classes (13).

The radioactivity in the separated lipid classes was measured by a Packard Tricarb 3385 using Liquiscint (13). For gas chromatographic analyses, duplicate aliquots of separated lipid classes were scraped from the thin layer plates and methylated with boron trifluoride. Gas liquidchromatographywas done using a Hewlett-Packard 5880A (14). A stainless steel column (3m long x 32 mm diameter), packed with 10% silar 1OC on Gas Chrom P (100/120) was employed. The temperature was pro- grammed to increase from 170C to 220C at 4°C per min.

Detection of Prostaglandins in Heart Perfusate. Immediately follow- ing t e co ection 0 hadfed (0.005%) and the contents were transferred to a round bottom flask and lyophilized. The powder was reconstituted with 15 ml of water, acidified to pH 4.0, and extracted twice with equal volumes of ethyl acetate. The pooled solvent layers were evaporated to dryness under N2, and the extract was applied to a silica gel G plate. The plates were developed twice to separate the PG's from FA's and other impurities in a solvent system composed of chloroform: isopropanol:ethanol:formic acid (45:5:0.3:0.2)

61",;, (PGE2, PGF2, The prostaglandins and AA bands corresponding to authentic stan-

, 6 keto-PGF1,) were scraped from the thin layer plates, the PG bands were pooled and the radioactivity was measured as described above.

421

Preparation of Sodium Palmitate and Sodium Arachidonate. The solu- ble sodium salts were used for perfusion studies. Fatty acid soaps were prepared by placing the appropriate amount of radioactive FA (105-lo6 cpm/ml) in a vial and evaporating the solvent under nitrogen. Sodium carbonate (100mM) and ethanol (150 ~1) were added, and the mixture was sonicated for 20 min at 30°C. Then the pH was adjusted to 7.0 and this solution was used as source of fatty acids for the perfusion experiments.

Results

Incorporation and Distribution of Radioactive Arachidonic and Palmitic Acid. This study was conducted to determine the distribution of infused radioactive fattv acids into cardiac lipids. The isolated heart was stabilized for 15-min and then the radioactive fatty acid was infused for 15 min. Aliquots (1 ml) of perfusate were collected every minute and the total radioactivity in these was determined. When infusion of labeled fatty acid was terminated, there was a sharp de- crease in radioactivity in the perfusate (Figure 1). Approximately 60% of the administered 20:4 was retained by the heart tissue.

Infusion Period Washout Period

??- radioactivity in perfusate A - incorporation into heart

T!ME (minutes)

Figure 1. The uptake of exogenous labeled arachidonic acid by perfused rat heart.

The radioactive arachidonic (2 x lo5 cpm) was infused into the heart at 0.34 ml/min for 15 min. The perfusate was collected. The radioactivity in 1 ml aliquots corresponding to l-min intervals were counted and samples were also taken at 16 and 25 min.

422

The absorbed fatty acids were predominantly esterified (Table 1). About 70% of the radioactivity in the neutral lipid (NL) was in the triglycerides (Table 1). The percentage radioactive AA esterified in the phospholipid classes and NL was proportional to the relative quan- tity of endogenous esterified arachidonic acid in these lipids, i.e. those classes containing a high amount of AA also contained a correspon- ding amount of the radioactive AA. However, this was not the case with phosphatidylethanolomine (PE) (Table 2) which contained a large amount of endogenous AA but relatively low quantities of the radioactive AA. An analysis of variance of the data showed a significant difference between the levels of endogenous AA and the levels of radioactive AA in the PE class (F < .005) (16). An isotope effect was ruled out because both 14C and 3H labels were used with identical results. Similar pat- terns resulted when palmitic acid was used to label the esterified lipid classes. These data indicated that within the time span of these exper- iments, all the lipid classes, except for PE, apparently had reached equilibrium with perfused AA.

Table 1. Oistribution of radioactive arachidonic acid in lipid classes extracted from the perfused rat heart.

