Release of prostaglandins and metabolites from guinea-pig lung: Inhibition by catecholamines

Release of Prostaglandins and Metabolites from Guinea-Pig Lung: Inhibition by Catecholamines Aleksander A. Math~ and Lawrence Levine +

Catecholamine Laboratory, Division of Psychiatry, Boston University School of Medicine, Boston, Massachusetts and Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts

ABSTRACT Prostaglandins E and F9~ and metabolites 13,14dihydro-15-keto-PGE 2 and 13~14-dihydro-15-keto-PGF2a were measured by specific radioimmunassay in outflows from isolated, perfused guinea-pig lungs~ Samples were collected from non-sensitized and sensitized guinea-pigs during baseline period and during challenge with ovalbumen or NaCI. Anaphylactic reaction greatly increased the release of both metabolites. Catecholamines, particularly epinephrine markedly reduced the content of metabolites in the outflow. Inhibition of prostaglandin release is parallel to that of histamine and points to the ~mportant role catecholamines may play in allergic reactions, specifically in modulating the release of mediators.

ACKNOWLEDGEMENTS Supported by grants from the National Heart and Lung Institute: SCOR Grant No. 15063 and Grant No. 15677, and by grants from the American Cancer Society, Grant No. IC-10L and the National Institute of Health, Grant No. HD-07966. We thank Barry Levine for excellent technical assistance. +American Cancer Society Professor of Biochemistry (Award PRP-21) Request for reprints should be addressed to: A.A. Math~, M.D., Boston University School of Medicine, 80 East Concord Street, Boston, Massachusetts 02118

Accepted October 10, 1973 PROSTAGLANDINS D E C E M B E R 1973

VOL. 4 NO. 6

877

PROSTAGLANDINS

INTRODUCTION Prostaglandins have been suggested to play a role in anaphylaxis and possibly in bronchial asthma (1,2,3). These assumptions are based on: observed actions of exogenous prostaglandins on the lung; inhibition of anaphylaxis by the same substance (polyphloretin phosphate) that inhibits effects of prostaglandins; and measurement of prostaglandin release from actively or passively sensitized lungs of various species, including human (2,3,4,5,6). However, the method of estimation was bioassay with its inherent difficulties in distinguishing between E and F group, as well as between prostaglandins and slow reacting substance and possibly other as yet unidentified mediators. Moreover, no measurement of metabolites was attempted in these experiments. Prostaglandins E and F can not, in all probability, be regarded as circulating hormones and when experimentally infused they are nearly completely inactivated during one single passage through the lung(7). Thus determination of metabolites is of great significance in assessing the processes occurring in this organ. Therefore, investigation of the release of prostaglandins from the lung using a more specific method of determination, namely radioimmunoassay, and measuring the main lung metabolites in addition to unchanged prostaglandins was undertaken. Furthermore, since catecholamines inhibit the release of histamine during anaphylactic shock (8,9) their possible antagonistic effect on the release of prostaglandins was also tested.

METHODS Ninety-six guinea-pigs weighing 500 ~ i00 g were sensitized with i.p. injection of 50 mg of ovalbumen (Sigma, A-5503) dissolved in 0.9% NaCI. A booster dose, 50 mg i.p., was given after a 48 hour interval. Control animals were injected with placebo (sodium chloride, 0.9%, was used as placebo throughout the whole experiment). Four weeks later the animals were randomly taken and stunned by a blow behind the head. The thorax was immediately opened, the animal bled and catheters inserted in situ via the right ventricle into the pulmonary artery and via the left atrium into the pulmonary vein. Perfusion was started with a Harvard pulsatile

878

D E C E M B E R 1973

VOL. 4 NO. 6

PROSTAGLANDINS

pump, model 1405, delivering 5 ml/minute of Tyrode solution kept at 37 ° C, pH 7.35 and bubbled with 5% CO 2 and 95% 02 . A special bubbletrap was constructed in order to prevent air emboli. A sidearm from the inflowing catheter was connected to a Statham pressure transducer P23 DC and the pressure in the pulmonary artery continuously monitored on a Grass polygraph. All animals with the pressure above 20 mm Hg at the end of the rinse period were discarded. After a ten minute rinse period the experiment was commenced and freely outflowing perfusate from pulmonary vein collected into chilled test tubes. Prostaglandins and metabolltes were measured in perfusates from the following groups of guinea-pigs: nonsensitized, sensitized non-challenged and sensitized challenged with ovalbumen. The experimental schedule is presented in Table I.

