THE JOURNAL OF BIOI.OGICAL CHEMWPRY Vol. 254, No. 5, Issue of March 10, pp. 1671-1676, 1979 Printed in U.S. A.

Stimulation of Human Foreskin Fibroblast Adenosine 3’:5’-Cyclic Monophosphate Levels by Prostacyclin (Prostaglandin 12)*

(Received for publication, August 14, 1978)

Robert R. Gorman, Ramon D. Hamilton, and Nancy K. Hopkins

From the Department of Experimental Biology, The Upjohn Company, Kalamazoo, Michigan 49001

An analysis of the prostaglandin stimulation of cyclic adenosine 3’:5’-monophosphate accumulation in hu- man foreskin fibroblasts showed prostaglandin [Z (PG12) to be the most potent agent followed by PGHz = PGEl > PGE2, while PGDz was inactive. The stimulation by PGHz was shown to be due to the synthesis of PGIz from PGHz by several biological and analytical tech- niques.

Incubation of [1-14C]PGHz with fibroblast homoge- nates resulted in the biosynthesis of radioactive prod- ucts that co-chromatographed with 6-keto PGF1, (the stable hydrolysis product of PGI2) as well as PGE2, PGFz~, and PGD2.

The synthesis of PGIz from PGHz and subsequent cyclic AMP accumulation, was blocked by the PGIs synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I), but not by a closely related analogue that is known not to inhibit the PGIz synthetase. The stimulation of cyclic AMP accumulation by PGH1, which cannot be converted to PGIZ, was not as potent as that induced by PGHs and was not influenced by the PGIz synthetase inhibitor.

Evidence that human foreskin fibroblasts synthesize a biologically active PGIz was obtained by showing that the unknown product synthesized from PGHz was labile to heat and acid treatment and could stimulate cyclic AMP accumulation and inhibit human platelet aggre- gation in a manner parallel to authentic PGI2.

Physical proof of PGIz biosynthesis from PGHz was obtained by demonstrating the presence of 6-keto PGF1, by gas chromatography-mass spectrometry analysis.

It is concluded that PGIz may be an important regu- lator of cyclic AMP levels in human foreskin fibro- blasts.

Prostaglandins modulate adenosine 3’S’-cyclic monophos- phate levels in a variety of mammalian cell lines in culture (l- 4). Prostaglandins of the E series and, to a lesser extent, of A series are stimulators of adenylate cyclase and cyclic AMP accumulation in cultured cells (5, 6), and the sensitivity of the cells to prostaglandins can be modified by other hormones (7-

9). Cultured cells produce prostaglandins (10, ll), but few

attempts have been made to correlate the actual biosynthetic products of a particular cell with the stimulation of cyclic AMP levels by the defined biosynthetic prostaglandin prod- ucts of those cells (12).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

The recent discovery of prostacyclin (PGI,‘) by Vane and co-workers (13) and the subsequent structure determination and total synthesis by Johnson et al. (14) opened a new area of prostaglandin research. PGI, is the most potent stimulator of human platelet adenylate cyclase ever reported (15, Iti), and its lability and high intrinsic activity relative to E pros- taglandins suggests that PGL may actually be a more impor- tant regulator of cellular cyclic AMP metabolism than E series prostaglandins.

The precursor for PGIz is arachidonic acid, which is also the precursor for PGE2. PGE, is formed from dihomo-y-lino- lenic acid, but most mammalian cells contain little, if any, measurable dihomo-y-linolenic acid (17, 18). To our knowl- edge, there are no published reports of the natural synthesis of PGEl in cell culture, so it is reasonable to confine our studies to prostaglandins derived from arachidonic acid, even though PGEl may mimic the actions of PGE,. In this report we show that human foreskin fibroblasts synthesize PGI:! and that the adenylate cyclase of these cells is at least 2 orders of magnitude more sensitive to PGL than PGEZ.

EXPERIMENTAL PROCEDURES

Materials-Prostaglandins El, E2, DZ, and I, and the nitrogen- containing prostaglandin analogues 9,11-azoprosta-5,13-dienoic acid and 9,11-iminoepoxyprosta-5,13-dienoic acid were obtained from the Experimental Chemistry section, the Upjohn Co. Prostaglandin HI, HZ, and [l-‘%]PGHz were biosynthesized according to Gorman et u/. (19). [:‘H]Adenine (52 mCi/mmol) and cholera toxin were purchased from Schwarz/Mann. Dowex AG 5OW-X4 was purchased from Bio- Rad Laboratories, and neutral alumina (WN-3) was purchased from Sigma Chemical Co. Plastic tissue culture flasks and plates were purchased from Falcon Plastics and Coster Plastics.

