Antagonistic actions of prostaglandins E2 and F2 alpha on the isolated lungs of the frog, Rana esculenta L.

Isolated lungs of the frog, Rana esculenta L., when incubated in amphibian Ringer solution for 30 min, produced a prostaglandin E2-like substance (27.1 +/- 3.8 ng/g w.w.), as determined by bioassay on the isolated rat stomach strip. The release of PGE2-like substance from skin, heart and bowel is also reported. The activity of synthetic prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha) on the muscular contractility of frog isolated lungs was investigated: PGE2 and PGF2 alpha relaxed and contracted respectively in a dose-dependent manner this preparation, a result similar to that obtained in mammals.


INTRODUCTION
The antagonistic actions of PGE, and PGF,alpha on mammalian bronchial muscle have been shown by several investigators: as a rule PGF,alpha contracts, and PGEz relaxes preparations from numerous species, including man (Anggard and Bergstrom, 1963;Cuthbert and Gardiner, 1981). Since lung tissue can synthesize and degrade both PGE, and PGF,alpha, a functional relationship between the two prostanoids and their involvement in the control of bronchial muscle tone has been suggested, but the physiological meaning of this relationship is still unclear (Sweatman and Collier, 1968).
During a survey on the presence and biological activities of prostanoids on isolated smooth muscle preparations of lower vertebrates, it was noticed that the isolated lungs of the common frog (Rana esculenta L.) both produced PGE,-like material and responded to PGE, and PGF,alpha in a fashion qualitatively identical to the one reported in mammals.

Animals
Frogs, Runa esculenra L., of either sex, weighing 4&50 g, were maintained at room temperature in slowly circulating tapwater till use. They were kept fasting prior to experimentation.
Male Wistar rats (20&250 g) were used for the bioassay.

Incubation, PG extraction and bioassay
The animals were pithed and lungs, heart, skin and intestine were rapidly removed and incubated at room temperature (20°C) in a saline (weight:volume I: I) of the following composition (g/l): NaC16.5; KCI 0.14; CaCl, 0.12; NaHCO, 0.2. After 30 &, I ml of the incubation medium was removed, 0.250ml of saturated NaCl solution added. acidified with 0.5 M citric acid and twice extracted with an equal volume of ethyl acetate.
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The organic phase was then evaporated in a stream of N,. The residue was dissolved in 0.2 ml of Krebs bicarbonate and PGE,-like activity tested by bioassay. The bioassay was performed by measuring the changes in isotonic tension (resting tension: 2 g) of a rat stomach strip superfused with Krebs bicarbonate (flow: 5 ml/min; Temp. 37°C). Synthetic PGEz (Upjohn, Kalamazoo) was used as reference standard.

Isolated lung preparation
The lungs were longitudinally cut and mounted into a 10 ml organ bath filled-with saline (see above) continuously bubbled with 95% 0, + 5% CO,. Contractions were recorded using an isotoiic transduce; connected to a polygraph.
The tissues were placed under an initial tension of 0.5 g and left to equilibriate for 6&90 min. PGE, and PG&alpha, dissolved in saline, were added to the bath and kept in contact with the tissue for 3 min; the strip was then gently washed by dilution. In all the preparations, rapid washing or any slight mechanical stimulation caused a sustained and prolonged increase in muscle tension. Results are expressed as per cent of the maximal response elicited either by PGE, or PGF,alpha in each preparation (& SEM). Regression analysis was carried out using the least-squares method.

RESULTS AND DISCUSSION
Frog lungs have been previously shown to generate prostanoids from endogenous polyunsaturated fatty acid stores (Christ and Van Dorp, 1974;Nomura and Ogata, 1976). The results shown in Fig. 1 confirm these findings and compare the PGEJike yields obtained in a few tissues under identical experimental conditions. Synthetic PGE, and PGF,alpha induced respectively relaxation and contraction of the isolated frog lungs (Fig. 2). This response was very rapid for PGE, and slower for PGF,alpha (latency of ca 1 min); in both cases it was concentration-related.
Arachidonate failed to elicit any motor response (not shown in figure).
The stimulatory action of PGF,alpha and the inhibitory one of PGE, demonstrate a close parallelism with the response exhibited by the mammalian lung in spite of the large structural and functional differences between the two classes.
Modern Anura possess sac-like lungs with ridged, highly vascularized internal walls, inside which smooth muscle elements are enclosed (Goldie et al., 1983). From a physiological standpoint this structure is essentially designed to supplement rather than to replace the exchange of gases through the skin. An important distinctive feature from mammalian lung is therefore the absence of a bronchial tree, which in mammals is the major control site of airway resistance and is very likely an important target of prostanoid action. Our findings show that the pattern by which smooth muscle respond to PGE, and PGF,alpha, whatever the function served, has been established early in evolution. As remarked by Johanssen and Reite (1967) this feature is shared by other neurohumoral substances such as acetylcholine, norepinephrine, epinephrine and serotonin.