Characterization of prostanoid receptors on rat neutrophils

Br. J. Pharmaol. (1994), 113, 581-587 Preparation of neutrophils Male, Sprague-Dawley rats fed ad libitum and weighing 19-23 g were used throughout this study. Peritoneal neutrophils were eliited by intraperitoneal injetion of 3 ml of NIH thioglyollate broth (.26 gml-') 19 h prior to harvesting the ells (Fisker et al., 199). The rats were killed by ervial disloation, and the leuoytes olleted from the peritoneal avity by lavage with 4 ml of ie-old HEPESff-". Mamillan Press Ltd, 1994 Charaterization of prostanoid reeptors on rat neutrophils 'Helen Wise & Robert L. Jones Department of Pharmaology, The Faulty of Mediine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Keywords: 1 The effets of various prostanoid agonists have been ompared on the inrease in intraellular free alium ([Ca2+]) and the aggregation reation of rat peritoneal neutrophils indued by N-formyl-Lmethionyl-L-leuyl-L-phenylalanine (FMLP). 2 Prostaglandin E2 (PGE2) and the speifi IP-reeptor agonist, iaprost, both inhibited the FMLPindued inrease in [Ca2+]J, (IC5o 33 nm and 18 nm respetively) and the FMLP-indued aggregation reation (IC5o 5.6 nm and 7.9 nm respetively). PGD2, PGF2,, and the TP-reeptor agonist, U 46619, were inative at the highest onentration tested (1 1AM). 3 The EPI-reeptor agonist, 17-phenyl-w-trinor PGE2, and the EP3-reeptor agonists, GR 63799X and sulprostone, had no inhibitory effet on FMLP-stimulated rat neutrophils. 4 PGE, (EP/IP-reeptor agonist) and iloprost (IP-reeptor agonist) inhibited the FMLP-indued inrease in [Ca2+], with IC5, values of 34 nm and 38 nm respetively. The EP2-reeptor agonists, butaprost and misoprostol (1 1AM), inhibited both FMLP-stimulated [Ca2+]i and aggregation. However another EP2-reeptor agonist, AH 1325, was inative in both assays. 5 Prostanoid reeptors present on rat neutrophils were further haraterized by measuring [3H]- adenosine 3':5'-yli monophosphate ([3H]-yli AMP) aumulation. Only those agonists apable of stimulating [3H]-yli AMP aumulation were able to inhibit both FMLP-stimulated [Ca2+]i and aggregation. 6 These results indiate that rat neutrophils possess inhibitory IP and EP-reeptors; the relative potenies of PGE2, misoprostol and butaprost are those expeted for the EP2-reeptor subtype. No evidene for DP, FP, TP or EPI and EP3-reeptors was obtained. Prostaglandin E2; prostaylin; prostanoid reeptors; yli AMP aumulation; intraellular free alium; FMLPstimulated aggregation; rat peritoneal neutrophils Introdution Neutrophils play a major role in the body's defene against infetion and trauma and ontribute towards pathophysiologial onditions suh as adult respiratory distress syndrome (ARDS) (Winder et al., 1993), myoardial infartion and rheumatoid arthritis (Carlos & Harlan, 199). Neutrophils an be reruited to areas of loal inflammation in response to a variety of hemotati stimuli suh as N- formyl-l-methionyl-l-leuyl-l-phenylalanine (FMLP), platelet-ativating fator (PAF), omplement fragment C5a, and leukotriene B4 (LTB4). Ativation of neutrophils by hemotati fators results in degranulation, NADPH oxidase-atalysed superoxide (2-) prodution, and a variety of other ellular responses (De Togni et al., 1984; Rossi, 1986). Neutrophil responses an be modulated by prostaglandins (Gryglewski & Salvemini, 1992) with prostaglandins having both pro- and anti-inflammatory roles (Goodwin, 1991; Kitsis et al., 1991; Phipps et al., 1991). Prostaglandins have been shown to have inhibitory effets on neutrophil responses in vivo (Fantone et al., 1981) and in vitro (Fantone & Kinnes, 