Eagles minimum essential medium (Earle’s base) was purchased from Kansas City Biological Co., and supplemented with sterile filtered fetal bovine serum (inactivated 56”C, 30 min) from Reheis Chemical Co. to a volume of 10% serum. This medium also contained 100 units of sodium penicillin G (The Upjohn Co.), 100 pg/ml of streptomycin phosphate (Eli Lilly Co.), and 6 pg/ml of fungizone (E. R. Squibb Co.), and will be referred to in the text as MEM-IO. Sterile isotonic saline was purchased from Gibco Inc., and Hepes buffer from Microbiological Services.

Cell Culture Conditions-Human foreskins were washed three times with sterile saline at room temperature and cut with scissors into small pieces. The tissue was then exposed to a 0.25%’ trypsin solution in Hank’s balanced salt solution and digested for 30 min at 37°C. The digested tissue was then filtered through a double layer of cheesecloth, and the filtrate was discarded. The partially digested tissue was then exposed to the same conditions of trypsin digest for two l-h periods, and the resulting cellular filtrates were pooled. The

’ The abbreviations used are: PGIa, PGEI, PGE,, PGDz, I’GHz, and PGH,, prostaglandins 12, El, Ez, Da, HP, and HI, respectively; azo analog I, 9,11-azoprosta-5,13-dienoic acid; 9,11-I.E.P., 9,11-iminoepox- yprosta-5,13-dienoic acid; 6-keto PGFI,,, 6-keto prostaglandin FI,,; HFF cells, human foreskin fibroblast cells; HHT, 12.L-hydroxy-5,8,1 Om heptadecatrienoic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineeth- anesolfonic acid.

1671

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1672 Prostacyclin Regulation of CAMP Levels

cells were harvested by centrifugation at 800 x g for 5 min at room temperature and washed once with a mixture of 3 ml of MEM-10 and 7 ml of Hank’s solution. This pool of cells was then added to 15 ml of MEM-10 and put in a single plastic Falcon culture plate (100 X 20 mm). After 48 h, and at 3-day intervals until confluent, the culture was refed with 15 ml of MEM-10.

Cells from a primary plate were split into three Falcon tissue culture bottles (75 cm”). For passage, the bottles were washed twice with isotonic saline and exposed to 0.25% trypsin in phosphate- buffered saline for 5 min at 37°C. Cells were washed and recovered by centrifugation as described above. After the first passage, the cells were routinely passed by dividing the bottles 1:5. The confluent cells were fed with 25 ml of MEM-2 (serum content reduced from 10% to 2%) every 5 days and passed every 13 to 14 days.

Cyclic Nucleotide Measurement-The [“Hladenine prelabeling technique was used to characterize the influence of prostaglandins and prostaglandin precursor molecules on cyclic AMP levels in human foreskin fibroblasts (20,21). Cells were resuspended in an appropriate volume of MEM-10, so that 2.0 ml contained 1.0 X lo” cells, and placed into 35-mm Coster wells. The cells were grown under an atmosphere of 95% air, 5% CO1 at 37°C in a humidified incubator, Belco Glass Inc. At confluency (3 to 4 days), 10 &i of [“Hladenine was added to each 35-mm well, and the cells were incubated for 1 h at 37°C. After the l-h prelabeling period, the media plus excess [ ‘Hladenine was aspirated and the cells were washed once with 2.0 ml of MEM containing 25 mM Hepes buffer (no fetal calf serum or phenol indicator). The cells were then allowed to equilibrate with 1.0 ml of the serum-free MEM/Hepes buffer for 5 min before challenging the cells with the appropriate prostaglandin or prostaglandin precur- sor molecule.

In experiments where prostacyclin synthetase inhibitors were used, the inhibitors were added to the cells 5 min before the addition of prostaglandin Hr. Reactions were terminated after the appropriate incubation period at 37°C with 0.5 ml of 25% trichloroacetic acid.

