enriched in parietal cells. [3H]Iloprost binding occurred

Journal of Physiology (1988), 405, pp. 39-55 39With 7 text-figuresPrinted in Great Britain

BINDING AND BIOLOGICAL ACTIONS OF PROSTAGLANDIN E2 AND I2IN CELLS ISOLATED FROM RABBIT GASTRIC MUCOSA

BY D. B. BARR, J. A. DUNCAN, J. A. KIERNAN, B. D. SOPERAND B. L. TEPPERMAN

From the Departments of Physiology and Anatomy, Faculty of Medicine,University of Western Ontario, London, Canada N6A 5C1

(Received 13 October 1987)

SUMMARY

1. We have investigated the binding of the tritiated forms of prostaglandin E2(PGE2) and a stable analogue of prostacyclin (Iloprost) to isolated cells of rabbitoxyntic mucosa.

2. The highest degree of specific [3H]PGE2 binding occurred in a cellular fractionenriched in parietal cells. [3H]Iloprost binding occurred predominantly in cellsidentified as mucous cells.

3. PGE2 binding to the parietal cell fraction was associated with its ability toinhibit histamine-stimulated aminopyrine accumulation by these cells. Iloprostbinding did not correlate with a biological action on the parietal cells.

4. PGE2 and Iloprost reduced Trypan Blue staining in cells exposed to 10% (w/v)ethanol. Iloprost (10-8 to 10-6 M) reduced Trypan Blue staining in cells identifiedas mucous and parietal cells. PGE2 (10-8M) significantly reduced Trypan Bluestaining in parietal cell-enriched fractions.

5. Cyclic AMP stimulation in response to either prostanoid occurred mostpotently on non-parietal cell fractions. However PGE2 or Iloprost binding affinitiesdid not correlate with cyclic AMP formation.

6. These data provide evidence for true PGE2 receptors on oxyntic mucosal cells.The receptors appear to mediate inhibition of acid secretion. Iloprost binds to siteswhich might mediate cellular protection.

INTRODUCTION

We have identified and characterized the binding of [3H]PGE2 to particulatefractions of porcine oxyntic mucosa (Tepperman & Soper, 1981, 1983). These studiesrevealed heterogenous populations of PGE2 binding sites in the tissue which wereprimarily associated the plasma membrane (Tepperman & Soper, 1983). Recentlyour initial demonstration of PGE2 binding sites in oxyntic mucosa and the optimalconditions for the binding assay have been confirmed by others (Woo, Roszkowski,Waterbury & Garay, 1986; Beinborn, Netz, Staar & Sewing, 1986). In all of thestudies described above the specific cellular type or types to which PGE2 bound werenot determined. Furthermore, PGE2 binding to porcine mucosa was not associated

D. B. BARR AND OTHERS

with a biological response of the tissue to exogenous PGE2 as determined by lack ofoverlap of the binding (Kd) and biological (Ki) affinity constants (Tepperman &Soper, 1981). However this lack of correlation between binding and biologicalactivity observed in previous studies may stem from the use of crude tissuehomogenate preparations which might preclude detection of a biological response. Inthe present study we have examined PGE2 binding capacity in various enrichedcellular fractions of rabbit oxyntic mucosa to determine the cellular locus of thePGE2 binding site. In addition we have examined various biological actions ofexogenous prostaglandin on these cellular fractions to determine whether arelationship exists between biological and binding activity of PGE2. Biologicalactions examined include: inhibition of histamine-stimulated aminopyrine accumu-lation, stimulation of cyclic AMP formation and protection against ethanol-inducedcellular death as determined by exclusion of Trypan Blue stain. A close quantitativeassociation between biological and binding activity in specific cellular types wouldimply that binding occurred at true PGE2 receptors on these cells. These studies havealso been done using a stable analogue of prostacyclin or PGI2 (Iloprost). BothPGE2 and PGI2 have been detected in gastric mucosa of a number of animals(Robert, 1981; Miller, 1983) and administration of PGE2, PGI2 or Iloprost exertpotent biological action on the stomachs of a number of animals and humans(Vischer & Casals-Stenzel, 1982; Miller, 1983). Furthermore this laboratory hascharacterized the binding of both prostanoids using gastric tissue from a number ofspecies (Tepperman & Soper, 1981, 1983; Tepperman, Soper & Emery, 1984).

