Prostaglandin D2 and endothelin-1 induce the production of prostaglandin F2α, 9 α, 11β-prostaglandin F2, prostaglandin E2, and thromboxane in capillary endothelium of human brain

PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS

Prostaglandin D2 and Endothelin-1 Induce the Production of Prostaglandin Fza, 9 a, lip-Prostaglandin FZ,Prostaglandin E2, and Thromboxane in Capillary Endothelium of Human Brain M. Spatz. D. Stanimirovic,

S. Uematsu,

*L. J. Roberts, II, **J. Bembry and R. M. McCarron

StrwkeBrcrwc~h. Nutional Institute qf Neurological Disorders and Stroke, Nutional lmtitutes oj’Hculth. Builditlg 30. Room 40-04. 9000 Rock\ille Pike. Bethesda. MD, 20892, USA. =The Johns Hopkins Hospital. Bultitm-e MD, 21287. USA. ‘Department oj’ Pharmm~log~ and Medicine. Vawderhilt Uk~ersit~, Ntrsh\?llc, TN 27232. USA (wpklt requests to MS) ABSTRACT. Endothelial cells derived from human brain capillaries (HBCEC) synthesize prostaglandin Dz (PGD2) which can be stimulated, among other prostanoids, by endothelin 1 (ET-l). Both the PGD, induced by ET-1 and the exogenously added PGDz to HBCEC are converted to 9a, lip-prostaglandin F2 (9a, lib-PGF?), a known potent vasoconstrictor. Exogenous PGD, also dose-dependently enhanced the production of vasoconstrictive PGF2,, thromboxane B, (TXB,), and the vasodilatory PGE2. as well as CAMP by HBCEC. The PGD,induced formation of PGF2,, PGE2, and TXBz was reduced by the cyclooxygenase inhibitors acetylsalicylic acid (.\SA) or indomethacin (Indo), indicating for the first time that PGDz may contribute to the formation of prostanoids in HBCEC. These results strongly suggest that PGDz may play an important role in the regulation of cerebral capillary function under physiologic and pathologic conditions.

INTRODUCTION

the endogenous (induced) PGD, is converted to 9a, 11P-PGF,. and stimulates the formation of PGF?,. PGE?. thromboxane B2 (TXB?), and CAMP in HBCEC.

Angiotensin II (Ang II)-or arginine vasopressin (AVP)stimulated production of endothelin I (ET-l) and prostaglandin D, (PGD,) has been shown by us to be mediated by their respective receptors (Ang II, AVPl) in human brain endothelial cells (1, 2). Previously, both synthesis and metabolism of PGD, were demonstrated in the human brain. mast cells, and other tissues (3, 4-14). An overproduction of PGD, and its metabolites was reported in patients with systemic mastocytosis. a disease characterized by increased proliferation of mast cells I IS). PGD? causes either vasodilation or vasoconstriction. inhibits platelet aggregation, and induces a variety of other biologic responses which may be linked to adenylate cyclase (AC) activity (3.4). Since cerebral mrcrovascular function could be influenced by PGD, or(ginating either from endogenous (endothelial) or exogenous sources, we investigated the response of human brain capillary endothelial cells (HBCEC) prostanoids and the AC system to PGD,. This report documents for the first time that both the exogenous and

MATERIALS

AND METHODS

Capillary EC used for this study were isolated from a small sample of human brain (surgically removed for the treatment of idiopathic epilepsy) cultivated and propagated by a modified technique of Gerhard et al ( 16). The purity of the HBCEC was >9X% as assessed by immunostaining for von Willebrand (Factor VIII)-related antigen, incorporation of acetylated low-density lipoprotein (Dil-Ac-LDL). and glial fibrillary acidic protein (GFAP). The effect of various concentrations of PGD, (l-100 nM) in the presence or absence of dexamethasone [(inhibitor of phospholipase Al and/or cyclooxygenase II). 50 PM] ( 17 ). cyclooxygenase blockers [acetylsalicylic acid (ASA) (500 pM) or indomethacin (Indo, 20 yM)] or ET-I (100 nM) (alone or with BQ133) was evaluated on the production of TXB?, PGF,,,. PGE,. and CAMP by HBCEC grown to conlluency in Petri dishes (34 mm) in the presence of ?-isobutyl-methyl-lxanthine (IBMX). After removal of the serum-containing medium, the HBCEC were washed with phosphate