Lipid Class Percent Radioactivity

Phosphatidylcholine (PC)

Phosphatidylinositol Phosphatidylserine $1

Phosphatidylethanolamine (PE)

Free Fatty Acids ( W

Triglycerides (TC)

Cholesterol Esters (CE)

68.5k1.5

13.6k2.1

0.523.1

0.622.6

19.424.5

8.6k3.7

The heart perfusion was initiated and stabilized for 15 min at a flow rate of 10 ml/min. Then the radioactive arachidonic acid (120 Ci/nmole) (2 x lo5 cpm) was infused at 0.34 ml/min for 15 min. After 1.5 hours of washout with buffer the perfusion was terminated and the lipids from the heart were extracted. The lipids were fractionated into the major lipid classes by TLC and the radioactivity associated with each lipid class was determined as described in the methods.

Release of Labeled Fatty Acids from Perfused Heart. The following studies were conducted to monitor the release of fatty acids and to determine if the quantity fusate was related to the

of prostaglandins recovered in the heart per- amount of AA released. For comparison, two

423

Tabl

e 2.

A

comp

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th

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di

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end

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and

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and

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arac

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and

palm

itic

ac

ids

in t

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ster

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of

th

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t he

art

(F < .0

05).

Arac

hido

nic

Acid

Pa

lmit

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id

(% t

otal

AA

in

eac

h li

pid

clas

s)

(% t

otal

pa

lmit

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id in

eac

h li

pid

clas

s)

Lipi

d Cl

ass

Endo

geno

us

Radi

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Endo

geno

us

Radi

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PC

51.7

+

5.5

58.6

+

11.5

36

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_ 7.

9 42

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8.4

PI,P

S 12

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9.8

10

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3.5

6.8

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0.8

% PE

21

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9.9

8.0

i 3.

8 18

.4 5

8.7

8.3

+ 0.

9

NL

20.9

+

8.5

24.8

+

9.2

38.0

+

14.7

47

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7.0

N=

9 12

9

3

- __

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Th

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art

perf

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at a

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10

ml/

min.

Af

ter

15 m

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st

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th

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tty

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was

term

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d th

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pids

fr

om th

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were

ex

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ted

and

two

samp

les

were

fr

acti

onat

ed

in p

aral

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by TL

C.

The

band

s fr

om on

e sa

mple

we

re sc

rape

d an

d th

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an

alyz

ed by

GL

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band

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we

re sc

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ity

coun

ted

in a

sci

ntil

lati

on co

unte

r as

de

scri

bed

in m

etho

ds.

(Leg

end:

PC

= p

hosp

hati

dylc

holi

ne;

PI =

pho

spha

tidy

lino

sito

l;

PS =

pho

spha

tidy

lser

ine;

PE

= p

hosp

hati

dyl-

et

hano

lami

ne;

NL =

neut

ral

lipi

d)

F r(

.005

treatments, both of which cause the mobilization of free AA, were evalu- ated. The first involved bradykinin (BK), a hormone which causes the release of PG's from heart tissue (17). The mechanism for activation of PG synthesis apparently involves a BK receptor in the plasma membrane with subsequent activation of phospholipase AZ, mobilization of AA and conversion to PG by cyclooxygenase (18). The other treatment was with bovine serum albumin (BSA), the natural transporter of free fatty acid (FFA) in serum. The level of FFA entering tissue cells is a function of the FFA:albumin ratio (19). A low FFA:albumin ratio results in a lower uptake of FFA by the cell. If defatted albumin is administered, there is an efflux of FA from tissues (7) and in that process, lipase is activated to replace the lost FFA (20). Therefore, the question posed was, does AA mobilized by BSA stimulate AA release and PG synthesis to the same extent as when BK is administered?

BK injection caused a 2.1 f l.O-fold increase in the release of radioactive AA in the perfusate (Figure 2). Defatted albumin caused a 2.5 + 0.5-fold increase. Therefore, defatted albumin and moderate doses of BK (20 Pg) were comparable in their ability to mobilize esterified radioactive AA from heart tissue.