METHODS

Cell isolationThe procedures for cell isolation and separation in the rabbit follow the techniques exactly as

described by Wollin (1984). Female New Zealand rabbits weighing 2-0-2-5 kg were used in allexperiments. The rabbits were killed by an overdose of sodium pentobarbitone. The stomach wasquickly removed then opened along the lesser curvature. The mucosa was rinsed with ice-coldsaline and the antrum and cardia were excised and discarded. The remaining mucosa was washedwith moist tissue paper to move food particles and mucus and gently stretched on a cork board.The tissue was kept moist with ice-cold saline. The mucosa was scraped from the underlying muscleusing a dulled scalpel blade and immediately placed in oxygenated Hanks' balanced salt solution(pH 7-4; Difco Laboratories). The procedures for dispersion and isolation followed those originallydescribed by Soll (1978) and modified by Wollin (1984) using rabbit mucosa. Nine cellular fractionswere collected using the Beckman JE-6 elutriator rotor. The number of cells in each fraction weredetermined using a haemocytometer counting chamber and the diameter of the cells wasdetermined using an eyepiece micrometer. The protein concentration of each fraction wasdetermined using the method of Bradford (1976). In some studies cells were pooled from the ninefractions collected from the elutriator rotor. The fractions were designed as follows: fraction A(pooled fractions 1 and 2), fraction B (pooled fractions 3 and 4) and fraction C (pooled fractions 7and 8). Elutriator fractions 5, 6 and 9 were mixed fractions consisting primarily of clumped cellsor cellular fragments and were therefore discarded.

Histological preparation and identification of cell fractionsEach of the pooled fractions (A-C) was diluted with 0-15 M-NaCl until just perceptibly turbid

(approximately 5 x 105 cells/ml), and 0 3-0{4 ml of each diluted suspension was spun for 5 min at1000 r.p.m. in a Shandon Cytocentrifuge (Sewickley PA). This provided a slide with the cells evenlydeposited in a spot. The slides were fixed, without being allowed to dry, for 30 min in Heidenhain's'Susa' (Kiernan, 1981) and then washed in 70% ethanol. After removal of mercury deposits with

40

PROSTAGLANDIN RECEPTORS ON RABBIT GASTRIC CELLS

an iodine-thiosulphate sequence (Kiernan, 1981), the slides were stained by a modification of theperiodic acid-Schiff-iron haematoxylin-Toluidine Blue-aurantia technique of Cook (1962). Themodification was the use of a 0-25% (w/v) solution of Metanil Yellow (in 0-25% (v/v) aqueousacetic acid) instead of the aurantia of the original procedure.

[3H]Prostaglandin binding assayFor PGE2 or Iloprost binding assays either the unfractionated cells or isolated fractions of cells

collected from the elutriator were dispersed in a medium consisting of 4 mM-Tris-HCl, 1 mm-dithiothrietol and 10 /ZM-indomethaciim. The assay conditions for [3H]PGE2 and [3H]Iloprostbinding to rabbit cells were found to be essentially similar to those previously described usinghomogenates of whole mucosa (Tepperman & Soper, 1981; Tepperman et al. 1984). The binding of[3H]PGE2 ([5, 6, 8, 11, 12, 14, 15, n-3H]-PGE2, specific activity 160 Ci/mmol; Amersham, Oakville,Ontario) and [3H]Iloprost (specific activity 14-5 Ci/mmol; Shering, Berlin) were assessed underconditions previously established in our laboratory (Tepperman & Soper, 1981; Tepperman et al.1984). These conditions have been shown to be optimal for demonstration of in vitro prostanoidbinding to gastric mucosal tissue (Tepperman & Soper, 1981; Woo et al. 1986; Beinborn et al.1986).