The acidified 1.5-ml extracts were added directly to Dowex 5OW- X4. The initial 1.5-ml eluate was discarded. The total ATP and ADP fractions were eluted by washing with 1.5 ml of 0.1 N HCl followed by 1.5 ml of HLO. These fractions were pooled and a I-ml aliquot was used for scintillation counting. The cyclic AMP was eluted by an additional 3.0 ml of Hz0 and collected directly into tubes containing 0.2 ml of 1.5 M imidazole buffer. The cyclic AMP fraction was further purified by passage over alumina columns. The per cent conversion of ATP to cyclic AMP was calculated using the following formula:

% conversion = (dpm CAMP) (100)

(dpm ATP + ADP) + (dpm CAMP)

The prelabeling assay was verified by a radioimmunoassay for cyclic AMP done according to Steiner et al. (22) with the incorpora- tion of the acetylation modification of Harper and Brooker (23). Although the two assays were qualitatively in agreement, the more rapid prelabeling assay was routinely used. Low passage cells (< seven passages) were found to be considerably more responsive to PGI, than high passage cells. HFF cells in response to 2.8 pM PGL showed per cent conversions ranging from 15 to 40%, which is considerably higher than prostaglandin stimulation in other reported cell lines.

Preparation of HFF Membranes, and Radio Thin Layer Chro-

matography of the Products of (1 -‘JC]PGH2 Metabolism-Cells were washed with 2.0 ml of cold 50 mM Tris-HCl, 0.15 M NaCl buffer, pH 7.50, and then harvested with a rubber policeman. The pooled cells were then centrifuged at 4°C for 15 min at 2000 x g. The cell pellet was resuspended in the above buffer and frozen and thawed three times in liquid Na, followed by 10 strokes of a Dounce homogenizer (glass/glass). The protein concentration of the homogenate was then adjusted to approximately 0.5 mg of protein/ml. The homogenate (0.5 ml) was then added to 0.5 ml of the Tris-HCl/NaCl buffer. [l- ‘“C]PGH, (0.60 pM) was then added to the homogenate and incubated at 22°C for 15 min. The samples were acidified to pH 3.0 with 1 N

HCl and extracted twice with diethyl ether followed by silicic acid thin layer chromatography in the organic phase of an ethyl acetate/ acetic acid/isooctane/H~0(110:20:50:100) solvent system. The devel- oped chromatograms were then scanned on a Vanguard model 930 TLC scanner, and the radioactive zones were scraped and quantitated by liquid scintillation counting.

Gas Chromatography-Mass Spectrometry-The thin layer prod- uct that co-chromatographed with 6-keto PGFI,, was eluted with methanol from the silica gel, taken to dryness, and resuspended in diethyl ether.

The unknown product was esterified by treatment with ethereal diazomethane and converted to the trimethylsilyl-0methoxime de- rivative according to Sun and Stafford (24).

Gas chromatographic-mass spectral analysis of 6-keto PGF,,, was done on a LKB-9000 (GC-MS) equipped with a 6-foot column of 3.8% UCW-98 on Gas-chrom Q operated at 240°C. The flash heater and separator were operated at 270°C and the column was operated with a helium flow of 30 ml/min. The electronic energy was kept at 22.5 eV and the trap current was 60 PA for both analyses.

Platelet Aggregation Studies-Platelet-rich plasma was prepared from fresh, human whole blood collected in trisodium citrate (1 part 3.8% citrate, 9 parts blood) by centrifugation at 200 x g for 10 min at 25’C.

Platelet aggregation was monitored at 37°C in a Payton aggrego- meter with constant stirring at 1100 rpm. Platelets were incubated for 2 min at 37°C prior to the initiation of aggregation with PGH?. In experiments where PGI, was used to antagonize PGHr, PGHa and PGI, were added simultaneously to the platelet-rich plasma.

