Formation of cysteinyl-containing leukotrienes by human arterial tissues

PROSTAGLANDINS

FORMATION OF CYSTEINYL-CONTAINING LEUKOTRIENES BY HUMAN ARTERIAL TISSUES. M. Edelmann2, G. Wittmannl B. M. Peskarl Th. Simmet4 ind B. A. Peskar'4 1

2

K. M. Miiller3,

Department of Experimental Clinical Medicine, Ruhr-University of Bochum, D-4630 Bochum 1, F.R.G. Department of Surgery, Knappschaftskrankenhaus Langendreer, Ruhr-University of Bochum, D-4630 Bochum 7, F.R.G.

3 Department of Pathology, Hospital "Bergmannsheil" Ruhr-University 4

of Bochum, D-4630 Bochum

Institute of Pharmacology and Toxicology, Ruhr-University of Bochum, D-4630, Bochum

1, F.R.G.

1, F.R.G.

ABSTRACT Human arterial rings incubated in modified Tyrode solution released small amounts of leukotriene (LT) C4-like material spontaneously and larger amounts upon stimulation with the ionophore A23187 as determined by radioimmunoassay. By reversed phase high pressure liquid chromatography (HPLC) LTC4-like material was found to comigrate with authentic LTC4, LTD4 and LTE4. Nordihydroguaiaretic acid (NDGA) significantly inhibited the ionophore A23187induced release of LTC4-like material,while indomethacin was without effect. Simultaneously the arterial rings released much larger amounts of 6-keto-prostaglandin (PG) which were significantly decreased by indomethacin. Fl a The rksults demonstrate that human arterial tissue has the capacity to synthesize cysteinyl-containing LT from endogenous arachidonic acid. INTRODUCTION As early as in 1960 Brocklehurst (1) has demonstrated that vascular tissue is an important source of slow-reacting substance of anaphylaxis (SRS-A), which is now known to consist of the cysteinyl-containing LTC4, LTD4 and LTE4 (2). Release of LT-like material from chopped vascular tissue of several animal species after stimulation with ionophore A23187 or after antigen challenge has been reported previously (3, 4). On the other hand, the vascular system is an important target organ for LT effects as LTC4, LTD4 and LTE4 exert potent vasoconstrictor actions in a variety of species both in vitro and in vivo and can

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enhance vascular permeability (2). Therefore, local formation of cysteinyl-containing LT might play an important role in the vascular system either alone or in combination with other vasoactive compounds. We have now investigated the capacity of human arterial tissue to synthesize cysteinyl-containing LT under basal and stimulated incubation conditions. MATERIALS

AND METHODS

Materials LTC4, LTD4 and LTE4 were generous gifts of Dr. J. Rokach, Merck Frosst Laboratories, Pointe-Claire, Dorval, Canada. Indomethacin, nordihydroguaiaretic acid and ionophore A23187 were from Sigma, St. Louis, USA. Tritiumlabelled eicosanoids were purchased from New England Nuclear Co., Dreieich, FRG. Methods Human arterial tissue was obtained from patients (age 29 - 83; 5 male, 10 female) undergoing surgery for colonic cancer or gastric ulceration. The arterial tissue used for the present experiments consisted of pieces from the right gastric or gastro-epiploic artery, the marginal arteries or from the superior rectal artery. Portions of 120 mg (wet weight) of washed arterial rings were incubated at 370C for 20 min in 0.5 ml modified Tyrode solution (composition (mmol/l): NaCl 137, KC1 2.7, CaC12 1.8, MgC12 1.1, NaHC03 11.9, NaH2P04 0.4, glucose 5.61, gassed with 95% 02 - 5% co2. After 20 min the incubation media were removed and replaced by fresh buffer containing ionophore A23187 (1.0 x IO-5 mol/l) or its solvent dimethyl sulfoxide (DMSO). Incubations in the presence or absence of ionophore were continued for 60 min. When tissues were incubated in the presence of indomethacin (2.8 x 10-6 mol/l) or NDGA (1.0 x IO-5 mol/l) the compounds were present in the incubation media both during the preincubation period and after addition of ionophore A23187. All tissue samples were fixed in formalin and examined histologically after the incubations. in the incuCysteinyl-containing LT and 6-keto-PGFla bation media were determined radioimmunologically (5, 6). The detection limits of the radioimmunoassays used were 33 fmolesltube for LTC4 and 35 fmolesltube for 6-keto, respectively. The anti-LTC4 antibodies used for PGFla the present experiments recognize mainly LTC4, but show 40% relative cross-reaction with both LTD4 and LTE4, while