1983; Ham et al., 1983; De Togni et al., 1984; Kato et al., 1986; Gryglewski et al., 1987; Ney & Shror, 1989; 1991; Nagata et al., 1992). Prostaglandins inhibit only responses mediated by diret reeptor ativation and do not inhibit phorbol ester or A23187-indued responses (Fantone & Kinnes, 1983; De Togni et al., 1984; Ney & Shror, 1991). Sine adenosine 3':5'-yli monophosphate (yli AMP) also inhibits neutrophil funtion and is thought to provide negative feedbak ontrol of neutrophil ativation (De Togni et al., 1984; Verghese et al., 1985; Nagata et al., 1992; Nielsen et al., 1992), it is presumed that the prostaglandin- ' Author for orrespondene. mediated effets may also be due to elevated neutrophil yli AMP resulting from ativation of prostaglandin reeptors. PGEI and PGE2 have been shown to be onsistently effetive in inhibiting human neutrophil responses and reent evidene suggests that their effet is mediated via an EP2-reeptor (Armstrong & Talpain, 1992; Wheeldon & Vardey, 1993) whih is oupled positively to adenylyl ylase (Coleman et al., 199). In ontrast, it has been reported that stimulation of EP3-reeptors (whih are negatively oupled to adenylyl ylase) by sulprostone leads to potentiation of opsonized zymosan-indued release of LTB4 from human neutrophils (Wheeldon & Vardey, 1993) and diretly stimulates hemotaxis (Armstrong, 1992). The aim of the present study therefore was to haraterize the major prostanoid reeptors present on rat neutrophils, and to examine the EP-reeptor in more detail. The effets of a omprehensive range of prostanoid reeptor-seletive agonists have been tested for potentiation or inhibition of neutrophil funtion, as measured by hanges in FMLPstimulated [Ca2eJ, and aggregation. These same agonists were also tested for effets on the prodution of yli AMP. Methods

582 H. WISE & R.L. JONES buffered saline (HBS:omposition, mm: NaCl 145, KCI 5, MgCI2 1, HEPES aid, 1, ph 7.55) supplemented with gluose (1mM) and bovine serum albumin (1%, w/v). The ells were entrifuged at 2C for 1 min at 3 g, and washed one in the same buffer. The ell suspension was then layered over Fioll-paque and entrifuged at 2'C for 2 min at 7 g in a swing-out rotor. The resulting ell pellet ontained 89 ± 1% neutrophils (assessed mirosopially using Wright's stain). The main ontaminating ell types were mononulear leukoytes (Fisker et al., 199). The viability of ells in the final ell pellet was 94 ± 1% as judged by the Trypan blue exlusion test (n = 19). Neutrophil aggregation Neutrophil aggregation was measured with a Chrono-log platelet aggregometer set at 37 C and 8 r.p.m. (Ford- Huthinson & Evans, 1988). The ells were resuspended in HBS supplemented with gluose (1 mm) to a ell density of 5 x 16 ells ml-'. The aggregometer was adjusted to reord % light transmission with unstimulated neutrophils and 1% transmission with 2.5 x 16 ells ml-'. The ell suspension (5 Al) was inubated in silionized uvettes for 1 min with the addition of CaCl2 (1 mm). Test drugs (5 tl) were then added for 2 min followed by FMLP (16 nm). Maximal aggregation was defined as the maximal hange in light transmission during the 1-2 min following addition of FMLP and is expressed in arbitrary units (mm defletion of pen reorder). Control responses to FMLP alone were tested regularly throughout the ourse of eah experiment. Measurement of ytosoli free alium onentration [Ca2+]i [Ca2+], was measured by resuspending the ells in HBS supplemented with gluose (1 mm), CaCl2 (1 mm) and bovine serum albumin (1%, w/v), to a ell density of 6 x 16 ells ml-' (MCarthy et al., 1989). The ell suspension was inubated