RESULTS

To establish the optimum incubation time for our cyclic AMP measurements in HFF cells, cells were stimulated with 2.8 pM PGI, and both total and extracellular cyclic AMP accumulation were simultaneously measured for 60 min (Fig. 1). The maximum PGIY stimulation occurs by 15 min at 37”C, and most of the cyclic AMP remains intracellular during the first 30 min of incubation. After 60 min of incubation, approx- imately equal amounts of cyclic AMP are intra- and extracel- lular. The 15-min incubation period also proved to be optimal for other agonists as well (data not shown). Since we had found PGI, to be the most potent stimulator of human platelet adenylate cyclase ever found (15), we compared the relative potency of PGL, PGEI, PGH%, PGE2, and PGD2 as stimulators of HFF cyclic AMP accumulation. Over the dose-response range of 28 nM to 28 PM, the potency order was PGL > PGEl = PGH, > PGEz and PGDz was essentially inactive (Fig. 2). This profile of activities suggested that either the unstable PGH2 was a direct stimulator of adenylate cyclase in HFF cells or it was being converted to some other active species.

Indirect evidence that HFF cells synthesize PGIz from PGHz is shown in Table I. When 0.60 pM [l-‘%]PGHr was incubated with HFF cell homogenates for 15 min at 22°C followed by radio thin layer chromatography, a prominent peak that co-chromatographed with authentic 6-keto PGFI,, was present (Table I). The 6-keto PGF1,, is a stable hydrolysis product of the labile PGI, (14) and can be used as an indirect indicator of PGIz synthesis. Approximately 16% of the PGH? was converted to 6-keto PGF1, (e.g. PGL) in this experiment. If the membranes were preincubated with 2.8 PM 9,11-azo- prosta-5,13-dienoic acid (azo analog I), a potent inhibitor of both thromboxane and PGI, biosynthesis (25, 26), there is essentially complete inhibition of 6-keto PGF,, biosynthesis and a concomitant enhancement of PGEz synthesis (Table I). A compound structurally related to azo analog I, 9,11-iminoe- poxy-prosta-5,13-dienoic acid, which does not inhibit hog aorta PGIz biosynthesis’ does not influence 6-keto PGFI,, synthesis in HFF cells (Table I). It is important to notice that, although PGE2, PGF2,, PGDZ, and the 12-OH fatty acid HHT are produced from PGHa in this system, boiled membranes can produce these compounds almost as well as viable mem- branes. The only compound that is not produced by boiled membranes is 6-keto PGFi, (Table I). No evidence of throm- boxane synthesis in HFF cells has ever been found.”

Our ability to inhibit 6-keto PGFI,, synthesis in isolated membranes with azo analog I, but not with 9,11-I.E.P. led US to use these compounds in intact HFF cells. HFF cells were

’ F. F. Sun, Oral Presentation, Winter Prostaglandin Conference, January 27 to 29, 1978, Sarasota, Fl.

‘I R. R. Gorman, unpublished observation.

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Prostacyclin Regulation of CAMP Levels 1 ti7:1

preincubated for 5 min with 28 nM to 2.8 pM azo analog I or 9,11-I.E.P. and then challenged with 2.8 PM PGHz. Azo analog I dose-dependently inhibited the PGHz-induced accumulation of cyclic AMP, but 9,11-I.E.P. produced only modest inhibi- tion at the highest concentration tested (Fig. 3). Neither azo analog I nor 9,11-I.E.P. at 2.8 pM inhibited a direct PGIg stimulation of cyclic AMP accumulation (Fig. 3). These data

0 10 20 30 40 50 60

MINUTES INCUBATION

FIG. 1. Time course of total and extracellular cyclic AMP accu- mulation in human foreskin fibroblast cultures. Cells were exposed to 2.8 PM PGIl and at the indicated time intervals both total (0) and extracellular (A) CAMP levels were measured. Data are presented as mean f S.E. of triplicate determinations.

35

30

f

$ 25

p‘

: 0 20

0

e---.-c PGlZ

- PGE,

o----o PGH2 - PGEl - PGDz

ss. I I

0 0.026 0.26 2.6 26

PROSTAGLANOIN CONCENTRATION pM

FIG. 2. Dose-response relationshlps for prostaglandin stimulation of cyclic AMP accumulation. Human foreskin fibroblasts were incu- bated for 15 min at 37°C with from 28 nM to 28 pM PGIY, PGEa, PGHz, PGE2, or PGD2 and the per cent conversion to cyclic AMP was measured. Data are presented as mean f S.E. of triplicate samples.

show that azo analog I, the prostacyclin synthetase inhibitor, blocks the PGHz stimulation of cyclic AMP but 9,11-1.X1’., which does not inhibit PGI, synthesis, does not block the elevation in cyclic AMP.