Date rece~vd 2 February lYY3 Date accepted 25 May 1993 7x9

790

Prostaglandins

Leukotrienes

and Essential Fatty Acids

buffer solution and preincubated in serum-free medium (Gibco M199) with or without inhibitors for 30 min at 37°C prior to the addition of the tested substances. The samples of the medium were collected and adjusted to pH 3.0 with 1 M citric acid (25 pi/ml) after 4, 8 or 24 h incubation, and were immediately stored at -70°C until assayed. PGD2, PGF2,, PGE,, 6-keto PG12, and TXBz levels were determined in unextracted acidified medium by radioimmunoassays utilizing respective specific (98% as indicated by Sigma Co. The estimated contamination of PGD,? with either PGF,,, PGE2 or TXB?, was < 1% as assessed by immunoassays with their respective antibodies after incubation (37°C) of PGD2 in the medium without cells. Statistical evaluation was performed using unpaired Student’s t-test or single factor analysis of variance Table 1

Control PGD2+ ET-l+

(ANOVA) followed by Fischer’s protected least significant difference test (PLSD), as indicated.

RESULTS PGD2 was the major prostanoid produced under control conditions by HBCEC (Table 1). A stimulated secretion of PGF,,, PGE,, and TXB2 was observed in the medium of HBCEC exposed to PGD2. Even though the level of the PGD,-induced prostanoids varied among different cell lines, the pattern of the prostanoid formation was similar. The levels of PGF*, and PGE2 induced by PGDl were similar regardless of the incubation period [(424 h); Fig. 11. The greatest PGD2 stimulation of TXB2 formation by HBCEC was observed at 8 h with subsequent reduction of TXB2 levels at 24 h. The PGD*induced formation of prostanoids was dose-dependent and the maximal effect of PGD? was seen at 100 nM (Fig. 2). Pretreatment with dexamethasone was not effective, but inhibitors of cyclooxygenase (ASA and Indo) blocked the PGD,-stimulated production of PGF,, TXB?, and PGE2 (Table 2). As may be seen from this table, the PGD,-induced prostanoids were inhibited by ASA to a greater degree than those by Indo. Complete

Effect of PGDz and ET-I on prostanoids

production

by HBCEC

PGD,

PGF?,

9o. 11 P-PGFZ

TXBL

PGE,

6.26 * 0.15 8.85 + 0.25*

1.48 f 0.05 57.90 f 5.42* 16.98 f 0.4.5*

n.d. 1.608 0.003

1.57 + 0.17 16.20 f 2.37* 4.08 * 0.39*

2.87 + 0.8 1 20.20 + 1.92* 2.94 + 0.29

Values for PGD,, PGFr,, TXBz and PGEz are given in ng/mg protein (mean f SEM) and obtained from three separate dishes of a representative experiment of three with similar results. The value for 9cr. 11P-PGF? (ng/mg protein) was obtained from pooled samples of the same experiment. n.d. - below detection limit. + - HBCEC were incubated for 4 h in the presence of 100 nM of either PGD, or ET- 1. * - significant difference from control group (p < 0.01; Student’s t-test).

= b s 0 5 E : z P

300 250 200 150 -

0 PGQ

100

I C

4

8

24

Time (hours) Fig. 1 Temporal effect of PGD, on the production of TXB,. PGFZa, and PGE,. The presented data expressed as the % of corresponding control values (100%) incubated for the same period of time represent the mean ? SEM of 3-5 individual experiments each performed on three separately cultured HBCEC.