Iloprost was chosen rather than natural PGI2 since it is a stable analogue which is relatively moreresistant to the incubation conditions used here than is native PGI2. To determine the optimal timefor incubation dispersed but unfractionated mucosal cells were incubated for 2-5, 5, 10, 20, 40, 80or 120 min with labelled prostanoid either in the presence or absence of unlabelled prostaglandins.The procedure for termination of incubation, separation of bound and free radioactivity anddetermining specifically bound prostanoid has been previously described for both PGE2 andIloprost (Tepperman & Soper, 1981; Tepperman et al. 1984). Total binding was determined byincubating cells with either [3H]PGE2 (6-3 x 10-1 M) or [3H]Iloprost (2 1 x 10-1 M) alone. Non-specific binding was determined by incubating cells in the presence of [3H]PGE2 or [3H]Iloprost anda 1000-fold molar excess of either unlabelled PGE2 or Iloprost. Specific binding was determined asthe difference between total and non-specific binding. Specific binding was expressed as the numberof femtomoles of PGE2 or Iloprost bound per milligram protein per 106 cells (fmol (mg protein)-'(106 cells)-') or per milligram protein (fmol/mg protein). Binding of PGE2 or Iloprost wasexamined using unfractionated material, the nine cellular fractions collected from the elutriatorrotor as well as pooled fractions A, B and C.

In some experiments the effects of increasing concentrations of [3H]PGE2 (5-3 x 1012 to2-3 x 10-7 M) or [3H]Iloprost (2-2 x 10-11 to 1-3 x 10-6 M) were examined in various cellular fractions.These studies were done using unfractionated cells as well as pooled fractions A, B and C.Equilibrium binding analysis studies were done as described previously (Tepperman et al. 1984)with and without a 1000-fold excess of unlabelled PGE2 or Iloprost. The binding to each fractionas well as unfractionated cellular material was analysed using the method of Scatchard (1949). Thedata were computer fitted using a weighted non-linear least-squares technique on an IBM-PCcomputer using an equilibrium binding data analysis (EBDA) program developed by McPherson(1983). This program is designed to convert the raw data obtained from the equilibrium bindingexperiments into a form suitable for use by a non-linear curve-fitting program (SCAFIT) developedby Munson & Rodbard (1980). The SCAFIT program analysed the converted data and calculatedthe dissociation constants (Kd) and binding site concentration (Bm.x). The computer-fitted plotswere examined for one or two saturable components. An F test comparison was done to comparea one-site fit to a two-site fit.

In an additional series of experiments the ability of Iloprost to displace [3H]PGE2 from theirrespective binding sites was examined. In the study aliquots of each fraction A, B and C wereincubated with [3H]PGE2 (2-4 x 10-9 M at 37 °C). Increasing concentrations of unlabelled Iloprost(1-4 x 10-12 to 1-4 x 10-4 M) were added to cells incubated with [3H]PGE2. The results wereexpressed as a percentage inhibition of total [3H]PGE2 bound in the absence of any unlabelledprostanoid.

Aminopyrine uptake['4C]Aminopyrine accumulation was determined in each of the pooled cellular fractions from

rabbit stomach using the technique and protocol described by Wollin (1984) for rabbit isolatedcells. Aminopyrine accumulation has been frequently used as an index of parietal cell activity (Soll

