PROSTAGLANDINS

THEINFLUENCEOFUNSATURATEDFATTYAC1DSONPROSTAGLAND1NRELEASEIN ISOLATED PERFUSEDGUINEA-PIGHEARTS P. Ments and W. Forster From the Research Association for Cardiovascular Diseases in the German Democratic Republic, Department of Pharmacology and Toxicology, Martin-Luther-University, Halle-Wittenberg, Halle/Saale , Leninalee 4, German Democratic Republic ABSTRACT Isolated perfused hearts of guinea-pigs continuously release a prostaglandinlike substance into the effluent. Polyunsaturated fatty acids, linoleic acid and arachidonic acid >linolenic acid, induced an increase in PO-efflux . Oleic acid was without effect. The results suggest that PG-formation is involved in the increase of contractile force and coronary flow after administration of linoleic, Hnolenic and arachidonic acids. ACKNOWLEDGEMENTS We wish to thank Dr. J . Pike, The Upjohn Comp . Kalamaeoo, for the gift of prostaglandins . The skillful technical assistance of Mrs. Voight and Mrs. Zobel is gratefully acknowledged. INTRODUCTION Recent investigations indicate that isolated perfused hearts continuously release prostaglandin-like substances (PLS) (1,2,3,4) . This output was inhibited by indomethacin (2). On the other hand infusion of noradrenaline or acetylcholine (4) , application of adenine nucleotides (5) or hypoxia (2,4) stimulated prostaglandin-release, Contradictory results were reported concerning PG-release, heart performance and coronary flow. Wennmalm (4) showed that a close correlation exists between hypoxia and PG-release which suggests that liberated PGs play a role in the development of reactive hyperemia, whereas changes in the mechanical activity of the heart did not induce PG-efflux . Block et al. (2,3) concluded from their experiments that PG-efflux is due to active heart contraction. Mechanical distortion and trauma or an Increase in pre- and afterload were regarded as important stimuli for prostaglandin-release . Furthermore the authors suggest that PGrelease does not contribute to the maintenance of coronary blood flow or the genesis of anoxic-induced vasodilator response. In order to explain these differences, further investigations on the influence of substrates and ion composition seem to be necessary. Little is known of the action of unsaturated fatty acids on PG-formation in isolated perfused hearts. Some of these substances serve as precursors for PGsynthesis. Studies in this field will yield further information on the enzymatic

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PROSTAGLANDINS

capacity of the heart for PG-synthesis . Therefore the influence of oleic , linoleic , linolenic and arachidonic acid on PG-formation in isolated perfused hearts of guinea pigs was studied. METHODS Perfusion. Experiments were carried out on guinea pigs of either sex ranging in weight from 300 - 500 g. The hearts were dissected and perfused according to Langendorff’s method viathe aorta with Tyrode solution of ihe following composition 6nM): NaCl 136,9, KC1 2,7, CaCl2 1,8, MgC12 l,O, NaHC03 11,9, NaH2P04 0,4, glucose 5 ;S. The solution was aerated with oxygen at 37oC, The isolated hearts were perfused with a constant pressure (50 mm Hg) . Isometric contractions were recorded by strain gauge transducers and coronary flow by photoelectrical drop counting. Superfused organ system. The PLS content of the effluent was determined by a modified superfusion technique of isolated smooth muscular organs according to Two stomach strips and the colon of the rat were used as assay organs. Vane (6,7,8). The initial resting load of the tissues was 2 g , the contractions were recorded by strain gauge transducers. The assay organs were made insensitive to acetylcholine , catecholamine, histamine and 5-hydroxytryptamine by infusing a mixture of antagonists: atropine , propranolol, phentolamine and antazolin in a concentration of 10-7 g/ml. Indomethacin (1 wg/ml) inhibited the PG-synthesis of the organs and increased their sensitivity to prostaglandin-like material. The PG-content was determined in aliquots of 16 ml of the effluent against PGEl and expressed as ng PGEl/ml, Fatty acids. The following unsaturated fatty acids were used in form of their sodium salts: oleic acid (OA) (Merck AG. Darmstadt) , linoleic acid (LA), linolenic acid (LLA) (Carl Roth OHG , Karlsruhe) and arachidonic acid (AA) (88%) (Unilever Research). The substances were tested as a bolus injection just proximal to the heart in doses from 0,l - 3,0 p.:g/O,l ml or as a continuous perfusion CO min) at a concentration of 0,2 .g/ml . Comparable concentrations could not provoke any effects in the assay organs. RESULTS Heart action and coronary flow. During the investigation period of about 40 minutes the control values of heart action and coronary flow did not show significant change. Applications of unsaturated fatty acids influenced the heart action in different ways. After single injections of LA, LLA and AA (0.1 - 3,O ug) a dose-dependent increase in contraction force was observed, especially with LLA (9 - 36%) and AA (0 - 32%)) LA (7 - 18%) was less marked. Continuous coronary perfusion of fatty acids (0,2 ug/ml) only caused a significant rise in contraction (38%) when AA was used. OA remained ineffective both in continuous and discontinuous administration. The heart rate was only influenced by the largest doses of LLA and AA and showed a trend of decrease (8 - 12%).