PGH2 is an obligatory precursor for PGI, and PGHl cannot be converted to PC12 (13, 14). PGH, can be converted to PGE,, but neither azo analog I nor 9,11-I.E.P. should influence this transformation. To test the specificity of the azo analog I inhibition, we challenged HFF cells with 28 nM to 2.8 pM

PGH, or PGH,. PGHz was a more potent stimulator of cyclic AMP accumulation than PGH, (Fig. 4). Azo analog I at 2.8 ,UM inhibited 65% of the PGH, stimulation but did not influ- ence the PGH, stimulation of CAMP accumulation. Subse- quent work has shown PGH, is converted to PGE:, in HFF cells, which in turn stimulates cyclic AMP accumulation, but this conversion is not influenced by azo analog I.”

Another indication that HFF cells produce biologically a(‘- tive PGI% from PGH2 is shown in Fig. 5. Membranes were prepared from six loo-mm plates of HFF cells, and the final membrane pellet was resuspended in 1.0 ml of 50 nlM Tris- HCl/NaCl buffer, pH 8.0. PGH?, 5.6 pM, was added to the suspension for a subsequent 15 min at 22°C and subsequentI> centrifuged at 4°C for 10 min at 5000 X g. The resulting supernatant was then removed and kept at 4°C in the pH 8.0 buffer for subsequent analysis. Two tests were made with the supernatant. In the first, the supernatant was added back to intact HFF cells and the stimulation of cyclic AMP was measured in response to 5, 10, 20, or 40 pl of supernatant, and the values were compared to a dose-response stimulation with authentic PGIz (Fig. 5). Ten microliters of supernatant was equivalent to approximately 28 nM PGIp and 20 ,~l were equivalent to 56 nM PGI,. It is important to mention that either boiling for 5 min or acid treatment of the supernatant destroyed essentially all of the increase in cyclic AMP levels. This lability is, of course, indicative of authentic PGI:! (13, 14). The second test was the simultaneous assay of the supernatant and authentic PGIz against a PGHZ-induced platelet aggre- gation in human platelet-rich plasma. These data are ex- pressed as per cent inhibition of the PGHz-induced aggrega- tion and are displayed in brackets directly above the cyclic AMP bar graphs. The degree of inhibition due to 28 and 56 nM PGIz were equivalent to 10 and 20 ~1 of supernatant, respectively (Fig. 5). As was the case with cyclic AMP levels, boiling or acid treatment also eliminated most of the inhibition of platelet aggregation.

The definitive proof that HFF cells produce PGI:! from PGH2 was done using gas chromatography-mass spectromet- ric analysis. The same radio thin layer chromatographic ex- periment described in Table I was repeated except on a lo- fold larger scale. The suspected 6-keto PGF,,, zone on the preparative thin layer plate was eluted and derivatized as previously described. Analysis of the unknown product pro-

TABLE I

[l-‘4C]Prostuglandin HZ metabolism in HFF cells HFF membranes were prepared as described under “Experimental of radioactivity that corresponded to known prostaglandin standards

Procedures.” [l-%]PGH2 at a concentration of 0.60 pM was added to were eluted and quantitated by liquid scintillation counting. Data 0.46 mg of membrane protein in 1.0 ml of 50 InM Tris-HCl/NaCl presented mean f S.E. Values are total disintegrations per min per buffer, pH 7.5, for 15 min at 22°C. The incubate was acidified, zone of triplicate samples with the per cent of total disintegrations extracted, and subjected to radio thin layer chromatography. Zones per min shown in parentheses.

Prostaglandin meas- 0.60 jm PGHz 0.60 PM PGHZ + am analog I 0.60 /.LLM PGHs+ 9,11-I.E.P. 0.60 QI PGHL + ked mcm wed branes

totaldpm/rone

6-Keto PGF,, 3067 + 377 (16.2) 269 f 18 (1.4) 3644 + 272 (15.7) 32 f 15 (0.16)

PGFz, 911 + 98 (4.8) 1157 + 77 (5.9) 1167 -c 59 (5.0) 1264 f 101 (6.7) PGE:! 10670 + 642 (56.4) 14372 + 789 (74.0) 13532 & 412 (58.3) 11643 f 411 (61.5) PGDz 1444 k 88 (7.6) 1309 z 62 (6.7) 1516 f 172 (6.5) 1357 rt; 43 (7.2) HHT 2835 f. 378 (15.0) 2324 f 112 (12.0) 3356 + 242 (14.5) 4645 f 298 (24.5)