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other eicosanoids do not interfere with the assay. Release of cysteinyl-containing LT was expressed as immunoreactive LTC4. Cysteinyl-containing LT were further characterized by reversed phase HPLC profiling of the immunoreactivity exactly as described previously (7). Means + S.E.M. were calculated and statistical analysis was performed using Student's t-test. RESULTS Washed human arterial rings stimulated with ionophore A23187 (1.0 x IO-5 mol/l) released 15.00 + 2.75 ng/g/60 min (n=15) of LTC4-like material, while in the absence of ionophore A23187 only 0.95 f 0.20 ng/g/60 min (n=7) were generated (Table 1). During the preincubation period 0.90 + 0.15 nglgl20 min (n=15) were spontaneously released into the incubation medium (Table I). The cyclooxygenase inhibitor indomethacin (2.8 x 10-6 mol/l) did not significantly affect the spontaneous or the ionophore A23187induced release of LTC4-like material (Table 1). On the other hand, the lipoxygenase inhibitor NDGA (1.0 x IO-5 mol/l) tended to decrease the release of LTC4-like material during the preincubation period and significantly inhibited the release in the presence of ionophore A23187 by 86.1 +- 4.8 "/o(P < 0.005, n=9) as compared to controls stimulated in the absence of NDGA (Table 1). The incubated arterial tissue released much larger amounts of 6-keto-PGF10 than of cysteinyl-containing LT (Table 1). However, contrary to cysteinyl-containing LT release of 6-keto-PGFla was not significantly stimulated by the ionophore A23187. While NDGA in the concentration used (1.0 x IO-5 mol/l) tended to decrease the release of 6-keto-PGF1 o during the preincubation period as well as during ionophore A23187 stimulation, indomethacin (2.8 x 10-6 mol/l) significantly inhibited release of 6-keto-PGF1 a in either period (P < 0.01, n=8). Characterization of the LTC4-like material generated by human arterial tissues by reversed phase HPLC demonstrated that the immunoreactive material consisted of a mixture of LTC4, LTD4 and LTE4 (Fig. 1). Release of these compounds was detected during the preincubation period as well as during ionophore A23187 stimulation (Fig. 1). Histological examination of the arterial tissues used did not reveal any morphological signs of inflammation. DISCUSSION In previous

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Table 1 Release of LTC4-like material and 6-keto-PCF1, by human arterial rings during a 20 min preincubation period followed by a 60 min incubation in the presence of ionophore A23187 or the solvent DMSO and the effects of indomethatin and NDGA. LTC4-LIKE MATERIAL Preincubation A23187 (1.0 x IO-5 mol/l) DMSO

6-KETO-PGF1,

0.90 t 0.15 (15)

68.43 + 13.50 (15)

15.00 + 2.75" (15)

72.48 + 16.01 (15)

0.95 + 0.20 (7)

68.69 + 20.04 (7)

1.06 + 0.39 (8)

13.83&3.33+

Indomethacin (2.8

x

10-6 mol/l)

Preincubation

18.62 + 4.84 (8)

8.52 + 1.12+ (8)

Preincubation

0.55 f 0.12 (9)

34.37 + 7.92 (9)

A23187 (1.0 x IO-5 mol/l)

2.08 + 0.72++ (9)

43.13 + 7.78 (9)

A23187 (1.0

x

IO-5 mol/l)

NDGA (1.0 x IO-5 mol/l)

"P < 0.005 as compared to tissues incubated in the presence of DMSO; ++ P ( 0.005, + P < 0.01 as compared to controls in the absence of indomethacin or NDGA. vascular tissue from sensitized guinea-pigs challenged with antigen in vitro released LTC4-like material as well as SRS-A-like biological activity (3, 4). Similarly, stimulation of chopped vascular tissue from guinea-pig, rat, rabbit, dog and pig with the ionophore A23187 resulted in release of LT-like material as established by bioassay and reversed phase HPLC (3, 8, 9). Piomelli et al.

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LTCl

LTD4

4

4

4

4

I

4

LTD4 LTE4

LTC4

0

LTE4

1

10

RETENTION

+

1

1

20

30

TIME

(min)

Fig 1. Reversed phase high pressure liquid chromatographic (HPLC) profile of immunoreactive cysteinyl-containing LT released from human arterial tissue during a 20 min preincubation period (upper panel) and during a 60 min incubation period in the presence of the ionophore A23187 (lower panel). Arrows indicate the retention time of appropriate standards. The figure shows the results of a representative experiment, which was performed three times with qualitatively identical results.