41


& Wollin, 1979; Soll, 1980; Wollin, 1984). ['4C]Aminopyrine (specific activity 120 mCi/mmol) waspurchased from Amersham, Oakville, Ontario. Cells from pooled fractions A, B and C werecollected in 1 ml of Earle's balanced salt solution containing 10 mM-HEPES and 0-2% bovineserum albumin (BSA) in 12 x 75 mm borosilicate test tubes. Cells were pre-incubated for 5 min inthe presence of saline, 10-5 M-histamine and PGE2 (10-5 M) or Iloprost (10-5 M). Aminopyrineaccumulation was also determined after addition of 10' M-histamine and increasing concentrationsofPGE2 or Iloprost ( 10-10-0-4 M). After addition of these agents the cells were incubated for 5 min.At the end of this incubation period ['4C]aminopyrine (02 #uCi in 10,l) was added and theincubation proceeded for a further 20 min. Each incubation took place at 37 °C under anatmosphere of 95% 02, 5% CO2' At the end of the final incubation period each tube wascentrifuged at 2000 g for 1 min and the supernatant discarded. The pellet was washed with coldEarle's solution and centrifuged again. The pellet was transferred to a scintillation vial andsolubilized overnight in 1 ml of Triton X-100 (Packard). The next day 5 ml of scintillation cocktailwas added (Beckman Redisolv -EP) and the samples were counted on an LKB Rackbetascintillation spectromoter. In response to increasing concentrations of PGE2 or Iloprost data wereexpressed as a percentage of [14C]aminopyrine accumulated in the absence of any prostanoid. Thedose of prostanoid which produced an inhibition of maximal aminopyrine accumulation (K.) wasdetermined by computer analysis using a non-linear curve-fitting technique (ALLFIT) as describedby DeLean, Munson & Rodbard (1978). The aminopyrine accumulation ratio for each of the cellfractions was calculated by the expression [(c.p.m. in the pellet/cell volume)/c.p.m. in thesupernatant], as described by Soll (1980). This equation was applied to each of the pooled fractionsA, B and C. To calculate the cell volume for each fraction the average cell diameters for fractionsA, B and C were used (Table 1).

Trypan Blue exclusion testTrypan Blue exclusion was used to determine the ability of PGE2 and Iloprost to preserve

viability of each type of enriched cell fraction in response to damage by ethanol (10% w/v). Thedye exclusion test is a widely used measurement of cell viability (Paul, 1975). Cells pooled fromfractions A, B and C were collected in 1 ml Earle's balanced salt solution containing 10 mM-Hepesand 0-2% BSA in 12 x 75 mm borosilicate tubes. Cells were incubated for 15 min with either 0 9%saline or increasing concentrations of PGE2 or Iloprost (10-10 to 10-4 M). Ethanol was then addedand the cells incubated for an additional 60 min under an atmosphere of 95% 02, 5% CO2 at37 'C. Control cells were incubated in the absence of added agents for various times up to 2 h todetermine the influence of time alone on cellular viability. At the end of the final incubation periodthe Trypan Blue exclusion test was performed according to a modification of the method of Phillips(1973). At the end of the incubation period Trypan Blue dye (100 ,u1 of a 1 % (w/v) solution) wasadded directly to the Earle's balanced salt solution and mixed. Within 5 min the number of stainedand non-stained cells in a 20,1 aliquot of the suspension was counted in a haemocytometerchamber at 400 x magnification. The percentage of stained cells was assessed by a naive observer.The effect of ethanol alone or prostaglandin and ethanol on Trypan Blue exclusion was assessedafter correction for the influence of time alone on cellular viability. All experiments were performedby counting 100-200 cells from each tube.

Cyclic AMP determinationsCells were incubated with 10-6 M isobutylmethyl-xanthine and PGE2 or Iloprost in the dose

