PROSTAGLANDINS STIMULATION OF PROSTAGLANDIN D 2 RECEPTORS ON HUMAN PLATELETS BY ANALOGS OF PROSTACYCLIN R. Alvarez, R.~. Eglen, L.F~K. Chang, J.J. Bruno, D.R. Artis ~, A.F. Kluge ~ and R.L. Whiting Institute of Pharmacology and Institute of Organic Chemistry 1 Syntex Research Palo Alto, California 94304
ABSTRACT RS-93427, a novel analog of prostacyclin, increased adenylate cyclase activity in human platelet membranes (ECs0 = 42 nM) to approximately the same maximum level as that produced by prostacyclin (ECs0 = 87 nM). The concentration-response curve for RS-93427 appeared to be monophasic. However, a selective prostaglandin D2 antagonist (BW A868C) significantly reduced the stimulation of adenylate cyclase produced by low concentrations of RS-93427 (3.2 to 32 nM). RS-93520, a stereoisomer of RS-93427, also stimulated adenylate cyclase activity but in a biphasic pattern. BW A868C reduced the activation produced by low concentrations of RS-93520 with a 100-fold shift in the response curve. Maximum stimulation by RS-93520 (4.5-fold) was less than that obtained with prostaglandin D2 (7.3-fold). Thus, the stimulation of adenylate cyclase activity by low concentrations of RS-93520 is due to an interaction with prostaglandin D2 receptors while the activation by RS93427 is mediated by both prostacyclin and prostaglandin D2 receptors. Additional data in support of these conclusions was obtained when these prostaglandins were tested as inhibitors of ADP-induced platelet aggregation in the presence or absence of BW A868C. The potent stimulation of prostaglandin receptors with chimeric molecules provides some insight into the structural features required for receptor activation. INTRODUCTION The mechanism of action of some prostaglandins on platelet function involves a receptor-mediated activation of adenylate cyclase [EC 4.6.1.1 ] and the subsequent increase in intracellular cyclic AMP (1-3). The activation process requires GTP and appears to involve the stimulation of a guanine nucleotide regulatory protein (Gs) coupled to adenylate cyclase AUGUST 1991 VOL. 42 NO. 2
105
PROSTAGLANDINS
on platelet membranes (4). Previous studies have demonstrated that human platelets contain distinct receptors for prostacyclin (PGI2) and prostaglandin D2 (PGD2) and that they are both coupled to adenylate cyclase (5,6). Kennedy et al (7), have proposed a classification system to describe the specific receptors that mediate the actions of several naturally-occurring prostanoids. In this system, IP and DP receptors are most sensitive to prostaglandins 12 and D2, respectively. The study reported here was designed to identify the receptor type on human platelets that mediates the effect of two novel structural analogs of PGI2. METHODS
Materials [ot32p]-adenosine 5'-triphosphate (10-30 Ci/mmole) and [2,8,-3H] adenosine 3',5'-monophosphate (30-50 Ci/mmole) were purchased from New England Nuclear Corp. (Boston, MA). PGI2, PGD2 and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). BW A868C (3-benzyl-5[6-carboxylhexyl]-l-[2-cyclohexyl-2hydroxyethyl amino]hydantoin) was kindly provided by the Wellcome Research Laboratories, Beckenham, U.K. RS-93427-017,Z-4- {(C3'S, 1S,2S,3R,6S)2-C3'-cyclohexyl-3'-hydroxyprop-1-ynyl)-3-hydroxybicyclo[4.2.0] oct-7ylidene }butyrate (calciumsalt)and RS-93520-007,Z-4- {(C3'S, 1R,2R,3S,6R)2-C3'-cyclohexyl-3'-hydroxyprop-1-ynyl)-3-hydroxybicyclo[4.2.0]oct-7ylidene}butyrate (Fig. 1) were synthesized according to published procedures (8). O
HO
HO
=
RS-93427
o.