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(IO) have suggested that human coronary artery rings obtained at cardiac transplantation can also synthesize LTC4, LTD4 and LTE4. However, no quantitative data were given. Our present results demonstrate that human arterial tissue from the gastrointestinal tract releases cysteinyl-containing LT upon stimulation with ionophore A23187, the absolute amounts being in the same order of magnitude as those released by porcine arterial tissue (8). By reversed phase HPLC the immunoreactive material was shown to coelute with authentic LTC4, LTD4 and LTE4. In contrast, after incubation of porcine pulmonary arteries for 60 min only LTE4 was detected in the incubation fluid, although at 30 min LTC4 and LTD4 could be detected as well (8). Thus, porcine vascular tissue seems to metabolize LTC4 to LTD4 and LTE4 more rapidly than human arterial tissue. While chopped vascular tissue of various species did not release measurable amounts of cysteinyl-containing LT before antigen challenge or ionophore A23187 stimulation (3, 41, human arterial rings released cysteinyl-containing LT already during the preincubation period. As confirmed by reversed phase HPLC the profile of this spontaneous release is qualitatively similar to that released in the presence of ionophore A23187. Similar to porcine vascular tissue (8) formation of cysteinyl-containing LT by human arterial rings was inhibited by the lipoxygenase inhibitor NDGA. The cyclooxygenase inhibitor indomethacin, however, which in the concentration used potentiated release of cysteinyl-containing LT from animal arterial tissues, probably by diversion of arachidonic acid metabolism away from the cyclooxygenase towards the 5-lipoxygenase pathway of arachidonic acid metabolism (3, 8, 9), did not significantly affect release of cysteinyl-containing LT from human arterial rings. The cellular site of cysteinyl-containing LT release from the arterial tissues is not known. Histological examination did not show signs of inflammation or the presence of mast cells in the tissues used. Studies using guinea-pig and porcine vascular tissue have demonstrated that the adventitia is a major source of cysteinyl-containing LT (3, 4, 8, 9). In addition, in porcine tissue the intima seems to contribute considerably to the total release of cysteinyl-containing LT (8). Only recently has it been demonstrated that the ionophore A23187 did not induce formation of cysteinyl-containing LT in human endothelial cells (II). However, incubation of these cells with the precursor LTA4 led to the formation of LTC4, LTD4 and LTE4.

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In conclusion our results demonstrate that human arterial tissue obtained from the gastrointestinal tract has the capacity to synthesize LTC4, LTD4 and LTE4 from endogenous precursor arachidonic acid. Release of cysteinylcontaining LT upon the rather mild stimulus of incubation in vitro in the absence of ionophore A23187 strongly supports the possibility that endogenous cysteinyl-containing LT synthesized locally in the arterial wall might be -involved in vasospastic and inflammatory reactions of the human vasculature. ACKNOWLEDGEMENT This work was supported meinschaft.

by the Deutsche

Forschungsge-

REFERENCES 1) Brocklehurst, W.E. The release of histamine and formation of a slow-reacting substance (SRS-A) during anaphylactic shock. J. Physiol. (London) 151:416, 1960. 2)

Samuelsson, B. Leukotrienes: Mediators of immediate hypersensitivity reactions and inflammation. Science 220:568, 1983.

3) Piper, P.J., L.G. Letts and S.A. Galton. Generation of a leukotriene-like substance from porcine vascular and other tissues. Prostaglandins 25:591, 1983. 4) Wolbling, R.H., U. Aehringhaus, B.M. Peskar and B.A. Peskar. Release of slow-reacting substance of anaphylaxis from layers of guinea pig aorta. Prostaglandins 25:823, 1983. 5) Peskar, B.A., Ch. Steffens and B.M. Peskar. In: Radioimmunoassay of Drugs and Hormones in Cardiovascular Medicine. (A. Albertini, M. DaPrada and B.A. Peskar, eds.) Elsevier/North-Holland Biomedical Press, Amsterdam, 1979, p. 239. 6) Aehringhaus, U., R.H. Wolbling, W. Konig, C. Patrono, B.M. Peskar and B.A. Peskar. Release of leukotriene C4 from human polymorphonuclear leucocytes as determined by radioimmunoassay. FEBS Lett. 146:111, 1982. 7) Peskar, B.M., K. Lange, U. Hoppe and B.A. Peskar. Ethanol stimulates formation of leukotriene C4 in rat gastric mucosa. Prostaglandins 31:283, 1986. 8) Piper, P.J. and S.A. Galton. Generation of leukotriene B4 and leukotriene E4 from porcine pulmonary artery. Prostaglandins 28:905, 1984.

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9) Samhoun, M.N. and P.J. Piper. Action and interactions of lipoxygenase and cycle-oxygenase products in respiratory and vascular tissues. Prostaglandins Leukotrienes Med. 13:79, 1984. IO) Piomelli,

D., S.J. Feinmark and P.J. Cannon. Leukotriene and hydroxy acid biosynthesis by canine and human coronary arteries. Clin. Res. 33:521A, 1985.

11) Claesson, H.E. and J. Haeggstrom. Metabolism of leukotriene A4 by human endothelial cells. Evidence for leukotriene C4 and D4 formation by leukocyte-endothelial cell interaction. Adv. Prostaglandin Thromboxane and Leukotriene Res., Vol. 17, Raven Press, New York (in press).

Editor: P. Piper

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Received 12-l-87

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Accepted: 2-26-87

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