range 10-1-10-4 M. Cells were incubated in 05 ml Earle's balanced salt solution for 20 min at37 'C under an atmosphere of 95% 02, 5% CO2. At the end of the incubation, the cells were flashfrozen in methanol-dry ice and stored at -80 'C for cyclic nucleotide determination. Cyclic AMPwas extracted and assayed as previously described (Sheppard, Spence & Kraicer, 1979). An equalvolume of 5% perchloric acid was added and the cells sonicated for dispersal and disruption. Cellswere centrifuged (10000 g for 15 min at 4 °C) and the supernatant was removed and neutralizedwith KOH. After centrifugation (10000 g for 15 min at 4 °C) the supernatant was layered on2-2 x 07 cm columns of Dowex (AP-1-X8, formate form, 200-400 mesh; Bio-Rad, Richmond,CA, U.S.A.) and washed with 10 ml distilled water. The column was eluted with 4 ml of 3 M-formicacid. Three millilitres of eluate were collected and these were evaporated in a Savant Speed-Vacevaporator (Emerston Instruments, Toronto). The dried eluate was reconstituted in assay bufferfor cyclic AMP measurement.

42


Traces of tritiated cyclic AMP were added during extraction to determine recovery rate.Recoveries were 70-90%. Cyclic AMP was measured by a commercial RIA kit (New EnglandNuclear, Boston). Results were calculated as fmol (106 cells)-' (#sg protein)-' and data werenormalized as a percentage of the cyclic AMP levels detected in cells of each fraction in the absenceof prostanoid. Samples from each experiment were measured within one assay. Intra-assaycoefficient of variation was less than 10 %.

Statistical analy8isThe statistical significance of differences was evaluated using analysis of variance and Duncan's

multiple range test or Student's t test for unpaired data. The accepted level of significance wasP < 0-05. Data are depicted as means+s.E.M. with n based on number of cell preparations eachfrom a different rabbit.

RESULTS

Histological identification of cell typesCell types were identified in the cytocentrifuge preparations by virtue of their

tinctorial properties. The following cell types were easily identifiable in the mucosa:

TABLE 1. Mean diameter and percentage of cell types in various fractionsCell types (% of total)

Cell diameterFraction (#m) Parietal Peptic Mucous Other

Unfractionated 36+3 27 +3 16+3 20+2A 9-7+0-6 1+0-6 17+3 17+4 62+7B 12-6+0 3 4+04 19+3 40+4 37+3C 23+0-9 58+4 18+2 3+1 21+2

Cell values are means (±S.E.M.) from each of ten to fifteen rabbits.

parietal cells (clear, bright yellow cytoplasm); peptic (chief) cells (blue, granularcytoplasm); mucous cells (cytoplasm with inclusions coloured pink to purple byperiodic acid-Schiff).However all the fractions contained many cells that could not be identified with

certainty. Most of these were small cells with yellow- or grey-stained cytoplasm; somewere recognizable as endothelial, because they formed parts of obvious fragements ofcapillaries. Many were probably damaged cells, including smooth muscle fibres, thathad lost some of their cytoplasm. Most could not be classified, so they have beencounted as 'other cells' in the quantitative study of the cellular compositions of thefractions.The proportion of these cell types is shown in Table 1. The large diameter cell

group (fraction C) was composed mainly of parietal cells with preparations rangingfrom 72 to 50% of the total cells present. Fraction A and B contained significantlysmaller proportions of parietal cells.

PGE2 and Iloprost binding studiesBinding of [3H]PGE2 and [3H]Iloprost to dispersed but unfractionated mucosal

cells is shown in Fig. 1. The optimal time for binding was found to be 40 min and thiswas used as an incubation time in all subsequent binding assays.

43

44 D. B. BARR AND OTHERS

Specific PGE2 binding within each elutriator fraction is shown in Fig. 2. Specificbinding occurred in all cell fractions. However the highest degree of binding as wellas the highest ratio of specific: non-specific binding was found to occur in the pooledfraction containing the largest diameter cells. In other fractions the degree of non-specific binding was greater than specific binding.

C

0L.

EE0

E

wa-I

c

._4.o 4(

o, 31.E

E34-

0.0 2(

a 1!0=U

a

~-L

5 10 20Time (min)

Fig. 1. Binding of [3H]PGE2 or [3H]Iloprost to dispersed but unfractionated cellsfrom rabbit oxyntic mucosa. Cells were incubated as described in Methods for 2-5, 5, 10,20, 40, 80 and 120 min. Total binding (O), non-specific (0) and specific binding (A) aredisplayed. The results are the mean (+S.E.M.) from four rabbits.

