LAMB DUCTUS ARTERIOSUS: EFFECT OF PROS’IXGLANDIN SYNTHESIS INHIBITORS ON THE MUSCLE TONE AND THE RESPONSE TO PROSTAGl&lDIN Es

Flavio

Research

Coceani,

Institute,

Peter

M. Olley

and Eva Bodach

The Hospital for Toronto, Ontario.

Sick

Children,

ABS’IRACT

The prostaglandin synthesis inhibitors, indomethacin and eicosa-5,8,11, 14-tetraynoic acid (ETA), have been tested on the isolated lamb ductus arteriosus at low and high POT levels. Both compounds produced a gradual contraction of the and at equal doses the effect of indmethacin was stronger. ‘Ihe hypoxic vessel, maximal tension output of the hypoxic tissue under indomethacin was equal to that of the oxygen-contracted control. ETA- and indmethacin-treated preparations contracted further upon transfer from a low to a high oxygen environment, and the response under indomethacin exceeded significantly control values. Control preparations were relaxed markedly by PGE, in low oxygen but showed little or no response in high oxygen. In contrast , preparations pretreated with the inhibitors retained their sensitivity to PGE, during exposure to high oxygen. The data are consistent with the idea that E-type prostaglandins play a role in the regulation of the intrinsic tone of the ductus arteriosus during foetal life. It is also suggested that the sensitivity of ductal muscle to E-type prostaglandins is controlled by the rate of endogenous prostaglandin formation.

ACKN’WLEDGEMEN’IS This work was supported by the Ontario Heart Foundation (Grant 4-8). The authors are indebted to Dr. G. Kent and Mr. F. Hamilton for assistance during surgery, and to Miss M. Jones for the statistical analysis. Prostaglandin Ea was kindly supplied by Dr. J. Pike (Upjohn Company, Kalamazoo, Michigan).

Accepted

January

20

PROSTAGLANDINS FEBRUARY

1975

VOL. 9 NO. 2

299

PROSTAGLANDINS

IN'JRODUCTION We have recently reported that prostaglandin El (PGEi) and Es (PGE,) relax markedly the isolated lamb ductus arteriosus in a low oxygen environment (POc between 10 and 14 mm Ug) but have little or no effect on the oxygen-contracted vessel (1). The reduction of prostaglandin activity under oxygen was not associated with changes in the contractile or relaxant properties of the muscle. The finding is interesting in two respects: first, it proves that relaxant and contractile actions of the prostaglandins have different requirement for oxygen (for the oxygen dependence of contractile responses, see 2-S); second, it indicates that the change in the reactivity of the ductus from a low to a high oxygen environment specifically concerns the prostaglandins. From this finding, we speculated that endogenous E-type prostaglandins may play a role in the regulation of ductus tone during the prenatal period and that suppression of their activity with increasing oxygen tension may be important to the initiation and/or the persistence of the functional closure at birth. In an attempt to provide some support to this idea, we have examined here the effect of the PG biosynthesis inhibitors, indomethacin and ETA, on the intrinsic tone of the ductus. We found that both compounds contract the vessel, the response being more marked with indcmethacin. Evidence has also been obtained suggesting that the sensitivity of ductal muscle to E-type prostaglandins is controlled by the rate of endogenous PG formation. METHODS General procedure. The experiments were performed with pregnant, Suffolk ewes of 134-144 days gestational age (term is 147 days). The surgical procedure, and the methods used for the dissection and preparation of circular strips of lamb ductus arteriosus under low ambient oxygen (POs cl4 mm Ug) have been described elsewhere (1). Ductal strips were set up in a lo-ml bath and left to equilibrate in Krebs solution gassed with 5% CO, in nitrogen. The organ bath was supplied from several reservoirs and this allowed rapid changes in the POe and/or the composition of the perfusion fluid. All solutions were pre-equilibrated with the appropriate gas mixture prior to reaching the organ bath and were gassed again in the bath. The perfusion rate was around 2 ml/min and the-temperature of the fluid was maintained at 37oc. Ambient light was constant throughout the experiment. The experimental protocol included the examination of control tissues (Group l), and tissues treated with ETA (Group 2) or indomethacin (Group 3). All tissues of Group 2 and 3 were equilibrated in a low oxygen medium prior to drug treatment. Cumulative dose-response curves for PGE, were obtained first at low and then at high oxygen levels in the presence or the absence of the PG synthetase inhibitors. In all instances, the compound was tested once at low P0e and from one to three times at high POe* Since the effect of PGEs fails commonly to reach a plateau especially

300

FEBRUARY

1975

VOL. 9 NO. 2

PROSTAGLANDINS

when the ductus is hypoxic rarily at 4 min. The total exceeded 0.35 ml D

(l),

application time for each of PGEs solution added

volume

dose was set arbitto the bath never

Muscle tension was measured by a force-disRecording of tension output. ulacement transducer (Grass Instrument Co. model FT.03C) coupled to a polywith a 7P122A amplifier graph unit (Grass Instrument Co. model 7-B, equipped rate of the transducer was 5 um per gram. The assembly) D The displacement initial tension of the preparation was adjusted at about 1.5 g by means of a micrometer. Measurement of oxygen tension. Oxygen tension of the bathing fluid was determined in l-ml samples using an Instrumentation Laboratory (mod. 213) gas Measurements were made whenever a new solution was introduced into analyzer. The analyses indicated a the bath and then, at random during the experiment. POs between 10 and 14 nsn Hg and between 479 and 613 mm Hg for media equilibrated respectively with zero and 95% oxygen. Solutions and drugs. The Krebs solution had the following composition (rmnol/l): NaCl, 118.1; KCl, 4.7; CaCls, 2.5; KHsPOPO,,1.0; MgS04, 0.9; dextrose, 11.1; NaHC4, 25. The pH was around 7.4 after equilibration with 5% COs in nitrogen or oxygen. Stock solutions pound in redistilled the experiment, the saline e