22

2.09 t 0.96 2.6 f 0.47

I I 1.47 t 0.47

I I

A S C

TREATMENTS

.a : 4.0

Fiqure 2. The release of previously esterified radioactive arachidonic acid from perfused heart after bradykinin stimulation and serum albumin infusion.

425

The heart perfusion was initiated and then radioactive arachidonic acid (2 x lo5 cpm) was infused for 15 min. After a 25-min washout per- iod, 70 ml of perfusate were collected. Then one of the following treatments were administered to the heart: A) bradykinin (20 pm); B) defatted bovine serum albumin (100 mg/ml) infused at 0.3 ml/min; C) fraction V BSA (100 mg/ml infused at 0.3 ml/min); or D) arachidonic acid bound to defatted BSA (6:l) infused at 0.3 ml/min (100 mg/ml).

After the treatment was administered, a perfusate sample (70 ml) was collected and both samples were analyzed for radioactive arachidonic acid. The results are expressed as treatment/control. A ratio above 1.0 signifies an increase, below 1.0 a decrease. Values represent means and standard deviation from 3-4 experiments (l p < 0.1, 2 p < 0.025).

To further characterize the release of AA caused by BSA, two addi- tional treatments were examined. Thus, fraction V albumin with a FFA: BSA ratio of 2:l (the FFA was a mixture of 16:0, 18:0, 18:l and 18:2) and BSA which contained bound AA (AA:BSA ratio of 6:l) was used. If defatted albumin mobilized AA by reversing the FFA flux, then fraction V should stimulate less FFA mobilization becasue its binding affinity is one order of magnitude less than defatted serum albumin (10). The BSA-AA treatment was administered because it was thought that some of the bound AA would be taken up by the cells and possibly stimulate PG biosynthesis.

The infusion of fraction V BSA resulted in a 1.47 i 0.5-fold in- crease in AA release. This was less than that obtained with the de- fatted albumin as expected because the ratio of FFA to albumin is 2:l while defatted albumin is virtually free of lipid. The BSA-AA caused a 22.8 + 4.0 fold increase in the amount of radioactive AA released into the perfusate. This apparently reflected the ability of the AA bound to BSA to enter the tissue and reduce, via dilution, the amount of labeled AA reesterified into the tissue lipids and hence more was released into the perfusate. This indicated the existence of an active deacylation-reacylation cycle in the heart tissue.

Whereas BK had a negligible impact on release of labeled palmitic acid (PA) from hearts prelabeled with 1-14C-palmitic acid defatted and fraction V BSA caused a 5 _+ 1.3 and 27 i 4-fold increase in release of labeled palmitic acid. These data indicated that whereas the effect of BSA was non-specific that of BK was selective for arachidonic acid.

Release of Labeled Prostaglandins from the Perfused Heart. If the release of PG's is controlled by the mobilization of free arachidonate, then the level of PG's in the perfusate should reflect the release of free AA. Thus ranking the effects of various treatments on AA release from the highest concentration to lowest, the expected PG levels in re- spective perfusates should be AA-BSA > defatted BSA > BK > fraction V

426

BSA. This pattern was not found. Only the BK treatment augmented the release of PG's with a 4.4 + 2.9-fold increase in the release of PG's (Figure 3). Most of the radioactivity was associated with 6-keto F,,. The other treatments did not stimulate nor inhibit the release of PG's. Hence the release of radioactive AA by albumin did not result in a con- comitant increase in release of PG's labeled.

4.44 f 2

A s C D

TREATMENT3

Figure 3. The release of radioactive prostaglandins from the perfused heart after infusion of bradykinin or serum albumin.

The heart perfusion was initiated and then radioactive arachidonic acid (2 x lo5 cpm) was infused for 15 min. After a 25-min washout period, 70 ml of perfusate were collected. Then one of the following treatments were administered to the heart: A) bradykinin (20 ug); B) defatted bovine serum albumin (100 mg/ml) infused at 0.3 ml/min; C) fraction V BSA (100 mg/ml infused at 0.3 ml/min); or D) arachidonic acid bound to defatted BSA (7:l) infused at 0.3 ml/min (100 mg/ml).