174

JULY 1977 VOL. 14 NO. 1

PROSTAGLANDINS

The coronary flow was increased by LA (5 - 12%) and AA (20 - 45%). LLA was without influence. At high concentrations GAalso showed an Increased coronary flow. (Table I) Release of prostaglandin-like substance. Isolated perfused hearts of guineapigs continuously release prostaglandin-like substances (PLS) . The average value of the control group reached a level of 0,83 ?O, 06 ng/ml PLS sufficiently detectable by the method of organ superfusion. This efflux showed no significant changes during the period of investigation as demonstrated by the following values: 0 min: 0,92*0, 08 ng/ml, 15 min: 0,78*0,06 ng/ml, 30 min: 0,71* 0,05 q/ml, 45 min: 0,82*0, 09 ng/ml (n = 12). Unsaturated fatty acids proved to influence the basic output of PLS . OA did not provoke any PLS-formation , higherdoses seemed to inhibit PG-synthesis . Contrary to this polyunsaturated fatty acids (LA, LLA , AA) induced an increase of PLS in the effluent of the heart. Direct application of 0,l - 3,O ‘pg results in an immediate and additionalPLSreleaseof2,5-5,9ng(L4),0-2,3ng(LLA)and1,1-7,8ng(AA) into the effluent of the heart. The comparison of the three fatty acids indicate that the least PLS formation results from LLA. The proportional conversion of fatty acids to PLS (FA/PLS (ng)) parallels the increasing doses of fatty acids which points to a lower conversion of the total amount of fatty acids to PLS with augmented doses. This proportion runs up to 83 (LA), 219 (LLA) and 96 (AA) after a dose of 0,3 ug, but 510 (LA), 1293 (LLA) and 388 (AA) after to 3 u g of the fat@ acids. This results in formation rates of between 0,l and 2,5%. (Table II) Continuous perfusion (Of2 u.g/ml) with exogenous PLS-forming fatty acids lead to the greatest amounts of PLS in the coronary effluent. Under these conditions the PLS content of the perfusates rose by Of72 ng/ml (LA) , 0,49 ng/ml (LLA) and 1,18 ng/ml (AA). The following average formation rates were obtained: 278 (LA), 405 (LLA) and 176 (AA) corresponding 0,4% (LA), 0,2% (LLA) and 0.6% (AA). The results are represented in Table II. DISCUSSION The evidence for the presence of prostaglandin-like substance in the effluent of isolated perfused hearts of guinea-pigs is in accordance with recent results obtained in rabbit hearts (1,2,3,4). The average concentration (0,8 ng/ml) in the perfusate of the guinea pig heart seems also to be similar to results from the rabbit heart. In the isolated rabbit heart Block et al. calculated the PLS-concentration in terms of PGE2, the values varying from 0,l - 1,O ng/ml. Wennmalm (4) also identified prostaglandins in the effluent of the rabbit heart. The initial spontaneous outflow in the perfusates ranged between 0,9 and 4,2 ng/min at a coronary flow of between 25 - 45 ml/min. Certain polyunsaturated fatty acids prove to be potent releasers of PLS from the heart, especial@ marked LA and AA, leas marked LLA. OA was without effect. At continuous perfusion the average formation rates amount to 0,2 - 0,6%. These results