with fura-2 aetoxy methyl ester (1 JAM) for 3 min at 37 C, then the ells were washed and resuspended in the same medium to a ell density of 2 x 16 ells ml-'. The ell suspension was kept at room temperature until required, and ould be kept for at least 5 h after loading without substantial leakage of fura-2 from the ells, inrease in basal [Ca2+]i, or derease in responsiveness to FMLP. In all experiments, a ontrol sample of ells was not inubated with dye; these ells were used to measure autofluoresene. Samples (.7 ml) of ell suspension were entrifuged in a Hettih mirofuge for 3 s at 11, g and then resuspended to 1 ml with HBS ontaining gluose (1 mm) and CaCl2 (1 mm). Fura-2 fluoresene of neutrophil suspensions (51 nm emission) was determined at 37 C with onstant stirring, using a Hitahi F-41 fluoresene spetrophotometer. [Ca2+], was measured as the ratio of fura-2 fluoresene at exitation wavelengths of 34 and 38nm aording to the method of Grynkiewiz et al. (1985). Cells were inubated for 1 min before the addition of test drug ( 1 l) whih was followed after 2 min with FMLP (1 nm). The FMLP-indued hange in [Ca2+], was alulated as the maximal hange in [Ca2+]i during the 3 s following addition of FMLP. Control responses to FMLP alone were tested regularly throughout the ourse of eah experiment. The various onentrations of prostanoids tested were added in a random sequene. There were no detetable solvent effets for any of the ompounds tested. Measurement of [3H]-yli AMP aumulation Cyli AMP prodution was assayed by measuring the onversion of [3H]-ATP to [3H]-yli AMP aording to Barber et al. (198). Rat neutrophils were inubated with [3H]- adenine (1 JCi ml-') for 6 min at 37 C in buffer (HBS supplemented with gluose (1 mm), CaCl2 (1 mm) and bovine serum albumin (1%, w/v)) at a ell density of 3 x I7 ells ml1 '. Labelled ells were washed and inubated at 1 X 17 ells ml' in buffer ontaining the type IV phosphodiesterase (PDE) inhibitor, rolipram (.1 mm), indomethain (3 AM) and adenosine deaminase (1 u ml-'). After 1 min inubation at 37 C, test drugs were added for a further 1 min when the reation was terminated by the addition of ie-old 7.5% trihloroaeti aid. [3H]-ATP was separated from [3H]-yli AMP by olumn hromatography aording to Salomon et al. (1974). [14C]- yli AMP (1, d.p.m.) was added to eah sample prior to olumn elution to orret for the perentage reovery of the olumns. The levels of [3H]-yli AMP and ['4C]-yli AMP were determined by liquid sintillation ounting. Results are expressed as either (1) relative to the basal ativity represented by [3H]-yli AMP generated in the absene of exogenous stimuli, or (2) relative to the response to iaprost (.1 AM). Data analysis EC5 values (onentration of FMLP produing 5% of maximal response) and IC" values (onentration of test ompound produing 5% inhibition of maximal response) were alulated using the four-parameter logisti urve fitting programme ALLFIT (De Lean et al., 1978). Results are expressed as means with standard error of the mean of (n) experiments. Statistial analysis was performed using Student's unpaired t test. Materials FMLP, fura-2 aetoxy methyl ester, PGD2, PGE2, PGFk, neutral alumina (type WN-3), and imidazole were supplied by the Sigma Chemial Co. (U.S.A.). PGEI, 17-phenyl-otrinor PGE2 and U 46619 (lss)-hydroxy-l la,9ax-(epoxymethano)prosta-5z,13e-dienoi aid) were purhased from Cayman Chemials (U.S.A.). NIH thioglyollate broth was purhased from Difo (U.S.A.) and Fioll-paque from Pharmaia (Sweden). 