:
RS-93520
0"
Figure 1. Structures of RS-93427 and RS-93520 106
AUGUST 1991 VOL. 42 NO. 2
PROSTAGLANDINS
Platelet Rich Plasma (PRP) Blood was obtained from female donors with informed consent for all studies performed with human platelets. The volunteers had not taken aspirin or similar medications for at least 2 weeks. The blood was collected by venipuncture from the antecubital vein into evacuated glass tubes [Vacutainer, Becton Dickinson, Rutherford, N.J.] containing EDTA (7.7 mM, final concentration) for the adenylate cyclase studies or sodium citrate (0.38% final concentration) for platelet aggregation studies. Platelet rich plasma was obtained by centrifuging the blood in polycarbonate tubes at 150 x g for 15 minutes at room temperature (-22oc). For the studies on rat platelets Sprague-Dawley rats (males 220-300 grams) were anesthetized with sodium pentobarbitol (50 mg/kg, i.p.). Blood was drawn from the descending abdominal aorta into a syringe containing sodium citrate (0.19% final concentration). The blood from 4-6 animals was pooled for each experiment. PRP was prepared as described above for human blood.
Washed Platelet Suspensions and Membrane Preparations A platelet pellet was obtained by centrifuging the PRP at 1000 x g for 15 minutes at -22°C. The pellet was resuspended in a volume of isotonic Buffer A (0.137 M NaCI, 12.3 mM Tris-HC1 buffer, pH 7.4 at 37oc, 1.54 mM EDTA and 20 mM glucose) equal to the original PRP volume. Platelet suspensions in Buffer A were centrifuged at 1000 x g for 15 minutes. The pellet was resuspended to the original PRP volume with an ice-cold hypotonic buffer (10 mM Tris-HC1 buffer containing 10 mM EDTA, pH 7.4). The suspension was preincubated for 30 minutes at 4°to remove tightly bound divalent cations (3) and then centrifuged at 6,000 x g for 15 minutes at 4°C. The lysed platelet pellet was suspended in a volume of cold 10 mM Tris-HC1 buffer equal to 1/10th the original PRP volume and used in the adenylate cyclase assay.
AUGUST 1991 VOL. 42 NO. 2
107
PROSTAGLANDINS
Adenylate Cyclase Adenylate cyclase activity was measured in accordance with a previously described method (9). The incubation medium contained 40 mM Tris-HC1 buffer, pH 7.4 at 37°C, 1 mM cyclic AMP, 0.1 mM GTP, 0.5 mM (0.1 ~tCi) [a32p]-ATP, 4 mM MgSO4, 20 mM phosphoenol pyruvate and 6.2 units of pyruvate kinase in a total volume of 200 ktl. The reaction was initiated by the sequential addition of 60 p.1 of the platelet membrane suspension. Incubations were performed at 37°C for 10 minutes. Reactions were terminated by the addition of 20 I.tl [3H]-cyclic AMP (0.005 ~tCi) in 2.2 N HC1 to monitor and correct for recovery of the nucleotide in the subsequent column chromatography step. The contents were mixed and the tubes placed in an ice bath. After all the tubes were processed, the rack was transferred to a boiling-water bath for 4 minutes. The tube contents were applied to disposable polypropylene columns (E & K Scientific Products, Inc.) containing 1.3 grams of neutral aluminum oxide. After the solution entered each column, cyclic AMP was eluted with 4 mls of 0.1 M ammonium acetate. Assays were performed in triplicate. Enzyme activity was linear for at least 10 minutes and proportional to enzyme concentration in the range of 30-120 I~g of protein. Protein content was measured colorimetrically (10) with bovine serum albumin as a standard. Stock solutions (10 mM) of RS-93427, RS93520 and PGD2 were prepared in 40% ethanol. Subsequent dilutions were made with 8% ethanol. PGI2 was dissolved in and dilutions were made with 10 mM Tris-HC1 buffer pH 9.1 at 25°C. The final concentration of ethanol in all of the enzyme reactions examined in this study was 0.4%.
