Release of prostaglandins from the passively distended wall of guinea pig small intestine.

The effects of the radial distension of the intestinal wall and the increased intraluminal pressure on the liberation of prostaglandins (PGs) were studied with the isolated guinea pig ileum. Both procedures were accompanied by an increased output of a prostaglandin-like substance and the release was proportional to the degree of distension or intraluminal pressure. Thin-layer chromatography coupled with bioassay suggested that this substance may be a mixture of PGF2 alpha, PGE2 and PGE1. The existence of the former two types of PGs were prominent. The distension-induced release of PG-like substance was assumed to be not mediated by nerve excitation since tetrodotoxin failed to inhibit the release. The increased output of the PG-like substance was not maintained after distension of the intestinal wall, despite continued application of stimulus, indicating that actual tissue deformation is an essential condition leading to increased PG output. It is concluded that distension of the intestinal wall to its circumferential direction is an appropriate stimulus for the release of PG-like substance from the small intestine. The present results favor the view that prostaglandins may participate in the peristaltic activity.

Abstract-The effects of the radial distension of the intestinal wall and the increased intraluminal pressure on the liberation of prostaglandins (PGs) were studied with the isolated guinea pig ileum. Both procedures were accompanied by an increased output of a prostaglandin-like substance and the release was proportional to the degree of distension or intraluminal pressure. Thin-layer chromatography coupled with bioassay suggested that this substance may be a mixture of PGF2 , PGEZ and PGEI. The existence of the former two types of PGs were prominent.
The distension-induced release of PG-like substance was assumed to be not mediated by nerve excitation since tetrodotoxin failed to inhibit the release. The increased output of the PG-like substance was not maintained after distension of the intestinal wall, despite continued application of stimulus, indicating that actual tissue deformation is an essential condition leading to increased PG output.
It is concluded that distension of the intestinal wall to its circumferential direction is an appropriate stimulus for the release of PG-like substance from the small intestine. The present results favor the view that prostaglandins may participate in the peristaltic activity.
Prostaglandins (PGs) of E and F types have been found in the wall of the intestine of different species (1,2) and the release of PGs is increased with transmural stimulation (3).
In view of the potent pharmacological activity on smooth muscle contractions and on neural excitation, modulation of peristaltic activity by PGs has been extensively demonstrated (4)(5)(6).
Distension of the intestinal wall is one of the most effective means of stimulation for the initiation of peristalsis. In guinea pig small intestine, many pharmacologically active substances are released during the distension of the intestinal wall and are considered to contribute to peristalsis (7,8). However, little is known of the release of PGs during dis tension of the intestinal wall. It has been suggested that PGs can be liberated by simple distortion of muscle cells (9). If such is indeed the case in the intestine, it can be expected that a significant amount of PGs would be release during distension of the intestinal wall.
The present work was carried out to clarify the characteristics of the release of PGs during distension of the intestinal wall and to determine if PGs act as modulators in peristalsis.