8-[3H]-adenine (speifi ativity 27 Ci mmolh') and [adenine-u-'4c]-yli AMP (speifi ativity 278mCimmol-') were purhased from Amersham International (U.K.). Dowex AG 5W-XA (2-4 mesh) was purhased from Bio-Rad (U.S.A.). The gifts of prostanoids are gratefully aknowledged: AH 1325 (trans-2-(4-(lhydroxyhexyl)phenyl]-5-oxoylopentaneheptanoi aid) and GR 63799X (13-oxa-1 3,14-dihydro-16-phenoxy-w-tetranor- PGE2-4-(benzoylamino)phenyl ester; Glaxo, U.K.); iaprost, iloprost, rolipram and sulprostone (Shering AG, Germany); butaprost (Bayer, U.K.); misoprostol (Searle, U.S.A.), and MB 28767 (ra 15S-hydroxy-9-oxo-16-phenoxy-w-tetranorprost-13E-enoi aid; Rhone-Poulen, U.K.). Results FMLP-indued neutrophil aggregation The addition of FMLP to suspensions of neutrophils produed a rapid onentration-dependent inrease in aggregation whih was maximal at 6 nm. The EC5 value for FMLP was 18.±3.4nM (n= 5). Effet ofprostanoids on FMLP-indued aggregation When tested at 1 JM, none of the fourteen ompounds tested had any diret effet of neutrophil aggregation. However, at the same onentration, PGE,, PGE2, butaprost, misoprostol, MB 28767, iloprost and iaprost all signifiantly inhibited aggregation stimulated by FMLP (16 nm) (Table 1). PGE2 and iaprost produed a onentration-dependent inhibition of FMLP-stimulated aggregation with maximal inhibition of approximately 5-6% (Figure 1). IC5 values

PROSTANOID RECEPTORS ON RAT NEUTROPHILS 583 for PGE2 and iaprost were 5.6 and 7.9nM respetively. PGD2, PGF2,, and U 46619 had no effet on FMLPmediated responses at.1 nm to 1 j4m (Figure 1); sulprostone was similarly inative (data not shown). Inlusion of indomethain (3plM) to inhibit any endogenous prodution of prostanoids had no effet on the onentration-response urve for PGE2 (data not shown) and was therefore not routinely inluded in this series of experiments. Effet of FMLP on rat neutrophil [Ca2+]j In the presene of external alium (1 mm), FMLP aused a rapid, onentration-dependent inrease in [Ca2+], whih Table 1 Effets of prostanoid agonists on the FMLPstimulated responses of rat neutrophils Prostanoid PGE2 Ciaprost PGD2 PGF2 U 46619 Iloprost PGEI 17-Phenyl-o-trinor AH 1325 Butaprost Misoprostol GR 63799X MB 28767 Sulprostone PGE2 Reeptor Aggregation [Ca2+ j rise speifiity (% ontrol) (% ontrol) EP IP DP FP TP IP/EP1 EP/IP EP1 EP2 EP2 EP2/EP3 EP3 EP3/TP EP3/EP, 38 ± 4*** 57 ± 1*** 99 ± 6 95± 12 15 ± 4 58 ± 6*** 52 ± 5*** 86 ± 8 11 ± 8 73 ± 7*** 48 ± 5*** 13 ± 5 76 ± 7** 11 ± 8 46± 5*** 61 ± 5*** 1±6 98 ± 5 99 ± 8 56 ± 3*** 6 ± 8*** 99 ± 5 99±3 78 ± 5* 63 ± 7** 87 ± 6 11 ± 3 12 ±4 Prostanoids (1 gm) were added 2 min before an EC" onentration of FMLP. Results are expressed as % ontrol response (means ± s.e.mean, n = 4-7). For the aggregation experiments, the ontrol response to FMLP (16 nm) was 16.3 ±.7 mm (n = 13). For the alium experiments, the ontrol response to FMLP (1 nm) was 126 ± 8 nm inrease in [Ca2+k (n = 2). *P<.5, **P<.1, ***P<.1, ompared to FMLP alone. 14 r inhibition of aggregation (r=.885, P<.1) exept that MB 28767 had no effet on FMLP-stimulated [Ca2+]J (Table 1). The onentration-response urves for the EP3-seletive agonists, GR 63799X and MB 28767, and the EP3/EP1- reeptor agonist, sulprostone, tended to lie above the ontrol response line, with the failitation of the FMLP response by 121- deayed to about 4% of its peak value within 1 min, after whih the deay was muh slower (Figure 2). A maximally effetive onentration of FMLP (1 nm) inreased [Ca2+]i by 297 ± 45 nm, from a basal level of 26 ± 1 nm, with an EC3 of 1.17 ±.8 nm (n = 3). Effet ofprostanoid agonists on the FMLP [Ca2+]J response None of the fourteen ompounds tested had any diret effet on rat neutrophil [Ca2+]i over the