Table I

Experimental schedule: isolated, perfused guinea-pig lungs Non-sensitized

Sensitized non-

Period lbaseline

0

0

Period 2treatment/ challenge

NaCI NaCI

Epi NaCI

challenged

challenged

0

0

0

0

0

0

NE NaCI

NaCI NaCI

Epi NaCI

NaCI Ovalb

Epi Ovalb

NE Ovalb

Each experiment consisted of 2 ten minute periods; the first serving as baseline control only, and the second being randomly either treatment and challenge or additional control period. Anaphylactic reaction was elicited with 0.5 and 5 mg ovalbumen in 0.5 ml of 0.9% NaCI successively injected into the pulmonary artery catheter at the beginning of the second ex-

D E C E M B E R 1973

VOL. 4 NO. 6

879

PROSTAGLANDINS

perimental period and 3 minutes later. All non-challenged animals received 0.5 ml of 0.9% NaCI at the same point in time instead. Most anaphylactic lungs registered 2 increases in the pulmonary artery pressure in response to the repeated application of antigen whereas injection of placebo had no effect. At the beginning Of the second period infusion of freshly prepared either 1-epinephrine or lnorepinephrine (both bitartarate, 10 ug/ml) or NaCI (0.9%) was started at the rate of 0.5 ml/minute. It preceded the challenge with ovalbumen or placebo by exactly 15 seconds and continued throughout the whole period. Harvard 940 syringe pump was used and infusion given via a separate needle directly into the pulmonary artery catheter. Prostaglandins E l and/or Eo and F~ and /or F. , and 13,14-dihydro-15-~eto-PGE 2 ~nd 1 3 , ~ - d i h y d r o - l ~ k e t o - P G F 2 ~ were determined by radioimmunoassay. The specificities of the antisera used in this study are described in detail elsewhere (10,11,12) and the cross reactions with antiserum to 13,14-dihydro-15-keto-PGF2~ are shown in Table II.

RESULTS Prosta$1andin E PGE (E 1 and/or Ep) or PGA (A 1 and/or A 2) was measured as PGB after treating-the perfusaEe for 5 mln at lO0°C at pH 12.5. None or less than i ng/ml perfusate was found. The results are therefore not presented quantitatively. In non" sensitized guinea-pigs, PGE was not detectable in any of the 13 control period samples (Table III). However, traces could be measured in 2 of the same 13 animals when treated with epinephrine. In sensitized animals, PGE was measured as follows: 2/40 controls and 2/11 epinephrine treated, 0/13 challenged with ovalbumen, 7/16 when both epinephrine treated and challenged with ovalbumen. Thus it appears that epinephrine, alone and possibly in combination with ovalbumen challenge might, at least in some instances, promote the release of PGE.

880

D E C E M B E R 1973

VOL. 4 NO. 6

o

==

27.0 670.0

13,14-dihydro-15-keto-PGE 2

13,14-dihydro-15-keto-PGE 2 pH 12.5, AlO0 °, 5 min i ,300.0

4.2

13,14-dihydro-15-keto-PGF2a pH 12.5, Al00 °, 5 min

PGF2~

4.2

(n$)

50% Inhibition

13,14-dihydro-15-keto-PGF2~

Inhibitor

13,14-dihydro-PGF2~

15-keto-PGE_ pH 12.5,A~00 °, 5 min

3,600

>5,000

36

55

15-keto-PGFo pH 12.5,A~0 °, 5 min 15-keto-PGE 2

i0

(n~)

50% Inhibition

binding

15-keto-PGF2~

Inhibitor

Inhibition of (3H)-15-keto-PGF2~ anti-13,14-dihydro-15-keto-PGF2~

TABLE II

>

©

PROSTAGLANDINS

Table III

Traces of prostaglandins E and F2~ found in perfusates from isolated guinea-pig lungs. Ratios represent number of animals in which prostaglandin was detected/total animals tested.

Non-Sensitized Control

NaCI

Sensitized Epi

Control

NaCI

Epi

Ovalb

Epi+Ovalb

PGE

0/13

-

2/13

2/40

-

2/11 0/13

7/16

PGF2~

4/23

3/10

1/13

19/53

4/13

2/11 13/13

2/16

Prostaglandin F2~ As with PGE, no quantitative results are presented because, when measurable, only traces were found. In nonsensitized animals PGF 2 was detectable in 4/23 control period samples. When t~ese guinea-pigs were treated with NaCI or epinephrine PGF2~ was present in 3/10 and 1/13 samples respectively. In sensitized guinea-pigs PGF2~ was detectable in 19/53 control perfusates and 4/13 NaCI treated and 2/11 epinephrine treated animals. However, when challenged with ovalbumen PGF 2 was found in each of the 13 perfusates, although at low ~evels only. In contrast, in the additional 16 ovalbumen challenged but also epinephrine treated guinea-pigs, PGF~ was detected in only 2 samples. Results suggest that ~ F 2 ~ may be released as a consequence of anaphylactic reaction and that epinephrine inhibits this process.