After the treatment was administered, a perfusate sample (70 ml) was collected and both samples were analyzed for radioactive prostaglandins.

The results are expressed as treatment/control. A ratio above 1.0 signifies an increase, below 1.0 a decrease.

427

(l Values represent means and standard deviation from 3-4 experiments

p < 0.025, 2 P < 0.0025).

Discussion

Bradykinin apparently causes the release of PG's from perfused organs via stimulation of phospholipase A2 (4-9).

Efforts by Isakson (6,7) and Hong and Levine (21) using isolated, perfused rabbit heart and cultured cells, respectively, whose esterified lipid pools were labeled with radioactive AA, showed that BK released the previously acylated AA only when defatted BSA was administered con- currently. Both investigators speculated that the acyltransferase systems were so active that the hormonal induced release of AA was not observed because of very rapid reacylation of the released AA.

lease The results showed that BK was quite specific for stimulating re- of AA. These results are in accordance with previous findings of

Hsueh (9) who found that after labeling the perfused rabbit heart with palmitic acid, BK did not release any radioactivity. Our study showed that BK caused an insignificant release of palmitic acid.

The present study showed that defatted BSA infused at physiological concentrations caused a 2.5-fold increase in the release of labeled AA. Other workers (7,9) using the perfused rabbit heart preparation reported a 25- and 14-fold increase, respectively, in release of labeled AA. The effect of serum albumin on FFA release was further examined by admini- stering fraction V albumin. If defatted albumin mobilizes AA merely by shifting it extracellularly, then fraction V should release less AA than defatted BSA. This was found to be true because the fraction V albumin in which only the low affinity binding sites were available, mobilized significantly less labeled AA. Evans (22) also reported that the per- fused heart released less FFA as the FFA:BSA was increased presumably because fewer binding sites were available on the BSA.

However, when AA bound to defatted albumin (6:lj was infused it caused a 22-fold increase in amount of labeled AA released. This was explained by the existence of an active deacylation-reacylation cycle. Thus, the BSA-bound AA upon entering the cell exchanged with the intra- cellular pool of labeled AA during the deacylationlreacylation cycle resulting in the release of more labeled AA in the perfusate.

Since the AA-BSA treatment (FFA:BSA, 6:l) caused a large increase in the release of labeled AA and fraction V BSA stimulated a comparable release of labeled palmitic acid (data not shown), it was suggested that there is preferential acylation of arachidonic acid. That is, the vari- ous fatty acids in the mixture of FFA bound to the fraction V BSA did not compete with AA for the acyltransferases, i.e. the acyltransferase for AA is apparently specific. Hiroko (23) has recently reported an AA specific acyltransferase isolated from liver microsomes and a similar selectivity for AA acylation has been observed in cultured kidney cells (24).

428

An objective of the present study was to test the hypothesis that the quantity of PG's synthesized is a function of the concentration of free AA in the system. Thus BK and BSA, both free fatty acid mobilizers, were infused into the perfused rat heart and compared for their ability to stimulate the release of PG's.

Bradykinin, the hormone, was an effective stimulator of PG synthesis while BSA, the FFA transporter, was not. This difference cannot be attributed to levels of substrate (i.e. AA) because both the treatments caused approximately a Z-fold increase in release of labeled AA. Speci- ficity was demonstrated in the BK treatment while the serum albumin infusions did not alter the concentration of PG's released. Several mechanisms could explain how BK selectively stimulates PG production They include increasinq phospholipase A, activity (8); decreasing acyl- transferase activity, i.e. retarding reacylation (25); compartmentali- zation of an active PG precursor pool (26,27); direct stimulation of the cyclooxygenase (28), and/or coupling of the deacylation-reacylation to prostaglandin synthetase (29). Further studies to determine the mech- anism(s) are warranted.

Acknowledgement

This work was supported by USDA-SEA Grants No. 5901-0410-9-0286 and 8200065.

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