Scatchard analysis of [3H]PGE2 binding to unfractionated cells as well as enrichedpooled fractions A, B and C revealed that a heterogeneous population of PGE2binding sites exist on rabbit oxyntic mucosal cells (Table 2). Both high- and low-affinity binding sites were detected using unfractionated material. F ratiodetermination of [3H]PGE2 saturation analysis revealed only one significant high-affinity binding site in fractions A and B. However fraction C contained both high-and low-affinity binding sites. The number of binding sites (Bmax) detected infraction C was greater than those determined in the unfractionated cells. Specific

-1----- 4

I.+--- ----+ -i

,I -

-

I II


401

30-

20-

45

Fraction number

1 2 3 4 5 6 7 8 9Fraction number

Fig. 2. Specific binding of [3H]PGE2 and [3H]Iloprost to each of the nine fractionsharvested from isolated rabbit oxyntic mucosal cells using the elutriator rotor. Data are

expressed as fmol (mg protein)-' (10" cells)-'. Data represents the mean (±S.E.M.) fromten rabbits. The numbers in parentheses are the average ratio of specific:non-specificbinding for that fraction. Elutriator fractions 1 and 2 were pooled for fraction A,elutriator fractions 3 and 4 were pooled to make fraction B and pooled fraction C was

comprised of elutriator fractions 7 and 8.

TABLE 2. Binding parameters (Kd and Bm.x) for PGE2-specific binding to isolated cells from rabbitoxyntic mucosa

Cell fraction

UnfractionatedABC

Kd, (M)

0-26+0-02 x 10-110.11+0-05x 10-10

0-21 +0-04 x10-100-31 +0-103 x 10-

Kd2 (m)

0-43+0-21 x 10-9

0-72 +0-35 x 10-9

Bmaxi Bmax2(fmol (mg protein)-'

(106 cells)-')2-7 + 0-2210-5+ 6.7*21-2+ 11.7*3-1+ 0-62

14-2+6-7

137 + 38*

Each mean (±s.E.M.) binding parameter is the result of Scatchard analysis of data fromexperiments done on six or seven rabbits.

* Significant differences (P < 0-05) from unfractionated cells by the unpaired t test.

-)

~0 uL

C00w -0'

C

I *i3

0.Ucnm~E

L-6)

c u

0._

._Z

0.-0I


binding of [3H]Iloprost to any of the nine fractions collected from the elutriator rotorwas greater than that observed for PGE2 (Fig. 2). The greatest degree of specificIloprost binding and the highest ratio of specific: non-specific binding was observedto occur in elutriator fractions 3 and 4. This corresponded to pooled fraction B. Other

TABLE 3. Binding parameters (Kd and Bm.x) for Iloprost-specific binding to isolated cells fromrabbit oxyntic mucosa

Bmax, Bmax2(fmol (mg protein)-1

Cell fraction Kd, (M) Kd2 (m) (106 cells) ')Unfractionated 011+0-06x 10-10 0-70+0-31 x 10-7 4-8+0-8 442+295

A 0-28+0-11 x 10-10 109+31*B 0-19+ 0 09 xI 10 0-58+0-38 x 10-7 84-9+ 23* 1690+ 284*C 0-22+0 01 x 10-10 282+ 74*

Each mean (±+.E.M.) binding parameter is the result of Scatchard analysis of data fromexperiments done on six or seven rabbits.

* Significant differences (P < 0 05) from unfractionated cells by the unpaired t test.