13,14-dihydro-15-keto-PGE 2 and 13,14-dihydro-15-keto-PGF2~ The perfusates were assayed with anti-13,14-dihydro-15keto-PGF2~ before and after heating at 100°C at pH 12.5 for 5 minutes. As shown in Table II, 96% of the serologic activity of 13,14-dihydro-15-keto-PGE 2 is destroyed by such treatment, while that of 13,14-dihydro-15-keto-PGF2~ is

882

D E C E M B E R 1973

VOL. 4 NO. 6

PROSTAGLANDINS

stable• Since the metabolite, 15-keto-PGF2~ , cross reacts appreciably with this antiserum, the perfusates were also assayed with a monkey antiserum to 15-keto-PGF2~ which cross reacts relatively poorly with 13,14-dihydro15-keto-PGE 2 and F 2 . With the use of both antiserums it was shown that t~e 13,14-dihydro-15-keto-PGE 2 and 13,14dihydro-15-keto-PGF2~ and not the 15-keto-PGmetabolites were being measured. In non-sensitized and sensitized but not challenged guinea-pigs <600 nanograms 13,14dihydro-15-keto-PGE 2 and 0 05) and 25.8% Of 13,14-dihydro-15-keto-PGF2~ (p~0.O01) weSe'found in the outflow• Moreover, the ratio ~f E 2 to F2~ increased to 13:1 showing a relatively higher degree of PGF2~ inhibition by epinephrine.

D E C E M B E R 1973

VOL. 4 NO. 6

883

== ==

OO

E:F metabolite

i00

6:1

2944~309 (13)

ng/perfusate N

% control

E2

metabolite

Control

I00

468!47 (13)

F2~

58.1 9:1

1717~344 (i0)

E2

NE

42.9

201!37 (I0)

F2~

52.8 13:1

1555!404 (16)

E2

Epi

Release of 13,14-dihydro-15-keto-PGE 2 and 13,14-dihydro15-keto-PGF2~ from isolated, perfused guinea-pig lungs challenged w~th ovalbumen. Results are expressed as mean ± SE nanograms/perfusate. Infusion of either sodium chloride (Control) or norepinephrine (NE) or epinephrine (Epi) was started 15 seconds before the challenge with antigen. Number of animals in parenthesis.

TABLE IV

25.8

121±12 (16)

F2~

>

>

PROSTAGLANDINS

FIGURE I

Isolated, perfused sensitized guinea-pig lungs challenged with ovalbumen. Mean changes in release of 13,14-dihydro15-keto-PGEo and 13,14-dihydro-15-keto-PGF~ from control to treatment/challenge perlods. Infuslon o~ sodium chloride (Ovalb + NaCI), norepinephrine (Ovalb + NE) or epinephrine (Ovalb + E) was started 15 seconds before the challenge with antigen. Number of animals in parenthesis.

E z metabolite

F2oc metabolite

mean

mean A

ng/ Perfusate

ng/Per fusate

15oo

2500

400

T

/m Ovalb ÷ NaCI

(13)

D E C E M B E R 1973

Ovalb + NE (lO)

Ovalb ÷ E (16)

VOL. 4 NO. 6

-300 - 200 - I00

I-=7 Ovalb ÷ NaC[

(i3)

Ovalb + NE (I0)

__0

Ovalb + E (t6)

885

PROSTAGLANDINS

DISCUSSION Several researchers have postulated that PGF2~ is one of the mediators of anaphylactic bronchoconstriction and that prostaglandins - predominantly of the E type modulate the anaphylactic reaction, since they are released during anaphylaxis from lungs of several species, including human (2,4,5,6). These conclusions were based primarily on bioassay methods (cf. Introduction). In our system with use of a specific radioimmunoassay method, little or no PGE and PGFo were detected. However, metabolites 13,14dlhydro-15-~eto-PGEp and 13,14-dihydro-15-keto-PGF2~, previously not measured, were found in relatively h~gh quantities. There was preponderance of E type metabolite; the E2:F 2 ratio being approximately 6:1, which confirms the suggestion (2,6) that predominantly E is released as a consequence of antigen-antibody reaction. •