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Prostacyclin Regulation of CAMP Levels 1674

z

45.0

0

z 37.5 0

::

9

B

30.0

s 22.5

i5

; 15.0

; F v) ( 7.5 N

H 0

I-

/ -

0

I 8

Y PGl2 PO12 + 9,ll 1.E.P

PGH2 PGl2 + AZ0 analog I

\ Am analog I

1 1 , I I ’ 0.026 0.26 2.6

INHIBITOR CONCENTRATION yM

FIG. 3. Inhibition of the PGH2 stimulation of cyclic AMP accu- mulation by azo analog I. Cells were preincubated for 5 min at 37’C with from 28 nM to 2.8 pM 9,11-azoprosta-5,13-dienoic acid (0) or 9,11- iminoepoxy-5,13-dienoic acid (A) and then challenged with 2.8 PM

PGH2 for an additional 15 min of incubation. The effect .of both agents at 2.8 pM was also measured against a direct stimulation by 2.8 pM PGI2. Data are presented as mean + SE. of triplicate determina-

-1

In a similar type of experiment, HFF cells were incubated for 24 h with 28 nM or 0.14 pM PGI2, PGEI, PGE2, or PGD2, washed and then rechallenged with 2.8 pM PGI2. Cells that were preincubated with the labile PGIz were not desensitized to subsequent PGIz stimulation (Table III). The less potent agonists PGEz and PGDz also did not desensitize the system. Only the stable agonist PGEl was capable of desensitizing the HFF cells to subsequent PGIz stimulation (Table III).

DISCUSSION

The stimulatory effects of prostaglandins on cyclic AMP accumulation in cell lines have been used by a number of different laboratories to study “receptor-adenylate cyclase” coupling (27,28), desensitization (29,30), and the mechanism of action of prostaglandins (31-33). Most of the previous work

tions.

3a , I

PROSTAGLANDIN CONCENTRATION PM

FIG. 4. Inhibition of PGH2 but not PGH1-induced cyclic AMP accumulation by azo analog I. The cells were challenged with from 28 nM to 2.8 PM PGHz (0) or PGH, (Cl), and the per cent conversion to CAMP was determined. Some cells were preincubated for 5 min with 2.8 ,uM azo analog I and then challenged with either PGHz (A) or PGHl (0). Only the PGHz-induced stimulation was attenuated by the azo analog. Data are presented as mean + S.E. of triplicate samples.

duced the mass spectrum shown in Fig. 6. The mass spectrum of the trimethylsilylmethoxime derivative of the unknown and derivatized authentic 6-keto PGFI, are identical. Prominent ions were found at m/e 115, m/e 378, m/e 418, m/e 508, and m/e 598, and the molecular ion at 629, which correspond exactly with authentic 6-keto PGF1, standard, indicating with- out question that HFF synthesize PGIz from PGH2.

We have completed some preliminary experiments to define the prostaglandin regulation of adenylate cyclase in HFF cells. An example of this is shown in Table II. HFF cells were incubated for 18 h in the presence or absence of 2.0 pg/ml of cholera toxin, washed and challenged with 2.8 PM PGI2, PGE2, or PGE,. The cholera toxin-treated cells had a higher basal rate of cyclic AMP synthesis and were much less sensitive to PGI*, PGE2, or PGEl stimulation of cyclic AMP accumulation than the control cells (Table II).

14 26 56 70 140 5 10 20 40 20 20 PGl2 nM )IL MICROSOMAL

SUPERNATANT

FIG. 5. Synthesis of biologically active PGIJike activity by human foreskin fibroblast membranes. Human foreskin fibroblast mem- branes (6.0 mg of protein/ml) were incubated for 15 min at 22°C with 5.6 pM PGHz. The membranes were rapidly centrifuged, and the resulting supernatant was used as either a stimulator of cyclic AMP accumulation in intact HFF cells or as an inhibitor of PGHz-induced human platelet aggregation. Data on the left side of the figure were generated with authentic PGI2, while the data on the right side were obtained with the experimental supernatant. Numbers inparentheses above the bar graphs represent per cent inhibition of platelet aggre- gation. Data are presented as mean + S.E. of triplicate samples.

