PROSTAGLANDINS

Release of Prostaglandins from Healthy and Sensitized Guinea-Pig Lung and Trachea by Histamine S.-S. Yen, A. A. Math6 and J. J. Dugan From the Laboratory of Biogenic Amines and Allergy Boston University School of Medicine, Boston, Mass. Abstract The effects of histamine and its antagonists on the release of (PGE and PGF2o) and the 15-keto-13,14prostaglandin E and F dihydro PGF2,/E (meta&elites) were examined in minced and whole perfused guinea pig lung. Lung fragments released considerable amounts of prostaglandins into the incubation media with time alone: parenchyma more PGF fo%d than PGE, trachea more PGE than PGF2,. The levels of PGF in the filtrates of both tissues on per gram basis were a2" out the same, whereas the concentrations of PGE were several fold higher in the media of incubated trachea. In contrast to lung, trachea released only trace amounts of metabolites. These differences in synthesis and turnover are probably of importance for maintenance of the adequate ventilation-perfusion ratios. The process of sensitization caused a significant increase in the outflows of PGF and metabolites from the lung fragments. The 10 was decreased in both parenchymal and tracheal ~~~s:~sPGF2a ra za . Increased spontaneous release of prostaglandins was also found in whole perfused sensitized lung. This was consistent with the hypothesis that sensitization with antigen alters the biochemical properties of the organism. Incubation of lung fragments with histamine had only a small additional effect on the liberation of prostaglandins, since the baseline release was high due to the trauma of mincing. However, histamine perfusion of whole lung caused severalfold increase in the outflows of prostaglandins. Pretreatment with pyrilamine (histamine receptor 1 antagonist) decreased the subsequent release of PGF2a by histamine. On the other hand, pretreatment with metiamide (histamine receptor 2 antagonist) diminished the subsequent release of PGE. It is suggested that stimulation of histamine receptor 1 is predominantly and stimulation (but not solely) related to the synthesis of PGF2 of the receptor 2 is related to the synthesis of PGi. This work was supported by USPHS, NHLI Grant HL-15677 and Pulmonary SCOR Grant HL-15063.

2-20-76

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INTRODUCTION Histamine was the first chemical mediator recognized to be released as a consequence of the antigen-antibody reaction (Bartosch et al, 1932; Dragstedt and Gebauer-Fuelnegg, 1932). While some phenomena of anaphylaxis are mimicked by its administration others are not. A search for additional mediators has resulted in discovery of slow reacting substance of anaphylaxis (SRS-A) and eosinophilic chemotactic factor of anaphylaxis (ECF-A). In addition to these, bradykinin'and 5-hydroxytryptamine are also considered to be the "primary" mediators of the antigen-antibody reaction (reviews by Kaliner and Austen, 1974 and 1975). Piper and Vane (1969) have discovered that prostaglandins were released during anaphylactic reaction as well. The major lung metabolites, 15-keto-13,14-dihydro-PGF2oIE, were subsequently also demonstrated in outflows from perfused, anaphylactic guinea pig lung (Math6 and Levine, 1973). Since some data, based on bioassay, showed that exogenous histamine might release prostaglandins (Bakhle and Smith, 1972; Orehek et al, 1973), it is conceivable that their liberation during antigen-antibody reaction is also secondary to that of the endogenous histamine. In order to further study these phenomena, effect of histamine on the prostaglandin release was examined. Since responses to histamine can be inhibited by blockers of the histamine receptor one or two, effects of pyrilamine and metiamide were also investigated.

MATERIALS AND METHODS Animal Model: Male guinea pigs weighing 400 f 100 grams were sensitized by injection of 100 mg ovalbumin (Sigma, St. Louis, MO.) divided into two equal doses and given I.P. and S.C. This was followed by a booster injection.of 50mg of ovalbumin I.P. on day three. Control animals received 0.9% NaCl. Five to seven weeks later, animals from both groups were randomly chosen, stunned, exsanguinated, the thoracic cavity opened and catheters inserted in situ via the right ventricle into the pulmonary artery and via the leEatrium into the pulmonary vein. The lung was continuously perfused with Tyrode's solution bubbled with 5% CO2 in O2 at a constant rate of 4 ml/min. Details of the experimental procedure were described previously (Math6 and Levine 1973; Math6 et al 1975a). After the rinse period, two types of experiments, one with lung fragments and the other using the whole perfused lung models, were conducted. In the first type of experiments trachea, large bronchi and blood vessels, and connective tissue were removed and the lung chopped into l-2 mm3 fragments which were then washed with 4OC Tyrode's solution and blotted with filter paper.