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175

I

0.1 6 0.3 6 1.0 6 3.0 6 0.2 ug/6 ml

perfusion)

0.1 6 0.3 6 1.0 6 3.0 6 0.2 w/5 ml

perfusion)

ml

23 23 23 20 20

-

-6+3.3 +2172.fl +29+4.4 >0.05 CO.01 CO.01

>o.os 0.05

+9+4.2

>o.os X.05 x.05 co.01 >0.05

>0.:5 >0.05 TO.05 )0.05 70.05

+24:4.5 +27T3.9 +36T4.5 +9T3.7 -

+7+3.1 +8+3.4 +11?4.2 +18r2.8 -4E2.5

Control effect (us) % dosage n rmn) 0. 15 20 +3+3.2 0.3 5 22 +G4.3 1.0 5 21 +4r3.0 3.0 5 21 +4F22.3 0.2 ug/s 20 +4+3.3 -

(continuous perfusion) arachidonic acid 0.1 8 0.3 7 1.0 8 3.0 6 0.2 g/ 6 ml

(continuous linolenic acid

(continuous linoleic acid

fatty acid oleic acid

force

235 226 222 222 226

241 230 232 226 226

243 246 246 252 242

contr./minl 236 234 230 230 222

eart rate ontrol

-2+2.7 -4F3.2 -211.6 -672.1 -12+_3.8

+1+1.9 -7T2.1 -6Fl1.9 -9r1.9 -9T3.0

-+0+2.a +2+1.7 +3+3.4 +4T2.5 +8+3.6 -

effect P +2+1.5 +lT2.6 +oT2.7 +4irz.O +9T4.3 -

of isolated perfused hearts of guinea f standard error) are given as au

t.wrtraction

Heart function and coronary flow unsaturated fatty acids.(results

Table under

>O.OS >o.os ,o.os (0.05 (0.05

>0.05 (0.05 0.05

P >0.05 >0.05 m.05 x3.05 x.05

pigs

of

6.4 6.4 6.2 6.2 6.5

6.8 6.8 6.2 6.4 5.4

7.3 7.0 7.2 6.8 7.1

+20+4.2 +37'c6.1 +45+5.6 +3874.3 +4254.8

-4+3-s -7F3.6 -2r4.1 -272.9 -13T5.3

+4+2.1 +12+3.3 +9r2.a +5T1.7 +lE22.6

0.05 >0.05 >0.05 >0.05 )0.05

>0.05 to.01 (0.05 0.05 6.6 F3Fl.9 )0.05 6.6 +5F2.6 >0.05 6.8 +10?2.7 (0.05 6.9 +13zl1.9 (0.05

flow

influence

oronary

the

PROSTAGLANDINS

Table II: Formation of proetaglandin-likesubstance (PL.9) in isolated perfused hearts of guinea pigs under the influence of unsaturatedfatty acids (FA) (resultsare given as mean & standard rror) -t PA induced PLS release rate of LS-formation (relatedto initial PLS formation control) fatty g/l0 ml ng/min ng acid dosage n PA/PC D oleic control 6 0.3+1.4 6.9+0.9 8.9+11.0 5.2FO.6 -1.4 >0.05 acid 0.1 M 6 >0.05 9.6T2.0 5.011.2 -0.7 0.3 ug bO.05 8.2T1.3 4.6Fl.l -2.1 1.0 w >0.05 7.251.5 4.050.8 -3.1 3.0 ug 0.2 w/ >0.05 8.6+2.3 4.1tl.l -1.7 Ccontrierf) linoleic acid control

6

0.1 0.3 1.0 3.0 0.2

m 6 ug 6 ug 6 ug 6 w/ ml 6 (cont.perf.

linolenic acid control 0.1 0.3 1.0 3.0 0.2

6

Irg6 ug 6 ug 6 ug 6 ug/ ml 16

6.521.8 4.550.6 +2.5 +3.6 +2.2 +5.9

CO.05 co.01 co.05 0.05 (0.05 0.05 2 0.01

219 1000 1293

0.5 0.1 0.1

LO.01

405

0.2