onentration range.1 nm to 1 gm. However, several of the prostanoids tested influened the subsequent response to a half-maximally effetive onentration of FMLP (1 nm) added 2 min after the test ompound (Figures 2 and 3). At onentrations of 1 nm, or less, PGE1, PGE2, the PGI2- mimetis, iaprost and iloprost, butaprost (an EP2-seletive agonist) and misoprostol (an EP2/EP3-reeptor agonist) tended to enhane the inrease in [Ca2+1. However, this enhanement was relatively small and rarely reahed the level of statistial signifiane. In ontrast, at onentrations greater than 1nM, PGEI, PGE2, iaprost and iloprost all inhibited the response to FMLP with maximal inhibition at approximately 44% of the ontrol response (P<.1 ompared with the ontrol response). The inhibitory phase of all four onentration-response urves were virtually superimposable, produing IC" values of 33, 18, 34 and 38 nm for PGE2, iaprost, PGE, and iloprost respetively. Butaprost and misoprostol also produed a statistially signifiant inhibition of the FMLP-stimulated response at 1 tlm (P<.5). All inhibitory ativities orrelated well with GR 63799X (1 nm) being statistially signifiant (P<.5). The thromboxane A2-mimeti, U 46619, PGD2, PGF2, and the EP2-reeptor agonist, AH 1325, had no effet on the FMLP-indued inrease in [Ca2+Jj. The EPI-seletive agonist 17-phenyl--trinor PGE2 (Lawrene & Jones, 1992) produed a slight inrease in response at 1 nm whilst being without effet at all other onentrations tested. I-._ o D 1-8 k 6 4 2 F 2 15 i -W 1 + o C- 5-1 -9-8 -7-6 [Prostanoid) (log M) Figure 1 The effets of prostanoid reeptor agonists on FMLPindued neutrophil aggregation. Rat neutrophils were inubated for 2 min with test ompound prior to the addition of an EC5 onentration of FMLP (16 nm). Control response to FMLP (16 nm) = 1 ± 6%, representing an aggregation response of 2.3 ±.7 mm (n = 13). Data shown are means with s.e.mean of 4 to 6 separate determinations: PGE2 (@); iaprost (); U 46619 (U); PGD2 (A); PGF2s (A). 2 3 Time (min) FIgure I The effet of PGE2 on FMLP-indued inrease in rat neutrophil [Ca2J]i. Results shown are the typial responses of fura-2- loaded neutrophils to buffer (solid line) or PGE2 (1 gm; dashed line) added at t = 1 min followed by FMLP (1 nm) at t = 3 min.

584 H. WISE & R.L. JONES C C.-O._i en ) CU ) C.) C 4- C C._ CU _._C o uc CU C.) IL a 14 12 1_ 6 4 8-2- _ 14 12 1_ 8 6 _ 4 2 L _ -1-9 -8-7 -6-1 -9-8 -7-6 [Prostanoid] (log M) L- C.' C.) ac L.)._ Co ; - a C. b 14 12 I 1 8 6 4 2 _ d 14 12 1_ 8 6 4-2 - -1-9 -8-7 -6-1 -9-8 -7-6 Figure 3 The effets of prostanoid reeptor agonists on FMLP-indued inrease in rat neutrophil [Ca2+],. Control response to FMLP (1 nm) = 1 ± 7% (n = 2). Basal [Ca2+]i was 21 ± 2 nm, and the inrease in [Ca2+]i due to FMLP (1 nm) was 126 ± 8 nm (n = 2). Data shown are means with s.e.mean of 5 to 7 separate determinations. (a) PGE2 (M); iaprost (); U 46619 (M); PGD2 (A); PGF2,t (A); (b) PGEI (@); iloprost (); 17-phenyl-w-trinor PGE2 (A); () GR 63799X (); sulprostone (); MB 28767 (A); (d) Butaprost (); misoprostol (A); AH 1325 (). [3H]-yli AMP aumulation PGE2 and iaprost produed a onentration-dependent inrease in [3H]-yli AMP aumulation, with iaprost being approximately 1 fold more potent than PGE2 (Figure 4a). PGD2, PGF2., and U 46619 had no effet on adenylyl ylase ativity at.1 nm to 1 JLM (Figure 4a). Of the EP-reeptor seletive drugs tested, 17-phenyl-o-trinor PGE2 and sulprostone were without effet at.1 nm to 1 glm, whilst butaprost produed a 2 fold inrease in [3H]-yli AMP aumulation at 1 JLM (Figure 4b). To determine if any of our seleted ompounds had EP3- like