120 -

o -~~~~~4.l1oo

e., =-0X,$> , _ Fraction B~80-,~

, X += s. Fraction A

60

I 40 Fraction C

20 - +; Fraction C (PGE2)

10-12 10-1o 1d-8 16-6 10iloprost or PGE2 (M)

Fig. 3. Dose-response relationship between concentration of Iloprost or PGE2 (M) in theincubation medium and total [3H]PGE2 binding expressed as a percentage of control levelin the absence of prostanoid. The results are the means (+ S.E.M.) of three or four rabbits.Experiments are done using fraction A (0), B (@) or C (A) using unlabelled Iloprost andfraction C (-) using unlabelled PGE2.

elutriator fractions demonstrated greater non-specific than specific [3H]-Iloprostbinding.

Saturation analysis of [3H]Iloprost binding to fraction B as well as unfractionatedmaterial suggested a heterogeneous population of binding sites (Table 3). Inunfractionated cells binding affinities at either the high-(O11+ 0-08 x 10-1 M) or low-(0 7 + 0'3 x 10-7 M) affinity sites were lower than those parameters detected forPGE2 binding in unfractionated cells. The affinities of [3H]Iloprost binding sites on

46

PROSTAGLANDIN RECEPTORS ON RABBIT GASTRIC CELLS 47

*

.° 244J

cC0

Eu

c 6-C:

0c 4-E

2

I

I2'1F raction A Fraction B Fraction C

Fig 4. Aminopyrine accumulation ratio in isolated mucosal cell fractions of rabbit oxynticmucosa under basal conditions (open bars), and in response to 10-5 M PGE2 (shaded bars),10-5 M-Iloprost (hatched bars) and 10-5 M-histamine (stippled bars). The results are themean (+ S.E.M.) of duplicate determinations from five rabbits for each group. Asterisks (*)indicate significant (P < 0-05) increases over control as determined by Duncan's multiplerange test.

120 -

100

80

60

40

20

0O

\

'N"N.\t

*

v t l

10-10 10 9 108 iO 10 i0 5 O4PGE2 or I loprost (M)

Fig. 5. Dose-response relationship between concentration of PGE2 (0) or lloprost (0) (M)in the incubation medium and [14C]aminopyrine accumulation expressed as a percentageof control levels determined in the absence of either PGE2 or Iloprost. The results are themean ( + S.E.M.) for six rabbits for each prostanoid. Fraction C demonstrated a significant(P < 0 05) reduction in histamine-stimulated (10- M) aminopyrine uptake in response toeach prostanoid as determined by an analysis of variance. Asterisks (*) indicatesignificant (P < 0 05) differences between PGE2 and Iloprost as determined by Student'st test for unpaired data.

26

04)c

0el.O-

00)

._

Q

-

L-

CL

E

N


fraction B were similar to those detected in the unfractionated material. Howeveronly one significant high-affinity binding site for Iloprost was detected in bothfractions A or C. The specific binding capacity (Bmax) for Iloprost determined in each

30- Fraction A

20

10'

0O

X 40- Fraction B0

2 30_

20-

10-p-

w 40- Fraction C

30-

20-

10- * *

10-10 10-8 10-6 10-4PG E2 or I loprost (M)

Fig. 6. The percentage of total cells stained with Trypan Blue dye. Cells were treated witheither saline (-) or increasing concentrations (M) of PGE2 (0) or Iloprost (@). Each setof data is the mean (+S.E.M.) for five or six rabbits. Asterisks (*) indicate significant(P < 005) differences from control as determined by Duncan's multiple range test.

of the enriched fractions was greater than that detected in unfractionated cells(Table 3).The ability of Iloprost to displace labelled PGE2 bound to each of the pooled cell

fractions is illustrated in Fig. 3. Unlabelled Iloprost reduced [3H]PGE2 binding to avariable degree in each of the three fractions. Displacement of [3H]PGE2 binding wasmost apparent at Iloprost concentrations greater than 10-6 M. The greatest degree of


displacement was observed in fraction C. Unlabelled PGE2 was more potent thanIloprost for the displacement of [3H]PGE2.