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Tissue aliquots weighing 500mg were used for subsequent incubation. Preparations of trachea were obtained in a similar manner. Tissues were incubated for 4 minutes in 3 ml of 37'C Tyrode's solution, pH 7.4, in a Dubnoff Metabolic Incubator (Precision Scientific, Chicago, Ill.) with continous aeration with 5% CO2 in 02. The reaction was stopped by passing the incubation fluid through Millipore filters (Millipore, Bedford, Mass.). The filter discs of the millipore apparatus did not bind any prostaglandins. Samples were frozen immediately and kept at -7OOC until analyzed for prostaglandins. To study the effects of histamine (histamine diphosphate, Aldrich Co., Milwaukee, Wis.) tissue aliquots were incubated with the following concentrations of the compound: 9 x 10-6, 4.5 x 105, 2.3 x 1O-4 and 1.3 x lo-%, calculated as histamine base. Control tissues were incubated with 0.9% NaCl. In the second type of experiment, lungs were continuously perfused with 3J°C Tyrode's solution, pH 7.4, bubbled with 5% CO2 in 02. Histamine or 0.9% NaCl was administered by a Harvard syringe pump (Model No. 340) at a rate of 0.5 ml/min. directly into the pulmonary artery catheter. The final concentration of histamine base in the Tyrode's solution perfused through the lung was 9 x 10-6, 4.5 x 105, and 9 x 10-5M. Samples were collected preceding, during and at various intervals after the end of histamine infusion. They were processed and later analyzed for prostaglandin content as in other experiments. The effects of histamine receptor 1 and 2 antagonists on the release of prostaglandins by histamine were examined by preinfusing pyrilamine maleate (2.0 x 10-4M, Hexagon Laboratories, Bronx, N.Y.) or metiamide (1.2 x 10-4M, a generous gift from Smith, Kline and French Laboratories, Philadelphia, Pa.). Infusion of histamine antagonists or vehicle I (0.9% NaCl) preceded that of histamine or vehicle II (0.9% NaCl) by 5 minutes and lasted until the end of the experiment. In one series of experiments histamine (4.5 x 105M) or NaCl were infused for 4 minutes and the perfusates collected. In experiments where anaphylaxis was provoked 50 mg ovalbumin was injected over a 15 second period commencing with infusion of histamine or vehicle II. Prostaglandins: Immunoreactive prostaglandins E2/El, F /Flu, and 15-keto-13.14-dihvdro-PGF,_/E were determined by the ra2" loimmunoassay procedure developed by Le$?ne et al (1971 and 1973). Each sample was assayed in duplicate and the means calculated. The specificities of the antibodies used were as follows. The rabbit antiserum used for determination of E-type of prostaglandins was not sufficiently specific to distinguish between PGEl and PGE2; thus 50% inhibition was obtained with 0.13 ng PGEl and 0.15 ng PGE2, Consequently, although it is probable that PGE2

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and not PGEl was predominantly released from the lung and actually measured in our samples, no attempt was made to distinguish between the two prostaglandins, and the notation immunoreactive PGE (I. PGE) is used. Specificity of PGF antiserum allowed for a reasonable differentiation betwe& PGF2, and PGFlo; 30 times more PGFlo than PGF2, was needed to achieve the same per cent of inhlbition. Moreover, a few samples treated also with Fla antiserum showed no presence of Flu. The notation rmmunoreactive PGF2p (I.PGF2,) is therefore used. As far as anti-15-keto,l3,14-dihydroPGF /E, 50X inhibition was achieved with 0.3ng of 15-keto-13, 14-&hydro-PGF 2o and 20ng of 15-keto,l3,14-dihydro-PGE2. Since both parent compounds are released from guinea pig lung and determinations were not carried out before and after the alkali treatment, the metabolite(s) measured is denoted as 15-keto,l3,14-dihydro-PGF20/E(I. metabolite). However, in view of equal or higher concentrations of PGF2o than PGE in lung outflows and the relatively smaller cross reactivity (0.3ng versus 20ng) it can be assumed that the bulk of metabolite measured is derived from the PGF2a parent compound. RESULTS Table 1:

EFFECT OF ANTIGEN-SENSITIZATION ON THE SPONTANEOUS ZELEASE OF PROSTAGLANDINS FROM GUINEA PIG PARENCHYMA AND TRACHEA

Parenchyma

Trachea

Healthy

Sensitized

6.2 ? 0.8

7.4 f 0.8

29.6 * 4.6

27.0 + 4.3

I. PGF20

15.0 f 1.2

25.2 f 2.7

21.5 ?r2.2

25.2 + 2.8

I. Metabolites

34.1 + 4.6

48.5 f 5.2

trace

I. PGE

Healthy

Sensitized

trace

Table 1: Lung fragments and slices of trachea were incubated for 4 minutes in Tyrode's solution at 3J°C and the spontaneous release of prostaglandins into the media measured. Results expressed as mean f S.E. total ng prostaglandin/g wet weight tissue. N = 12fgroup.

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There was a considerable amount of prostaglandins released from the lung fragments and slices of trachea into the incubation media with time alone (Table 1.). Lung parenchyma released more PGF2u than PGE, whereas trachea released more PGE than PGF Lung parenchyma also released significant amounts of tfU' e metabolites. In contrast, only traces of metabolites were found in the media incubated with trachea. Calculated per gram tissue, about 4 times higher concentrations of PGE were found in the media incubated with trachea than parenchyma. While the concentrations of PGF2, were about equal,the mean ratios of PGE to PGF2u were .43 f .05 and .35 f .04 in the parenchymal media and 1.53 + .23 and 1.05 f -33 in tracheal media in the healthy and sensitized preparations. The process of sensitization caused an increase in the outflow of PGF and the metabolite from the parenchyma, but it did not pro%a uce a change in trachea. Moreover, it also led to decreased PGE/PGF2, ratio in both kinds of tissues. In parenchyma the PGE to PGF2o ratio was decreased secondary to an increase in PGF2,, while in the trachea a small decrease in PGE together with a small increase in PGF2u led to the same results. Table 2:

EFFECT OF HISTAMINE ON THE RELEASE OF PROSTAGLANHINS FROM HEALTHY AND SENSITIZRD GUINEA PIG LHNG FRAGMENTS Histamine(ug/ml)

Healthy 0 4.5 f 0.5

1 5.4 + 1.1

5 5.3 r 0.8

25 5.6 f 1.4

125 7.8 f 1.7

11.7 ? 1.5

11.6 f 1.6

12.2 + 1.7

12.2 + 1.2

12.3 2 1.0

I. metab- 17.0 it4.0 olites.

21.6 f 4.0

32.9 f 6.1

35.9 +_7.6

50.4 + 15.5

I. PGE I. PGF2,

Sensitized 0

1

5

25

125

6.4 ? 1.0

6.3 f 0.8

7.4 + 1.0

6.1 + 0.9

5.8 + 0.3

I. PGF2u

17.2 ? 1.3

18.2 f 0.6

18.8 f 1.0

18.7 f 1.9

19.0 f 1.0

I. metabolites

31.0 f 4.5

34.0 + 5.5

42.3 f 6.0

44.0 f 5.5

53.0 f 10.

I. PGE

Table 2: Lung fragments were incubated with NaClor 1, 5, 25 or 125 ug/ml histamine for 10 minutes in Tyrode's solution at 37OC and prostaglandins determined in the media. The results expressed as mean f S.E.ng prostaglandins/gwet weight. N = 6 animals/group.

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Effects of histamine on the release of prostaglandins from healthy and sensitized lung fragments is shown in Table 2. In healthy, histamine, only at the highest concentrations, increased the spontaneous release of PGE2 (p< 0.05). The spontaneous outflows of PGF2u did not change with any of the histamine concentrations used. Regression analysis showed that the release of 15-keto-13,14-dihydro_PGF2,/E was enhanced (p< 0.01). Histamine had no significant effect on the release of PGE and PGF from the sensitized lung fragments. However, the level of t?? e metabolites found in the medium was elevated by histamine (p< 0.01). Baseline control outflows of PGE, PGF2u and the metabolites were significantly higher in the media containing sensitized lung fragments than those in the healthy control (p = 0.06,

Release of prostaglandins from healthy and sensitized guinea-pig lung and trachea by histamine.

The effects of histamine and its antagonists on the release of prostaglandin E and F2alpha (PGE and PGF2alpha) and the 15-keto-13,14-dihydro PGF2alpha...
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