ativity, we inubated the ells with 1 gm test drug in the presene and absene of an adenylyl ylase stimulant to look for inhibition of adenylyl ylase ativity. Ciaprost (.1 sm) was hosen to ativate adenylyl ylase as it was onsiderably more effetive (237 25% ontrol, n = 6) than either forskolin (1 gm, 133 27% ontrol, n = 4) or isoprenaline (1 gm, 186 ± 11% ontrol, n = 4). When tested alone, PGE2, PGEI, iloprost, butaprost and misoprostol all inreased [3H]- yli AMP aumulation (Figure 5a,b), with the response to PGE2, PGEI and iloprost being statistially signifiant (P <.5). None of the remaining eight ompounds had any effet alone. GR 63799X, MB 28767 and sulprostone, i.e. the EP3-reeptor agonists, all failed to inhibit iaproststimulated [3H]-yli AMP aumulation. Sulprostone was tested further at.1 nm to 1 gm and had no effet on the response to iaprost (data not shown). Similarly none of the remaining ompounds tested at 1 gm had any inhibitory effet on the response to iaprost (Figure 5). Disussion In this investigation we have haraterized the prostanoid reeptors present on rat neutrophils by using a mixture of funtional and biohemial assays, and a wide range of prostanoid agonists of varying reeptor seletivity. The primary response of neutrophils to ativation by FMLP is the rapid elevation of [Ca2+]i (MCarthy et al., 1989) losely followed by adherene to, and migration aross, the endothelial ell layer (Winder et al., 1993). Neutrophils stimulated by hemotati fators undergo a transient hange in surfae properties that permits the ells to adhere more readily to surfaes suh as endothelial ells, and to eah other (Hoffstein et al., 1982). Neutrophil aggregation in vitro has therefore been used in the present study as a measure of the adhesion response. Of the five major prostanoid agonists (PGE2, iaprost, PGD2, PGF2., and U 46619) that define the EP, IP, DP, FP and TP-reeptors (Coleman et al., 199), only PGE2 and iaprost inhibited FMLP-stimulated elevation of [Ca2+]i and aggregation. The log onentration-response urves for eah agent in the two assays were parallel. Other prostaglandins with IP-agonist ativity, i.e. PGEI and iloprost, were also similarly potent in inhibiting FMLP-stimulated elevation of [Ca2+], with IC" values of approximately 36 nm. PGE2 and IP-reeptor agonists suh as PGI2 and beraprost have also been reported to be equipotent in inhibiting FMLP-indued hemotaxis in rat neutrophils (Kainoh et al., 199). Ciaprost is onsiderably more speifi than iloprost as an IP-reeptor agonist (Dong et al., 1986, Armstrong et al., 1989), and its profile in our experiments indiates the presene of IP-reeptors on the rat neutrophil. Our observations therefore onfirm the presene of both EP and IPreeptors apable of inhibiting rat neutrophil funtion and ontrast with the relative lak of poteny of IP-reeptor agonists in assays of human neutrophil funtion (Heker et al., 199, Wheeldon & Vardey, 1993). Our data also onfirm the observations of Ham et al. (1983) who noted that rat neutrophils were unresponsive to PGD2, whih was in marked ontrast to the high poteny of DP-reeptor agonists in inhibiting opsonized zymosan-indued release of LTB4 from human neutrophils (Wheeldon & Vardey, 1993). In this present paper we show that at the level of the initial response to FMLP, i.e. the elevation of [Ca2J]1, prostaglandin