The site of synthesis, the mechanism of increased release and the role of prostaglandins in anaphylaxis are not well understood. Piper and Vane (2) have hypothesized that prostaglandins are released whenever cells are damaged and/or mechanical changes, such as distortion or stretching of the cell membrane induced. The latter was probably not the major source of prostaglandins in our experiments since lungs were not ventilated nor mechanically interfered with. During the baseline period the pressure in the pulmonary artery was constant and well below 20 mm Hg. Anaphylaxis always caused a rise in the pulmonary artery pressure and it is probable that there was a simultaneous contraction of the bronchial smooth muscle. In a separate investigation it was found that histamine in concentrations sufficient to elicit bronchoconstriction also released prostaglandins (Math~ and Levine, unpublished data). If prostaglandins are released secondary to the contraction of bronchial smooth muscle, the possibility that contraction of other smooth muscle, for instance in the vasculature has the same effect should also be considered. Finally, changes in the mast cells concommitant to and/or as a consequence of exocytosis of histamine could be an additional significant source of these compounds.

886

D E C E M B E R 1973

VOL. 4 NO. 6

PROSTAGLANDINS

The constantly much greater ratio of metabolites to unchanged prostaglandins suggests that, regardless of the site of synthesis and mechanism of release, prostaglandins are mainly metabolized within the cell and only subsequently released as metabolites. Such a view is supported by the fact that we could not detect 15-hydroxy prostaglandin dehydrogenase or Al3,14-reductase in a number of perfusates where they were measured. This is in line with the previously reported extreme efficiency of the lung in inactivating the infused prostaglandins (7), and - from a broader physiologic viewpoint - with the postulate that prostaglandins of F and E type are primarily acting locally at the site of synthesis and are not, at least under physiologic circumstances, circulating hormones. Stimulation of sympathetic nerves in certain intact tissues promotes release of predominantly E prostaglandins (13), and in homogenates of the rat stomach fundus catecholamines increase synthesis of prostaglandin E but not F type (14). In this investigation epinephrine markedly decreased release, following anaphylactic reaction, of both metabolites. Since it also changed the E~:F~ ratio from 6:1 to 13:1 more Z Z~ s p e c l f z c i n h i b i t i o n o f PGFpa s y n t h e s i s / r e l e a s e can be a s s u m e d . This is now being investigated. In view of the opposite actions of PGE and PGF in the lung, such property of epinephrine would seem plausible and consistent with its clinical effects in bronchial asthma. The mechanism of inhibition of prostaglandins by catecholamines is not known. Influence on the antigen-antibody binding and effects on the cell membrane were not studied. Inhibition of bronchoconstriction, although probably significant, does not seem to constitute in our system the predominant mode of action. Another possibility: increase in cyclic A~P could be an important mechanism, since cyclic AMP might regulate the expression of immediate hypersensitivity .

.

.

.

.

.

(15,16). Schild and co-workers (8,9) found that catecholamines inhibit the release of histamine in a variety of anaphylactic systems. Since they inhibit both histamine and prostaglandins, as demonstrated in this investigation, it is conceivable that at least some of the prostaglandin synthesis/release

D E C E M B E R 1973

VOL. 4 NO. 6

887

PROSTAGLANDINS

is concommitant to or induced by the release of histamine and distortion of the mast cell membrane. The same authors further hypothesized that the inhibitory effect may be by increase in the levels of cyclic AMP and that circulating epinephrine might play a role as a physiologic regulator of anaphylactic reactivity in the organism. Previously, we have shown that asthmatic patients are hypersensitive to PGF ~ (3) and, independently, that they .2 4 . , excrete less free eplnephrlne in the urine when compared to the healthy controls (17). It was suggested that complementary to and interacting with the antigenantibody reaction a dysfunction of the autonomic nervous system, specifically, decreased availability of catecholamines locally in the lung (18), is contributing to the pathogenesis of bronchial asthma. The results presented - increase of prostaglandin release by anaphylaxis and decrease by catecholamines - seem to be consistent with that hypothesis and constitute further supportive evidence for the important function catecholamines may exert in allergic reactions. Such a role is probably not only postjunctional, that is direct action on the effector organ, but also prejunctional, that is modulation of the release of mediators. The mechanism of such modulation remains to be elucidated. In conclusion, the data presented demonstrate significant release of prostaglandins during anaphylactic reaction and show that they are predominantly found as specific E 2 and F 2 metabolites. Catecholamines markedly inhlblted the release of prostaglandln metabolltes, more so F2~ type. T h e s e findings perhaPs point to the important role circulating catecholamiNes may p l a y i n therapy - and possibl e unavailability of catecholamines in pathogenesis - of certain allergic disorders.