MATERIALS AND METHODS
Male guinea pigs weighing 300 to 600 g were killed by a blow on the head and bled.
Three to five segments of ileum about 6 cm long were excised excluding the most 10 cm distal portion and weighed after blotting both sides of the wall with filter paper. These segments were then incubated separately at 38'C in the organ-baths containing 10 to 20 ml Tyrode solution aerated with mixture of oxygen (95 %) and carbon dioxide (5 %) under any one of the procedures described below.
The procedures tested were a) Radial distension: The segment was distended to its circumferential direction by inserting a glass rod of proper diameter, usually 7 mm, into intestinal lumen and then the segment was fixed on the rod by binding at both ends of the segment. Special care was taken to avoid any longitudinal distension. In these series All the samples collected were kept in an ice cold situation until biological estimation of the PGs.
Identification of prostaglandins: Two to five samples of bath fluid were combined, acidified to pH 3 immediately with hydrochloric acid and extracted three times with an equal volume of peroxide-free ether. The combined ether phase was evaporated to dryness at a room temperature below 30°C under a stream of nitrogen gas. The dried material was dissolved in 0.5 ml ethanol and then subjected to thin-layer chromatography on silica gel G containing three per cent silver nitrated. The A II solvent system of Green and Samuelsson (11) was used as a developing solvent. Authentic PGE1, E2 and Fed were treated similarly, chromatographed concurrently and made visible by spraying the plate with 10 per cent molibdeic acid followed by heating. Prior to visualization, consecutive centimeter bands of the plate to which the fluid extract was applied and then developed, were extracted with 5 ml of 50 per cent chloroform in methanol. One gram of NaCI was added to this extract to precipitate the silver nitrate therein. After separation from solid matter, the solvent was again evaporated to dryness as described above and the residue was dissolved in 1 ml Tyrode solution for biological assay.
Assay of prostaglandins: PGs in a sample of bath fluid were assayed on a rat fundus strip suspended in oxygenated Tyrode solution at 37"C. To increase the specificity of the preparation for PGs, hyoscine, phenoxybenzamine, pyrilamine (0.1 ag/ml each), propranolol 2 ,ug/ml and methysergide 10 ng/ml were added to the Tyrode solution. The contractions of the muscle strip to the bath fluid were compared with standard doses of PGE1. The amount of released PGs was expressed a ng PGE1 equivalent/g tissue/min, although the bath fluid contained PGEI, E2 and F2a, as shown in results. When the intestinal segment was exposed to tetrodotoxin, the bath fluid was compared with standard PG solutions to which the drug concerned was added to give the same final concentration in the assay bath.
A stock solution of PGEI was prepared by dissolving 10,ag PGE1 in 1 ml ethanol and kept at -18'C until use. The standard solution was made by diluting the stock solution with an appropriate volume of Tyrode solution, immediately before assay. On thin layer chromatography using the A II solvent system of Green and Samuelsson, PGF1. and some of PGF211 metabolites such as 13,14-dihydro-oxo PGF2, 15-oxo PGF2. are indistinguishable from PGE2 since they have bery closed Rf values (11,12). These metabolites and PGF1. are, however, only weakly active on the rat stomach strip (13,14). Therefore, it is likely that the fraction tentatively expressed as PGE2 is com posed mainly of PGE2. PGD2 is not distinguishable from PGF2,,, with respect to the Rf value and biological potency to rat fundus strips (12,15). Thus, the activity residing in the zone which corresponded to the zone PGF20 was probably a PGF2, like substance.
Although it is unlikely that unstable PGG2i PGH2, PGI2 or thromboxane A2 would be present in the collected bath fluid, since these compounds are metabolized very rapidly in the tissue (16), the contribution of other substances, such as 6-keto-PGF1 or thromboxane B2, to the biological activity is not known.
In the present study, samples were assayed on rat fundus strips and the amount was expressed in terms of PGE1, which is about equipotent with PGE2 (12) but about five times more potent than PGF2as and PGD2 (14,15). The total amount of PGs in the collected bath fluid is therefore probably underestimated, but correction cannot be made as the relative amount and the potencies of all the types of PGs are unknown.
In the gastrointestinal tract, considerable amounts of PGs are released from the mucosa during incubation (12,14). Thus, it may be that the increased output of PG-like substance seen in the present experiments was derived from the mucosal PG. However, this is unlikely since both ends of the intestinal segment were ligated and PGs hardly diffuse from the mucosal to the serosal side (4). PGs detected in the bathing fluid, therefore, are assumed to originate from the muscle of the intestinal wall. The presence of two types of PGs, PGE2 and PGF2 in guinea pig longitudinal muscle preparation has been reported by Ambache et al. (1).
Concerning the release of PGs from the tissue, Botting (17) suggested that the pro staglandin-like activity from isolated guinea pig ileum during field stimulation was caused by the release of noradrenaline from the intramural sympathetic nerves. Involvement of nervous activity in the release of PGs has also been reported in the case of other organs such as heart (18), stomach (14) and vas deferens (19). Ramwell  In the present experiments, the increased output of PG-like substances was not maintained after distension of the intestinal wall, despite continued application of stimulus, indicating that actual cell deformation was the essential condition which led to the augmented output of PGs. This finding parallels that seen in the case of other tissues (23) where increased output of PG from the spleen, as induced by stimulation of splenic nerves or by the application of adrenaline declined almost to the basal level after completion of contraction of the tissue, despite continued application of stimulus. The decline of PG release during sustained stimulus may reflect the generally accepted idea that PGs are stored to a limited extent in tissues.
The augmented release of a PG-like substance during radial distension favors the view that these substances may participate in peristaltic activity (4-6) for the following reasons.
a) Both types of PGs, PGE2 and PGF211, have relatively high stimulating activity on the guinea pig ileum (24,25), b) PGE2 increased the output of acetylcholine from the intestine, and this compound presumably plays an important role in initiating peristalsis (26) and c) radial distension is an effective physiological stimulus for peristalsis.