50. 1 i 173 ' ' 1 196 322 353 I 'm+.,5

I 2”

24

i,i 6l4

-A..+.--L

ii) ,,,,, ;/,, ,;liil, I.':- '

I m+ / 829

50 100 150 200 250 300 350 400 450 500 550 600 650

FIG. 6. Gas chromatographic-mass spectrometric analysis of un- known product. Human foreskin fibroblast membranes (0.66 mg of protein/ml) were incubated with 60 jtM PGHl for 15 min at 22°C. The reaction was stopped by lowering the pH to 3.0 with 1.0 N HCl, followed by extraction with diethyl ether. The ether extract was subjected to silicic acid preparative thin layer chromatography. The area of the chromatogram that corresponds to authentic 6-keto PGFI, was eluted, derivatized, and analyzed by gas chromatography-mass spectrometry as described under “Experimental Procedures”. Prom- inent ions of the unknown material and authentic 6-keto PGFI, proved to be identical.

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Prostacyclin Regulation of CAMP Levels 1675

1’AHLF. 11

Attenuation ofprostaglandin-stimulated cyclic AMP accumulation by cholera toxin

HFF cells were incubated in MEM-10 for 18 h with and without 2.0 pg/ml of cholera toxin. The cells were washed twice with 2.0 ml of MEM-10 and allowed to equilibrate for 15 min with 2.0 ml of MEM- 10. The cells were then prelabeled with 10 &i of [“Hladenine, washed with serum-free MEM/Hepes and challenged with either 2.8 pM PG12, PGE,, or PGE,. Data presented as mean + SE. Values are per cent conversion to CAMP per 15 min of triplicate samples.

‘% conversion cAMP/15 min Prostaglandin ___- ___-

additions Control 18 h with 20 pg/ml cholera toxin

None-basal 2.8 PM PGEl 2.8 PM PGE2 2.8 PM PGIa

0.37 -t 0.01 1.19 + 0.12 35.78 f 1.42 13.57 3~ 1.26 4.22 +- 0.32 2.83 f 0.06

37.13 + 0.48 17.31 f 0.31 -.~

TABLE III Desensitization of PGI,-stimulated CAMP accumulation in HFF

cells by prostuglandins HFF cells were preincubated in MEM-IO for 24 h with either 28

nM or 0.14 pM PGIa, PGE), PGE,, or PGD,. The cells were washed twice with 2.0 ml of MEM/Hepes buffer and allowed to equilibrate for 5 min. The cells were then rechallenged with 2.8 pM PGIz. Data are presented as mean f SE. of triplicate samples.

Additions - % conversion cAMP/l5 min during 24-h when challenged with

q

preincubation 2.8 pv PC& desensitization ~--.

None 33.68 f 0.24

0.028 PM Ia 34.62 rt 0.54 0.140 /JM Ia 34.51 t 1.72

0.028 ,uM E, 27.39 +- 1.54 19 0.140 /LM E, 12.40 5~ 0.62 63

0.028 /dM DY 34.98 +- 0.39 0.140 FM D:! 34.30 + 0.97

0.028 PM E:! 34.35 + 0.43 0.140 PM Er 34.21 f 0.14

has been done with the so-called parent prostaglandin mole- cules PGE, or PGE,. There have been few attempts t,o link the actual synthesis of prostaglandins in a particular cell line with the stimulation of adenylat,e cyclase in that same cell (12).

Because we found PGIz to be the most potent stimulator of human platelet adenylate cyclase ever studied, we felt it was important to evaluate PGI, as a potential stimulator of cAMP accumulation in other homogeneous cell populations as well. The profile of prostaglandin-stimulated cyclic AMP accumu- lation in the HFF cells proved to be very similar to that initially observed in the human platelets (15). PGIz was about 10 times more potent than PGE, and approximately 100 times more potent than PGE,. The endoperoxide PGHZ was equi- potent to PGE,, and PGDz was inactive. Two differences between HFF cells and human platelets are pertinent. In platelets, PGHz is not converted to PGI,; it is converted to TXAz which inhibits CAMP accumulation (32), and PGDz is a slightly more potent stimulator of adenylate cyclase than PGE, (33).