ADP-Induced Platelet Aggregation Human platelet aggregation studies were performed with a Payton dual channel aggregometer according to the turbidimetric method of Born (11). The prostaglandin stock solutions (10 mM) were dissolved in ethanol. Subsequent dilutions were prepared with distilled water. Various concentrations of the prostaglandins (10 txl) were added to aliquots of PRP (0.97 ml) in siliconized cuvettes. Ten t~1 of BW A868C
108
AUGUST 1991 VOL. 42 NO. 2
PROSTAGLANDINS
(100 I.tM) or water were added to the PRP immediately after the addition of the prostaglandins. The mixtures were incubated in the aggregometer at 37°C with stirring at 500 rpm for 5 min before addition of the inducer, 5 ~tM adenosine 5'-diphosphate (ADP). The maximum change in light transmission (AT) in chart units was recorded from each aggregation tracing. The percent inhibition of the maximum change in light transmission for each concentration tested was calculated using a control sample (no prostaglandin addition). Concentration-response curves for RS-93427, RS-93520 and prostaglandin El (PGE~) with or without BW A868C (1 ~tM) were established with PRP from five donors. The concentration-response relationship for PGD2 was established with PRP from four donors. The IC5o values (concentration of a compound that produced 50% inhibition) for each test compound were obtained by nonlinear regression techniques. Approximate 95% confidence intervals were obtained for IC50 and shift parameters (ratios of ICs0s) using the asymptotic standard errors from each regression. The significance of the change in IC50 values produced by BW A868C was determined using the 95% confidence intervals of the ratio of the two IC50 values (with and without BW A868C). The change was significantly different when the 95% confidence interval of the ratio of the IC5o values did not contain 1.0. Rat platelet aggregation studies were performed with diluted rat blood PRP. PRP was diluted (2.5-fold) with Tyrode's buffer (KC1, 0.2 g/l; NaHCO3, 1 g/l; NaH2PO4, 0.05 g/l; NaC1, 8.5 g/l and dextrose, 1 g/l). The aggregation of rat platelets was induced with ADP (1-5 ~tM) and examined in the presence of various concentrations of the prostaglandins as described (above) for human platelets.
Computational Studies The calculations described here were carried out using SYBYL 5.32, available from Tripos & Associates, with default parameters except as noted. The Tripos force field was used with gradient termination. Semiempirical molecular orbital calculations were carried out using the MNDO hamiltonian in MOPAC 4.0 as supplied. The global energy minimum for the central ring system of each molecule was determined for a simple model structure. The side-chains of PGD2 and RS-93520
AUGUST 1991 VOL. 42 NO. 2
109
PROSTAGLANDINS
were then added in low energy, extended conformations. The carbonyl group and ring of PGD2 were next aligned with the ring hydroxyl and six-membered ring of RS-93520. The co side-chain hydroxyl groups of the two molecules were then aligned via side-chain bond rotations. The oxygens of the a side-chain carboxylic acids were finally aligned, primarily through rotation of the more flexible PGD2 linkage. Each structure was refined using molecular mechanics and the structures from the best overlap between the two compounds were submitted to full geometry optimization with MOPAC 4.0. RESULTS
Effect of BW A868C on Prostaglandin-Stimulated Adenylate Cyclase PGI2 and PGD2 increased adenylate cyclase activity in human platelet membranes with ECso values of 87 and 180 nM, respectively (Fig. 2). PGI2 exhibited greater i n t r i n s i c activity than PGD2 (15- versus 7 . 3 - f o l d stimulation). These results are in agreement with previous studies (3). The activation by PGI2 was unaffected by 1 ktM BW A868C, a selective and competitive PGD2 antagonist (Ki = 0.78 nM; ref. 12). In contrast, stimulation of enzyme activity by PGD2 was inhibited by BW A868C with a large (200-fold) shift in the ECso value (Fig. 2). A structural analog of PGI2, RS-93427 (Fig. 1), increased adenylate cyclase activity to approximately the same maximum level as PGI2 with an ECs0 value of 42 nM (Fig. 3). Although the concentration-response curve appeared to be monophasic, BW A868C (1 I.tM) reduced the stimulation of adenylate cyclase produced by low but not high concentrations of RS-93427, thereby exposing a biphasic curve (Fig. 3). Antagonism by BW A868C was statistically significant (Student's t test for paired samples, P < 0.01) in the presence of 3.2, 10 and 32 nM concentrations of RS-93427. This relatively small change in the concentration-response curve suggested that RS-93427 may interact with both IP and DP receptors. RS-93520, a stereoisomer of RS-93427 (Fig. 1), alone stimulated adenylate cyclase activity in a biphasic pattern (Fig 3). BW A868C (1 ktM) markedly reduced the activation produced by low concentrations of RS-93520 with a 100-fold shift in the response curve. Since the biphasic
110
AUGUST 1991 VOL. 42 NO. 2
PROSTAGLANDINS
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100
Figure 2.