Prostaglandin

D, and Endothelin-

I 79 I

800 TxB2

PGFzu

PGEz

600

[PGD2], nM Fig. 2 The dose-dependent effect of PGD? on the formation of PGF,,. PGE,, and TXB?. The values represent the mean + SEM of 3-5 individual experiments each performed on three separately cultured HBCEC. *indicates a significant increase as compared to controls (P < 0.01 ANOVA). Table 2

stimulated

The effects of Indo (20 PM). ASA (500 wM). and Dxm (50 PM) on PGD,production of TXBI, PGF:,, and PGE, in HBCEC

Treatment

TXBz

PGFzc,

PGE,

Ml99 PGD, (10 nM) PGDz (100 nM)

3.29 f 0.34 (3) 5.45 * 0.48 (3) 15.03f 1.28(3)

4.85 -t 0.69 (3) 18.13* I.60 (3) 41.53 + I.98 (3)

4.58 IL 0.68 (3 I 9.47 k 0.96 (3) 28.68 + 1.49 (3)

lndo +PGDL (10 nM) +PGD> I 100 nM)

2.87 + 0.25 (3) 3.84 ?I0.16 (3)* II.53 f 1.03(3)*

4.20 + 0.40 (3) Y.04? 0.84 (3)* 33.32 f I.21 (3)*

2.54 f 0.29 (3) 5.10 * 1.49 (3,* IX.13 kO.98 (3,*

ASA +PGD, (100 nMJ

3.03 f 0.21 (3) X.56f 0.15 (3)*

3.78 k 0.42 (3)28.07 +I 2.24 (3)*

3.61 + 0.49 (3) l6.69+ I.72 (31*

3.97 !I 0.34 (6) 16.76 + 1.54 (6) 39.45 + 2.45 (6)

3.74 It 0.45 (6) 7.86 * 0.38 (5 1 26.14 f 4.20 (6)

Dxm +PGD, (IOnM) +PGD, (100 nM)

3.01 ? 0.19 (6) 6.13 f 0.56 (5) 14.98* 0.80 (6)

Values are given in ng/mg protein (mean f SEMI obtained from three separate dishes of a representative experiment out of three with similar results. - indicates significant difference of drug alone (p < 0.05, ANOVA) from M 199. *_‘ Indicates significant difference (p < 0.01 1ANOVA) from PGD, alone.

inhibition of PGD,-stimulated PGFLu, PGE2, and TXB? was seen after HBCEC exposure to 1 nM PGD, in two additional experiments not shown here. Addition of ET-l stimulated the endothelial production of PGD?, PGF,, and TXB, (Table 1). The secretion of ET- 1-induced prostanoids except for PGF,, was completely blocked by pretreatment with dexamethasone (data not shown). The possible presence of the PGD? metabolite 9a, 11 P-PGF, derived either from exogenous or endogenously formed PGD:, was detected by mass spectroscopy (Table I). The data indicate that the 1.1% of exogenous and 0.13% of endogenously formed PGD, were converted to 9cq 1 I@PGF,. Both PGD, and ET- 1 dose-dependently stimulated the formation of CAMP (Fig. 3). Pretreatment with dexamethasone neither affected the basal nor the PGD,- or ETstimulated production of CAMP (data not shown). To determine whether the activation of adenylate cyclase by

PGDz was mediated by either its own or TXA, receptors, we compared the PGDz formation of CAMP with that of TXA? alone and in combination. Each of the prostanoids similarly augmented the accumulation of CAMP (Fig. 3). However, TXA: did not enhance the PGD+timulated accumulation of CAMP. The selective ET-l A antagonist, BQ123 ( 1 PM), almost completely inhibited the ET-l stimulated production of CAMP and the prostanoids (except for PGF& (Fig. 3).

DISCUSSION The results indicate that PGD, can be converted into its metabolite, 9a, 11 P-PGF? (a known potent vasoconstrictor) and stimulates vasoconstrictive (PGF,, and TXA,) as well as vasodilatory (PGE,) prostanoids in HBCEC. The PGD, transformation into the PGF, isomer 9a,

792

Prostaglandins

Leukotrienes

and Essential Fatty Acids

5

*

0

10

+TxAz,lOO nM

100 250

0

100

[PGDz],nM

5

*

f

4 3 2 1 0

0

10

0

100

100

[ET-I], nM The dose-dependent effect of PGD, and ET- 1 on CAMP production. The data also show CAMP production by TXA, and BQ123 in the presence and absence of PGD, and ET-l, respectively. The values are the mean f SEM of a representative of 3-5 individual experiments each performed on three separately cultured HBCEC. *-indicates a significant difference from respective controls (p < 0.01 ANOVA). *-indicates a significant decrease of ET-l-stimulated CAMP by BQ123.