The demonstration that HFF cell membranes could readily convert [ l-‘%]PGH2 into 6-keto PGF1, indicated that not only was the adenylate cyclase of HFF cells most responsive to PGI1, but PGI, could be synthesized by these cells as well. None of the other prostaglandins were produced at levels sufficient to significantly influence cyclic AMP levels.

In isolated membranes, and in intact cells, the prostacyclin

synthetase inhibitor, azo analog I, blocked PGI? synthesis from PGH, and the PGHP-induced elevation in cyclic AMP. A structurally similar compound, 9,11-I.E.P., which does not inhibit PGIz synthesis, could not attenuate the PGHz stimu- lation. Neither compound influenced direct PGIz stimulation.

The inhibition by azo analog I was also shown to be specific. Azo analog I could block the PGHa stimulation, but it could not block the stimulation due to PGH,. Since PGHl cannot be converted to PGI, (13), but can be converted to the weaker agonist PGEI, azo analog I would not be expected t,o interfere with a PGH, stimulation. It is appropriate to note that when the PGHz and PGH, stimulations of cAMP accumulation are compared, PGH, is about 10 times more potent than PGH,. which is the differential between PGL and PGE,.

HFF cell membranes were shown to produce a biologically active “PGL-like” activity from PGH,. This biological activity stimulated cyclic AMP accumulation in intact HFF cells and inhibited human platelet aggregation in a completely parallel manner to authentic PGI2. The unknown biological activity was also heat- and acid-labile, which are known physical properties of PGIz (13, 14).

Finally, the gas chromatography-mass spectrometry analy- sis of the unknown compound, whose synthesis was inhibited by the azo analog I and that co-chromatographed with au- thentic 6-keto PGF,,, proved without equivocation that HFP cells produce PG12 from PGH,.

Our initial studies with PGIz stimulation following exposure to cholera toxin or desensitization by PGE, suggest HFF cells behave in an analogous manner to other cell lines (29,30,34). However, the observation that the labile PGI, did not desen- sitize the cells suggests that sustained exposure to a prosta- glandin agonist may be necessary for prostaglandin-mediated agonist-specific desensitization, at least in HFF cells. Prelim- inary experiments indicate that it is very difficult to induce PGIz-mediated desensitization in HFF cells. If this finding can be expanded to other cell lines, many of the published pros- taglandin-mediated desensitization experiments may require re-examination.

We feel that the biosynthetic capacity of a part,icular cell is an important consideration in studies of the regulation of adenylate cyclase by prostaglandins. Even though PGE, is a potent agonist in HFF cells, we have never found PGE, in HFF cells by gas chromatography-mass spectrometry analy- sis, unless PGH, was added artificially to the system.” PGE, and PGFza are produced by HFF cells, but at concentrations that have negligible effects on adenylate cyclase, and PGD, is inactive.

At present we do not know what role(s) PGI, may play in the regulation of HFF metabolism or growth, or both. How- ever, a recent report has shown that elevat,ions of cyclic AMP in HFF cells inhibit collagen production (35). Our findings do show that PGI, biosynthesis is not confined to endothelial cells or smooth muscle cells (36, 37) and suggest a widespread distribution of PGI, biosynthesis in cells.

The capacity to supply unstable prostaglandin precursor molecules to cells and to selectively inhibit PGI, or throm- boxane biosynthesis, or both, allow specific questions to be asked. In particular, we plan to investigate the effects of specific PGI, synthetase inhibitors on the growth character- istics of HFF cells. Previous attempts to evaluate the influence of prostaglandins on cellular growth have been made with prostaglandin cycle-oxygenase inhibitors (38) which can have direct effects on adenylate cyclase (39). We are now trying to inhibit selectively the synthesis of PGIz in HFF cells and to follow the subsequent growth of these cells. This approach may greatly increase our understanding of the role that pros- taglandins play in cellular growth and metabolism.

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1676 Prostacyclin Regulation of CAMP Levels

Acknowledgment-We thank Dr. F. F. Sun for his assistance during the gas chromatography-mass spectrometry experiments.

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R R Gorman, R D Hamilton and N K Hopkinslevels by prostacyclin (prostaglandin I2).

Stimulation of human foreskin fibroblast adenosine 3':5'-cyclic monophosphate

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