Stimulation of adenylate cyclase by PGI2 and PGD2 in the presence (e) or absence (o) of 1 I.tM BW A868C. The symbols represent the-mean values + standard error (n = 4). AUGUST 1991 VOL. 42 NO. 2
111
PROSTAGLANDINS
0.5 .~
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RS-93427, I.tM • i,,,,ll
ABSTRACT RS-93427, a novel analog of prostacyclin, increased adenylate cyclase activity in human platelet membranes (ECs0 = 42 nM) to approximately the same maximum level as that produced by prostacyclin (ECs0 = 87 nM). The concentration-response curve for RS-93427 appeared to be monophasic. However, a selective prostaglandin D2 antagonist (BW A868C) significantly reduced the stimulation of adenylate cyclase produced by low concentrations of RS-93427 (3.2 to 32 nM). RS-93520, a stereoisomer of RS-93427, also stimulated adenylate cyclase activity but in a biphasic pattern. BW A868C reduced the activation produced by low concentrations of RS-93520 with a 100-fold shift in the response curve. Maximum stimulation by RS-93520 (4.5-fold) was less than that obtained with prostaglandin D2 (7.3-fold). Thus, the stimulation of adenylate cyclase activity by low concentrations of RS-93520 is due to an interaction with prostaglandin D2 receptors while the activation by RS93427 is mediated by both prostacyclin and prostaglandin D2 receptors. Additional data in support of these conclusions was obtained when these prostaglandins were tested as inhibitors of ADP-induced platelet aggregation in the presence or absence of BW A868C. The potent stimulation of prostaglandin receptors with chimeric molecules provides some insight into the structural features required for receptor activation. INTRODUCTION The mechanism of action of some prostaglandins on platelet function involves a receptor-mediated activation of adenylate cyclase [EC 4.6.1.1 ] and the subsequent increase in intracellular cyclic AMP (1-3). The activation process requires GTP and appears to involve the stimulation of a guanine nucleotide regulatory protein (Gs) coupled to adenylate cyclase AUGUST 1991 VOL. 42 NO. 2
105
PROSTAGLANDINS
on platelet membranes (4). Previous studies have demonstrated that human platelets contain distinct receptors for prostacyclin (PGI2) and prostaglandin D2 (PGD2) and that they are both coupled to adenylate cyclase (5,6). Kennedy et al (7), have proposed a classification system to describe the specific receptors that mediate the actions of several naturally-occurring prostanoids. In this system, IP and DP receptors are most sensitive to prostaglandins 12 and D2, respectively. The study reported here was designed to identify the receptor type on human platelets that mediates the effect of two novel structural analogs of PGI2. METHODS
Materials [ot32p]-adenosine 5'-triphosphate (10-30 Ci/mmole) and [2,8,-3H] adenosine 3',5'-monophosphate (30-50 Ci/mmole) were purchased from New England Nuclear Corp. (Boston, MA). PGI2, PGD2 and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). BW A868C (3-benzyl-5[6-carboxylhexyl]-l-[2-cyclohexyl-2hydroxyethyl amino]hydantoin) was kindly provided by the Wellcome Research Laboratories, Beckenham, U.K. RS-93427-017,Z-4- {(C3'S, 1S,2S,3R,6S)2-C3'-cyclohexyl-3'-hydroxyprop-1-ynyl)-3-hydroxybicyclo[4.2.0] oct-7ylidene }butyrate (calciumsalt)and RS-93520-007,Z-4- {(C3'S, 1R,2R,3S,6R)2-C3'-cyclohexyl-3'-hydroxyprop-1-ynyl)-3-hydroxybicyclo[4.2.0]oct-7ylidene}butyrate (Fig. 1) were synthesized according to published procedures (8). O
HO
HO
=
RS-93427
o.
:
RS-93520
0"
Figure 1. Structures of RS-93427 and RS-93520 106
AUGUST 1991 VOL. 42 NO. 2
PROSTAGLANDINS
Platelet Rich Plasma (PRP) Blood was obtained from female donors with informed consent for all studies performed with human platelets. The volunteers had not taken aspirin or similar medications for at least 2 weeks. The blood was collected by venipuncture from the antecubital vein into evacuated glass tubes [Vacutainer, Becton Dickinson, Rutherford, N.J.] containing EDTA (7.7 mM, final concentration) for the adenylate cyclase studies or sodium citrate (0.38% final concentration) for platelet aggregation studies. Platelet rich plasma was obtained by centrifuging the blood in polycarbonate tubes at 150 x g for 15 minutes at room temperature (-22oc). For the studies on rat platelets Sprague-Dawley rats (males 220-300 grams) were anesthetized with sodium pentobarbitol (50 mg/kg, i.p.). Blood was drawn from the descending abdominal aorta into a syringe containing sodium citrate (0.19% final concentration). The blood from 4-6 animals was pooled for each experiment. PRP was prepared as described above for human blood.