11 P-PGF, was shown to occur by an NADPH-dependent 11-keto-reductase in many human and animal tissues (7-10). The detected low level of PGD,? conversion to 9a, I Ifl-PGF;? by HBCEC may be a result of the HBCEC incubation under conditions suboptimal for the activity of this enzyme. Nevertheless, the conversion of PGD, to 9a, IlP-PGF2 which was observed in human coronary arteries (14) had not been described in either peripheral or cerebrovascular endothelium until now. The induction of PGF,, and PGE2 in HBCEC by PGD, is of special interest since different biosynthetic pathways have been reported to contribute to PGF*, formation: 9-keto reduction of PGE2 and 9-l 1 peroxide reduction of PGH, (6, 11, 12). A NAD-dependent conversion of PGF2, to PGE, was also demonstrated in rat heart homogenate (5). The observed inhibition of the PGD,-stimulated production of PGF,,, PGE2, and TXB2 by blockers of cyclooxygenase (ASA and Indo) in HBCEC represent the first documentation of PGD,inducible formation of cyclooxygenase products in any tissue. However, based on these results, it is not possible to ascertain whether the PGD,-induced release of prostanoids represents a direct effect of PGD2 or whether

it is mediated by 9a, 11 P-PGF*. The ineffectiveness of dexamethasone to inhibit the PGD,induced formation of PGF2, strongly suggests that this prostanoid is not a product of PLAz or cyclooxygenase II stimulation by PGD2. On the other hand, ET-l-induced PGF?, formation was markedly reduced by pretreatment with dexamethasone (data not shown) indicating that ET-lstimulated secretion of PGF,, primarily occurs by different mechanisms. Thus PGD,, the main constituent and peptide-induced prostanoid in HBCEC, can modulate the production of prostanoids and in this way contribute to the secretion of PGF,, PGE2, and TXB, by the cells. The role of PGD2 and its metabolites in HBCEC is unknown. Based on pharmacologic studies, it has been postulated that the dual vascular effect (dilatation and vasoconstriction) of PGD2 may be mediated by separate PGD2 (DP) receptors (3). However, other investigations suggest that the vasoconstrictive response to PGD, is mediated by TXA2 (TP) receptors. The absence of an additive effect of TXA2 (which also increases the formation of CAMP) on PGD*stimulated CAMP production suggests that the PGD2induced CAMP formation occurs through the DP receptor. Both the ET- 1-stimulated prostanoid and CAMP formation is inhibited by the specific ET, receptor antagonist, BQ123. Taking into consideration all the described observations, the PGD,-induced prostanoid and CAMP production is mediated by receptors and mechanisms different than ET- 1. The DP receptor-mediated vascular relaxation induced by PGD2 is thought to be associated with CAMP formation. The observed PGDl stimulation of CAMP production in HBCEC suggests that this PGD2 effect on the EC may mediate cerebral capillary dilatation through PGDZ receptors. Moreover, the demonstrated PGD,-stimulated formation of both vasoconstrictive and TXBJ and vasodilatory (PGF,,, 9a, 1 lp-PGF,, (PGE,) prostanoids as wells as CAMP strongly suggest that PGD2 may play a role in maintaining the tone and homeostasis of the microvascular bed in the brain. These findings may have important implications concerned with PGD2 function in cerebral capillaries under normal and disease conditions.

Acknowledgements The authors thank Mrs Nancy Merkel for excellent sistance, and D. Schoenberg, MS, for editorial help.

technical

References Bacic F, Uematsu S, McCarron R M, Spatz M. Secretion of immunoreactive endothelin-1 by capillary and microvascular endothelium of human brain. Neurochem Res 1992; 17: 699. Bacic F, Uematsu S, McCarron R M, Spatz M. Prostaglandin Dz in cultured capillary and microvascular endothelium of human brain. Prostaglandins Leukotrienes Med 1992; 46: 231. Giles H, Leff P. The biology and pharmacology of PGD,. Prostaglandins 1988: 35: 277.