PROSTANOID RECEPTORS ON RAT NEUTROPHILS 585 a 4 r U 3-1 4- I 2 - - - C._ 1 F Con PGE2 PGD2 F2. U46619 L- 4- o ' b 4 r- 3-1 -9-8 -7-6 2 9: Con 17-PGE2 AH But Miso C._ U 2 15 1.I 1 ' -1-9 -8-7 -6 [ProstanoidJ (log M) Figure 4 The effets of prostanoid reeptor agonists on [3H]-yli AMP aumulation in the presene of rolipram (.1 mm), indomethain (3 ftm) and adenosine deaminase (I u ml-). Rat neutrophils were inubated for 1 min before the addition of test ompound for a further 1 min. Data represent the means with s.e.mean of 3 experiments performed in tripliate. (a) PGE2 (); iaprost (); U 46619 (U); PGD2 (A); PGF2 (A); (b) Butaprost (); 17-phenyl-w-trinor PGE2 (A); sulprostone (). analogues ative at the EP2-reeptor, e.g. PGE1, PGE2, butaprost and misoprostol, all have an inhibitory ation. This is unlikely to be due to any ytotoxi effet sine there was no obvious fura-2 loss from the ells on the addition of prostanoids. These same ompounds, tested at I JIM, also inhibited the aggregation response to FMLP, thus identifying an inhibitory role for EP2-reeptors in rat neutrophils. MB 28767 was the only ompound tested whih inhibited FMLPstimulated aggregation yet had no effet on FMLPstimulated [Ca2+],. MB 28767 has previously been shown to exhibit EP2-like ativity by relaxing rabbit jugular vein with an equi-effetive molar ratio of approximately 4 when ompared with PGE2 (Lawrene & Jones, 1992). Therefore it is probable that we are able to detet an EP2-like effet of MB 28767 due to the relatively high sensitivity of the aggregation assay to PGE2 (IC5 5.6 nm) when ompared with the sensitivity of the alium assay to PGE2 (IC5o 33 nm). In the same study by Lawrene & Jones (1992), the relaxant poteny of butaprost on the rabbit jugular vein was found to be surprisingly weak (about 7 and 8 times less than PGE2 and misoprostol respetively) and this led to the first suggestion of the existene of subtypes of the EP2- reeptor. In our present study, the EP2-seletive agonist AH 1325 (Nias et al., 1993) at 1 JAM had no effet on FMLPmediated ativation of the rat neutrophil or on the aumulation of [3H]-yli AMP. In ontrast, AH 1325 was only 16 fold less potent than PGE2 in inhibiting opsonized zymosan-indued release of LTB4 from human neutrophils (Wheeldon & Vardey, 1993). More reently AH 1325 has been used to distinguish the EP2-reeptor from a reently Con GR MB SuIp 1io PGE1 Figure 5 The effets of prostanoid reeptor agonists on [3H]-yli AMP aumulation stimulated by iaprost. Rat neutrophils were inubated in the presene of rolipram (.1 mm), indomethain (3 JM) and adenosine deaminase (I u ml-) for 1 min before the addition of test ompound (1 AM), in the presene (hathed olumns) and absene (open olumns) of iaprost (.1 AM). The reation was stopped after a further 1 min. Data represent the means with s.e.mean of 3 experiments performed in tripliate. Ciaprost (.1 JAM) elevated [3H]-yli AMP levels to 223 ± 38% ontrol (n = 3). Abbreviations: Con (ontrol), 17-PGE2 (17-phenol-w-trinor PGE2), AH (AH1325), But (butaprost), Miso (misoprostol), GR (GR 63799X), MB (MB 28767), Sulp (sulprostone) and T1o (iloprost). identified subtype, the EP4-reeptor, present in pig saphenous vein where AH 1325 was reported to be 1l,O fold less potent than PGE2 (Coleman et al., 1994). It is oneivable that AH 1325 may have shown an effet in our study at a higher onentration, but we hose to limit the onentration of ompounds tested to 1 JAM in order to maintain their speifiity of ation. Sine in the present study the poteny of butaprost is muh loser to that of misoprostol, it is probable that our results support the presene of onventional EP2- reeptors. The inhibitory ation of prostaglandins on neutrophils is onsidered to be due to stimulation of yli AMP prodution sine hemoattratants suh as FMLP indiretly elevate intraellular yli AMP levels as part of a negative regulatory system (Verghese et al., 1985), and prostaglandinindued inhibition of neutrophil funtion tends to be paralleled by inreases in yli AMP (Ney & Shror, 1991). We have found that only those ompounds whih stimulated adenylyl ylase ativity were able to inhibit FMLPstimulated [Ca2+]i and neutrophil aggregation. Whilst the drugs whih an elevate neutrophil [3H]-yli AMP learly inhibit neutrophil ativity and might be onsidered as antiinflammatory, one rarely observes more than 6% inhibition of any measure of neutrophil funtion (see also Wheeldon & Vardey, 1993). Clearly there are important regulators of neutrophil funtion other than adenylyl ylase. The lak of effet of prostaglandins alone in stimulating an inrease in [Ca2+]i and aggregation also suggests the lak of

586 H. WISE & R.L. JONES prostaglandin reeptors diretly oupled to phospholipase C. In partiular, the lak of effet of the EPI-seletive agonist 17-phenyl-w-trinor PGE2 would indiate the absene of EP1- reeptors on rat peritoneal neutrophils. Prostanoids with EP3-like ativity suh as PGE1, PGE2, GR 63799X, MB 28767, misoprostol and sulprostone all tended to enhane the inrease in [Ca2+]i due to FMLP, although rarely by more than 2% of the ontrol response in the absene of prostanoid. Although the apparent potentiation of FMLP-mediated inreases in [Ca2+]i by various prostanoids is relatively small and rarely reahed a level of statistial signifiane, the response ould not be attributed to an artifat of the experimental design and was of a similar magnitude as the potentiation by sulprostone (1 SM) of opsonized zymosan-indued release of LTB4 from human neutrophils reported by Wheeldon & Vardey (1993). Any potentiation of FMLP-mediated inreases in [Ca2+J1 might be expeted to result in an enhaned aggregatory response to FMLP. However, sulprostone (.1 nm to 1 g4m) had no potentiating effet on FMLP-mediated neutrophil aggregation. Ativation of EP3-reeptors would also be expeted to inhibit adenylyl ylase ativity. GR 63799X, MB 28767 or sulprostone did not inhibit iaprost-stimulated [3H]-yli AMP aumulation, thus providing further evidene for the lak of EP3-reeptors on rat neutrophils. It is not lear at present why low onentrations of EP3 and some EP2- reeptor agonists appeared to failitate FMLP-mediated inrease in [Ca2+]i. The absene of speifi reeptor antagonists learly hampers the interpretation of the results here. In onlusion, we have shown that the ability of FMLP to inrease [Ca2+]i and to stimulate an aggregation response in rat neutrophils is not affeted by DP, FP or TP-reeptor agonists, but an be inhibited by EP and IP-reeptors agonists. The lak of DP-reeptors and the presene of funtionally ative IP-reeptors ontrast with reported studies on human neutrophils. Rat neutrophils appear to lak EP1- reeptors whilst possessing an EP2 or EP2-like reeptor whih appears to mediate the inhibitory effet of PGE2. We have found no evidene for an EP3-reeptor apable of failitating the hemotaxi responses of rat neutrophils. The authors would like to aknowledge the exellent tehnial support provided by Mr Kevin Chow Bing Shui. This projet was supported by UPGC Grant SP-92/9. Referenes ARMSTRONG, R.A. (1992). PGE2 and EP3 agonists indue hemotaxis of human neutrophils in vitro. Br. J. Pharmaol., 15, 45P. ARMSTRONG, R.A. & TALPAIN, E. (1992). Charaterisation of the PGE reeptor on human promyeloyti leukemia (HL-6) ells differentiated with dimethylsulfoxide into neutrophil-like ells. Br. J. Pharmaol., 17, 91P. ARMSTRONG, R.A., LAWRENCE, R.A., JONES, R.L., WILSON, N.H. & COLLIER, A. (1989). 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