Washed Platelet Suspensions and Membrane Preparations A platelet pellet was obtained by centrifuging the PRP at 1000 x g for 15 minutes at -22°C. The pellet was resuspended in a volume of isotonic Buffer A (0.137 M NaCI, 12.3 mM Tris-HC1 buffer, pH 7.4 at 37oc, 1.54 mM EDTA and 20 mM glucose) equal to the original PRP volume. Platelet suspensions in Buffer A were centrifuged at 1000 x g for 15 minutes. The pellet was resuspended to the original PRP volume with an ice-cold hypotonic buffer (10 mM Tris-HC1 buffer containing 10 mM EDTA, pH 7.4). The suspension was preincubated for 30 minutes at 4°to remove tightly bound divalent cations (3) and then centrifuged at 6,000 x g for 15 minutes at 4°C. The lysed platelet pellet was suspended in a volume of cold 10 mM Tris-HC1 buffer equal to 1/10th the original PRP volume and used in the adenylate cyclase assay.
AUGUST 1991 VOL. 42 NO. 2
107
PROSTAGLANDINS
Adenylate Cyclase Adenylate cyclase activity was measured in accordance with a previously described method (9). The incubation medium contained 40 mM Tris-HC1 buffer, pH 7.4 at 37°C, 1 mM cyclic AMP, 0.1 mM GTP, 0.5 mM (0.1 ~tCi) [a32p]-ATP, 4 mM MgSO4, 20 mM phosphoenol pyruvate and 6.2 units of pyruvate kinase in a total volume of 200 ktl. The reaction was initiated by the sequential addition of 60 p.1 of the platelet membrane suspension. Incubations were performed at 37°C for 10 minutes. Reactions were terminated by the addition of 20 I.tl [3H]-cyclic AMP (0.005 ~tCi) in 2.2 N HC1 to monitor and correct for recovery of the nucleotide in the subsequent column chromatography step. The contents were mixed and the tubes placed in an ice bath. After all the tubes were processed, the rack was transferred to a boiling-water bath for 4 minutes. The tube contents were applied to disposable polypropylene columns (E & K Scientific Products, Inc.) containing 1.3 grams of neutral aluminum oxide. After the solution entered each column, cyclic AMP was eluted with 4 mls of 0.1 M ammonium acetate. Assays were performed in triplicate. Enzyme activity was linear for at least 10 minutes and proportional to enzyme concentration in the range of 30-120 I~g of protein. Protein content was measured colorimetrically (10) with bovine serum albumin as a standard. Stock solutions (10 mM) of RS-93427, RS93520 and PGD2 were prepared in 40% ethanol. Subsequent dilutions were made with 8% ethanol. PGI2 was dissolved in and dilutions were made with 10 mM Tris-HC1 buffer pH 9.1 at 25°C. The final concentration of ethanol in all of the enzyme reactions examined in this study was 0.4%.
ADP-Induced Platelet Aggregation Human platelet aggregation studies were performed with a Payton dual channel aggregometer according to the turbidimetric method of Born (11). The prostaglandin stock solutions (10 mM) were dissolved in ethanol. Subsequent dilutions were prepared with distilled water. Various concentrations of the prostaglandins (10 txl) were added to aliquots of PRP (0.97 ml) in siliconized cuvettes. Ten t~1 of BW A868C
108
AUGUST 1991 VOL. 42 NO. 2
PROSTAGLANDINS
(100 I.tM) or water were added to the PRP immediately after the addition of the prostaglandins. The mixtures were incubated in the aggregometer at 37°C with stirring at 500 rpm for 5 min before addition of the inducer, 5 ~tM adenosine 5'-diphosphate (ADP). The maximum change in light transmission (AT) in chart units was recorded from each aggregation tracing. The percent inhibition of the maximum change in light transmission for each concentration tested was calculated using a control sample (no prostaglandin addition). Concentration-response curves for RS-93427, RS-93520 and prostaglandin El (PGE~) with or without BW A868C (1 ~tM) were established with PRP from five donors. The concentration-response relationship for PGD2 was established with PRP from four donors. The IC5o values (concentration of a compound that produced 50% inhibition) for each test compound were obtained by nonlinear regression techniques. Approximate 95% confidence intervals were obtained for IC50 and shift parameters (ratios of ICs0s) using the asymptotic standard errors from each regression. The significance of the change in IC50 values produced by BW A868C was determined using the 95% confidence intervals of the ratio of the two IC50 values (with and without BW A868C). The change was significantly different when the 95% confidence interval of the ratio of the IC5o values did not contain 1.0. Rat platelet aggregation studies were performed with diluted rat blood PRP. PRP was diluted (2.5-fold) with Tyrode's buffer (KC1, 0.2 g/l; NaHCO3, 1 g/l; NaH2PO4, 0.05 g/l; NaC1, 8.5 g/l and dextrose, 1 g/l). The aggregation of rat platelets was induced with ADP (1-5 ~tM) and examined in the presence of various concentrations of the prostaglandins as described (above) for human platelets.