as-

Prostaglandin Higashida H, Nakagawa Y, Miki N. Facilitation of synaptic transmission by prostaglandin D? at synapses between NGlOXXlS hybrid and muscle cells. Brain Res 1984. ,_395. _. 113. Leslie C A, Levine L. Evidence for the presence of a prostaglandin I?,-9-keto reductase in rat organs. Biochem Biophys Res Comm 1973: 51: 717. Lin Y-M. Jarabak J. Isolation of two proteins with 9ketoprostaglandin reductase and NADP-linked 15. hyhdroxyprostaglandin dehydrogenase activities and studie\ on their inhibition, Biochem Biophys Res Comm 1978: Xl: 1227. Liston T E. Roberts L J II. Transformation of prostaglandin D? to 9a. I ID-(lSSl-trihydroxyprosta-(5Z, 13E)-dien- I -oic acid (9a. I I B-prostaglandin F21: A unique biologically active prostaglandin produced enzymatically in viva m human. Proc Nat1 Acad Sci USA 1985; x2: 6030. Pugliese Cr. Spokas E G, Marcinkiewicz E. Wong P Y-K. Hepatic transformation of prostaglandin D, to a new prostanoid. 9a. I Ip-prostaglandin F1. that inhibits platelet aggregation and constricts blood vessels. J Biol Chem .9X5: 160: 14611. Roberts 1. J 11. Seibert K. Liston T E. Tantengco M V, Robertson R M. PGD, is transformed by human coronary arteries to 9~. I I B-PGF,. which contracts human coronary artery irings. Adv Prostaglandin Thromboxane Leukotriene Res 1987; 17: 427. Seibert K. Sheller J R, Roberta L J II. (52. 13E)-( 15Sl9rx. I 1p, I S-trihydroxy-prosta-5.13-dienI-oic acid 9. I I B-prostaglandin F?): formation and metabolism by numan lung and contractile effects on human bronchial \mooth muscles. Proc Nat1 Acad Sci USA 19X7: 84: 256. Watanabe K. Iguchi Y. lguchi S. Arai Y. Hayaishi 0, Roberts L J, II. Stereo-specific conversion of pro\taglandin D, to (52. 13El-( ISSl-9a.-I lB.lS-tri-

12.

13.

14.

15.

16.

17.

1X.

19.

20.

D, and -Endothelin-

hydroxyprosta-5.13-dien-1-oic acid (9a. I I pprostaglandin F,) and of prostaglandin H, to prostaglandin F, by bovine lung prostaglandin F syntha\e. Proc Nat] Acad Sci USA 1986: X3: 1583. Watanabe K. Shimizu T. Hayaishi 0. Enzymatic conversion of prostaglandin D2 to F,,, in the rat lung. Biochem Int. 1991: 7: 603. Wolfe L S, Rostworowaki K. Pellerin L. Sherwin A. Metabolism of prostaglandin D? by human cerebral cortex into 9a. I 1P-prostaglandin F1 by an active NADPHdependent 1 I-ketoreductase. J Neurochem 19X9: 53: 64. Wong P Y-K. Purification and partial characterization ol prostaglandin D1 1 I-ketoreductasc in rabbit liver. Biochem Biophys Acta I9X 1: 659: 169. Roberts L J II. Sweetman B J. Lewis R A. Austen K F. Oates J A. Increased production of prostaglandin D, in patients with systemic mastocytosis. N Engl J Mcd IUXO: 303: 1400. Gerhard D Z. Broderius M A. Drewes L R. Cultured human and canine endothelial cells from brain microvessels. Brain Res Bull 1988: 71: 7X5. Bailey J M, Makheja A N, Pash .I, Vcrma M. Corticosteroids suppress cyclooxygenase messenger RNA levels and prostanoid synthesis in cultured vascular cells. Biochem Biophya Res Comm 1988: 157: 1159. Wendelbom D F. Morrow J D, Roberts L J II. Quantification of 9a. I lb-prostaglandm F, by stable isotope dilution mass spectrometric assay. Methods Enzymol 1990; 187: 5 I. Kamushina I, Spatz M. Bembry J. Cerebral endothelial cell culture. Il. Adenylate cyclase response IO prostaglandins and their interaction with the ndrenergic system, Life Sci 1983: 32: 1477. Lowry 0 H. Rosenbough N J, Farr A L. Randall R J. Protein measurement with folin phenol reagent. J Biol Chem 1951: 193: 76.5

I 793