888

DECEMBER 1973

VOL. 4 NO. 6

PROSTAGLANDINS

REFERENCES

i.

Horton, E.W., Hypotheses on Physiological Roles of Prostaglandins. Pysiological Reviews 49: 122, 1969

2.

Piper, P.J. and J.R. Vane. The Release of Prostaglandin from Lung and Other Tissues. N.Y. Acad. Sci. 180: 363, 1971

3.

Math~, A.A., P. Hedqvist, A. Holmgren, and N. Svanborg. Bronchial Hyperreactivity to Prostaglandin F, and Histamine in Patlents wlth Asthma. Br. Med. 5. i: 193, 1973 •

•

•

L

4.

Piper, P.J. and J.R. Vane. Release of Additional Factors in Anaphylaxis and its Antagonism by Anti-Inflammatory Drugs. Nature 223:29, 1969

5.

Strandberg, K., A.A. Math~ and B. Fredholm. Protective Effects of Polyphloretin Phosphate in Anaphylaxis in the Guinea-Pig; Life Sci. ii, Part I: 701, 1972

.

Piper, P.J. and J.L. Walker. The Release of Spasmogenic Substances from Human Chopped Lung Tissue and its Inhibition. Br. J. Pharmac. 47: 291, 1973

7.

Ferreira, S.H. and J.R. Vane. Prostaglandins: Their Disappearance from and Release into the Circulation. Nature 216: 868, 1967

8.

Schild, H.O. Histamine Release and Anaphylactic Shock in Isolated Lungs of Guinea Pigs. Quart. J. exp. Physiol. 26: 576, 1971

9.

Assem, E.S.K. and H.O. Schild. Inhibition of the Anaphylactic Mechanism by Sympathomimetic Amines. Arch. Allergy 40: 567, 1971

Int.

i0.

Levine, L., R.M. Gutierrez-Cernosek and H. Van Vunakis. Specificities of Prostaglandins BI, FI and F~ AntigenAntibody Reactions. J. Biol. Chem. 2 ~ : 6782~1971

Ii.

Levine, L., R.M. Gutierrez-Cernosek. Levels of 13,14Dihydro-15-Keto-PGF~ in Biological Fluids as Measured by Radioimmunoassay7 Prostaglandins 3: 785. 1973

D E C E M B E R 1973

VOL. 4 NO. 6

889

PROSTAGLANDINS

12.

Levine, L., Antibodies to Pharmacologically Active Molecules: Specificities and Some Applications of Antiprostaglandins. Pharmacological Reviews 25: 293, 1973

13.

Ramwell, P.W., and J.E. Shaw. Biological Significance of the Prostaglandins. In Recent Progress in Hormone Research, Vol 26 (E.B. Astwood, Editor), p. 139, 1970

i4.

Pace-Asciak, C.R., Catecholamine Induced Increase in Prostaglandin E Biosynthesis in Homogenates of the Rat Stomach Fundus. In Adv. in the Biosciences, Vol. 9 (S. BergstrSm and S. Bernhard, Editors), Pergamon Press-Vieweg, Oxford, 1973

15.

Lichtenstein, L.M., E. Gillespie, H.R. Bourne, and C.S. Henney. The Effects of a Series of Prostaglandins on in vitro Models of the Allergic Response and Cellular Immunity. Prostaglandins 2: 519, 1972

16.

Orange, R.P., M.A. Kaliner and K.F. Austen. The Immunological Release of Histamine and Slow-Reacting Substance of Anaphylaxis from Human Lung. III Biochemical Control Mechanisms Involved in the Immunologic Release of the Chemical Mediators. In Biochemistry of the Acute Allergic Reactions (K.F. Austen and E.L. Becker, Editors), Blackwell Scientific Publications, Oxford, 1971

17.

Math~, A.A. and P.H. Knapp. Decreased Plasma Free Fatty Acids and Urinary Epinephrine in Bronchial Asthma. New Eng. J. Med. 281: 234, 1969

18.

Math~, A.A. Decreased Circulating Epinephrine, Possibly Secondary to Decreased Hypothalamic-Adrenal Medullary Discharge; A Supplementary Hypothesis of Bronchial Asthma Pathogenesis. J. Psychosom. Res. 15: 349, 1971

890

D E C E M B E R 1973

VOL. 4 NO. 6