Computational Studies The calculations described here were carried out using SYBYL 5.32, available from Tripos & Associates, with default parameters except as noted. The Tripos force field was used with gradient termination. Semiempirical molecular orbital calculations were carried out using the MNDO hamiltonian in MOPAC 4.0 as supplied. The global energy minimum for the central ring system of each molecule was determined for a simple model structure. The side-chains of PGD2 and RS-93520
AUGUST 1991 VOL. 42 NO. 2
109
PROSTAGLANDINS
were then added in low energy, extended conformations. The carbonyl group and ring of PGD2 were next aligned with the ring hydroxyl and six-membered ring of RS-93520. The co side-chain hydroxyl groups of the two molecules were then aligned via side-chain bond rotations. The oxygens of the a side-chain carboxylic acids were finally aligned, primarily through rotation of the more flexible PGD2 linkage. Each structure was refined using molecular mechanics and the structures from the best overlap between the two compounds were submitted to full geometry optimization with MOPAC 4.0. RESULTS
Effect of BW A868C on Prostaglandin-Stimulated Adenylate Cyclase PGI2 and PGD2 increased adenylate cyclase activity in human platelet membranes with ECso values of 87 and 180 nM, respectively (Fig. 2). PGI2 exhibited greater i n t r i n s i c activity than PGD2 (15- versus 7 . 3 - f o l d stimulation). These results are in agreement with previous studies (3). The activation by PGI2 was unaffected by 1 ktM BW A868C, a selective and competitive PGD2 antagonist (Ki = 0.78 nM; ref. 12). In contrast, stimulation of enzyme activity by PGD2 was inhibited by BW A868C with a large (200-fold) shift in the ECso value (Fig. 2). A structural analog of PGI2, RS-93427 (Fig. 1), increased adenylate cyclase activity to approximately the same maximum level as PGI2 with an ECs0 value of 42 nM (Fig. 3). Although the concentration-response curve appeared to be monophasic, BW A868C (1 I.tM) reduced the stimulation of adenylate cyclase produced by low but not high concentrations of RS-93427, thereby exposing a biphasic curve (Fig. 3). Antagonism by BW A868C was statistically significant (Student's t test for paired samples, P < 0.01) in the presence of 3.2, 10 and 32 nM concentrations of RS-93427. This relatively small change in the concentration-response curve suggested that RS-93427 may interact with both IP and DP receptors. RS-93520, a stereoisomer of RS-93427 (Fig. 1), alone stimulated adenylate cyclase activity in a biphasic pattern (Fig 3). BW A868C (1 ktM) markedly reduced the activation produced by low concentrations of RS-93520 with a 100-fold shift in the response curve. Since the biphasic
110
AUGUST 1991 VOL. 42 NO. 2
PROSTAGLANDINS
0.5 .~
A
0.4
0.3 0.2 0.1 ~h
0
-
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.
.001
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, ....
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,
.
:
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.01
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. . . . . . . .
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1
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I , P , , .
100
10
Prostacyclin, pM
• ~
o
B
0.5 0.4 o.3
=
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0
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,
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,
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100
Figure 2.
Stimulation of adenylate cyclase by PGI2 and PGD2 in the presence (e) or absence (o) of 1 I.tM BW A868C. The symbols represent the-mean values + standard error (n = 4). AUGUST 1991 VOL. 42 NO. 2
111
PROSTAGLANDINS
0.5 .~
0.4
0.3 .~
0.2 0.1
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RS-93427, I.tM • i,,,,ll