(20

Indcmethacin mg/ml; see 6)

of pG!& were prepared by dissolving the crystalline comethanol-water (95:5, by vol; 1 or 10 mg/ml). On the day ethanol solution was diluted with appropriate volumes of

(Sigma) was and aliquots

prepared as a nearly saturated solution were diluted with Krebs medium.

of

in ethanol

Eicosa-5,8

,11,14-tetraynoic acid (Abbott) was recrystallized from benzene. were used to solubilise the compound. 1) In four experiments, ETA as the ammonium salt, which had been formed by bubbling with mania was tested of the free acid in diethyl ether. 2) In three experiments, ETA gas a solution was dissolved first in 0.2 M borate buffer at pH 9.0 (10 n&ml; see 7) and this solution was then diluted with Krebs medium. With either procedure, ETA did not dissolve completely in Krebs solution, and this may explain the apparent lack of dose-dependent effects (see Results).

TbOprocedures

For

all

FEBRUARY

compounds,

1975

doses

are

given

VOL. 9 NO. 2

as molar

concentrations

in

the

bath

fluid.

301

PROSTAGLANDINS

RESULTS The pattern of mechanical activity of the ductus arteriosus was similar to that reported in previous experiments (1). As shown in Table I, control tissues (Group 1) contracted during the equilibration period in a low oxygen medium and developed an additional, stronger contraction when exposed to high oxygen. ETA, in concentrations between 5 x 10-6 and 3 X 10m5M, caused a sustained contraction of the hypoxic ductus in six of the seven experiments (Group 2). A similar but more pronounced response was consistently obtained with indomethacin 2.8 X 10m5M With ETA, the effect started after a mean latency of 10 min (range (Group 3). 5-14) and reached the maximum in 24 to 61 min. A comparable time course was observed with indomethacin, although in this case the latency of onset of the contraction appeared to be shorter (mean, 3 min; range, 2.4-5). Interestingly,

200

r 180-

q 0 X

q

X

0

q 0

X

0

X

X”x X

t

0

n A

0

x x

x x

a#

OL x% I

1o-1’

a X

0 X

X

+

I

I

1o-10

10-9

x 8 h

10-E

CONCENTRATION

10”

0

; 1

10-b

1

1o-5

OF PGE;, (M)

Figure 1. Isolated circular strip of ductus arteriosus. Dose-response curve for PGEa at high POs (479-613 mm Rg) before and after treatment with the inhibitors of PG biosynthesis. For each condition, the response is expressed as percent of the relaxant response under low oxygen (POs between 10 and 14 mm Rg). In those instances where PGEs had been tested more than once at high POs (see for example Fig. 2)) the first dose-response curve was used for plotting purposes. Each point applies to a single test o In this and the subsequent figure, doses are molar concentrations in the bath fluid.Control (0);ETA 5 X lo-6to3xlO-5W (x);Indomethacin 2.8 X 10m5M (0).

302

FEBRUARY

1975 VOL. 9 NO. 2

PROSTAGLANDINS

the maximal tension output of hypoxic, indomethacin-treated tissues did not differ significantly from that of oxygen-contracted, control tissues. Table I also shoos that both ETA- and indcmethacin-treated preparations contracted further upon transfer from a low to a high oxygen medium, but only with indomethacin the response exceeded significantly the normal values. Control experiments proved that the vehicle for indomethacin or ETA (i.e. Krebs solution containing ethanol or borate buffer) is without effect on the intrinsic tone of the ductus. In the course of these experiments, we have also analyzed the effect of FCE, on the ductus. We have confirmed (see 1) that the relaxant action of PCE,, while intense and persistent with the hypoxic.ductus, is markedly reduced or abolished after transfer of the tissue to a high oxygen environment. As shown in Fig. 1, the response of the vessel in a high POs is at the most 44% of that occurring in

10 min

Figure 2. Isolated circular strip of ductus arteriosus. Indomethacin was added to the fluid after equilibration of the tissue at low POa9 PCE, was tested on the hypoxic and oxygen-contracted ductus (contact time for each dose is 4 min). Note the increase in muscle tension as result of indomethacin treatment, and the intense and consistent response to PCE, at high POa. Recovery of the initial tension after PGE, required 160 and 120 min respectively at low and high POs. The record covers the whole experiment, except for a 15-min interval prior to exposure of the ductus to the high POaO During this interval, the muscle tension dropped suddenly, which did not occur with the other indomethacin-treated preparations.

FEBRUARY

1975

VOL. 9 NO. 2

303

PROSTAGLANDINS

TABLE I. IAMB DUCTUS

ARTERfOSUS.

ON THE TENSION

OUTPUT

EFFECT OF PG SYNTHESIS

INHlRfTORS

AT LOW AND HIGH PD,lEVELS. PO2 OF PERFUSION

CONDITION

479-613

10-14 mm Hg

GROUP

1

Control

33~0.5

(9)

GROUP

2

Before ETA

3.3M.5

(7)

After

4.1kO.3

(7p

ETA’

MEDIUM mm Hg

9.6kO.4 (10)

9.1kO.4

(7p

(5x1o-6- 3xlO_5M)

GROUP

3

Before

indomethacin

3.BkO.8

(4)

After

indomethacin

9.7kO.4

(4)O

(2.8~16’

Values

(91 or.

Tension

l~uponrer and

ore

consequently

aStotirticolly bDiffw.nce ‘Si9nificontly

304

means

is corrected

SE.

+ for

not

for

the

related

the

initial to

number basal

the

dose

all

results

or.

pooled

significant

using

the

paired

from

control

different

(Group (P