I4C]Aminopyrine accumulationBasal accumulation of [14C]aminopyrine in any fraction examined was not

significantly affected by addition of either 1o-5 M-PGE2 or Iloprost. Accumulation in

250

0

0

0~

LI

0

Fraction AFraction B

Fraction C

PGE2 (M)

04)c

e04-

0

a.

LI

1~

1:

Fraction A

Fraction B

Fraction C

1 0000s0 10-1° 10-9 10-8 10-7 10-6 10-5 10-4

Iloprost (M)Fig. 7. Cyclic AMP levels in cellular fractions from rabbit oxyntic mucosa in response toincreasing concentrations of PGE2 or Iloprost (M). Data are expressed as percentagestimulation from control as determined in the absence of PGE2. The data are the meanpercentage increase (± S.E.M.) for five rabbits.

response to 10' M-histamine was significant only in fraction C (Fig. 4). Therefore theeffect of increasing the concentration of prostaglandins was tested only on fraction C(Fig. 5). A dose-related inhibition ofhistamine-stimulated aminopyrine accumulationwas observed in response to both prostanoids. PGE2 Was a more potent inhibitorthan was Iloprost. PGE2 produced a significantly greater effect on aminopyrineuptake compared to Iloprost in the concentration range 10-10 to l0-8 M. Thecomputer-estimated Ki for PGE2-mediated inhibition was determined to be


012 +0413 x 10-9 M whereas the Ki for Iloprost was determined to be 0-36 +016 x10-8 M.

Effect of prostaglandins on cellular staining with Trypan Blue dyeFigure 6 demonstrates the effect of PGE2 and Iloprost on the Trypan Blue dye

exclusion test in response to 10% (w/v) ethanol. In the absence of prostaglandin,ethanol resulted in a cellular mortality ranging from 17-3 + 1P4% for fraction A to28-2 + 3-1 % for fraction C. Analysis of variance revealed that PGE2 at aconcentration of 10-8 M resulted in a significant reduction in the percentage of cellsstained with Trypan Blue only in fraction C. Some concentrations (10-8 to 10-6 M) ofIloprost significantly reduced Trypan Blue staining in both fractions B and C.

Prostaglandin stimulation of cellular cyclic AMP formationIn the absence of PGE2 or Iloprost cyclic AMP levels were greatest in fraction C

(4-5 + 1-6 fmol (106 cells)-' (,ug protein)-'. Basal cyclic AMP levels in fractions A andB were 0 57 + 0-11 and 3-1 +0 43 fmol (106 cells)-' (/,tg protein)-' respectively. Rabbitisolated mucosal cells were more responsive to PGE2 than Iloprost (Fig. 7). Eachfraction demonstrated a dose-related increase in cyclic AMP levels in response toPGE2. The greatest responses were observed in fractions A and B. The estimatedEC50 for these fractions were 1-7 x 10-6 and 6-4 x 10-7 M respectively. Fraction C wasnot as responsive to PGE2 with an EC50 of 1-2 x 10-5 M. In response to Iloprost therewas no increase in cyclic AMP formation in the cells of fraction C. Both fraction Aand B demonstrated concentration-related increases in cellular cyclic AMP. TheEC50 for Iloprost in fractions A and B was 7-2 x 10-5 and 5-3 x 10-6 M respectively.

DISCUSSION

In the present study both [3H]PGE2 and [3H]Iloprost bound to isolated fractionsof cells from rabbit oxyntic mucosa. The greatest degree of specific PGE2 binding wasassociated with the fraction containing the largest diameter cells. This fraction ofcells has been determined histologically to be enriched with parietal cells. Our dataindicates that the large cell fraction contains approximately 60% parietal cells. Anumber of arguments may be made for concluding that it is the parietal cells in thisfraction that are responsible for the observed biological and binding activity.Parietal cell content increases significantly in fraction C and the degree of PGE2binding follows suit. The proportion of peptic cells is the same in all three pooledfractions while the proportions of mucous and unidentified cells in fraction C are lessthan in fractions A and B. Therefore the increase in binding occurs in a fraction inwhich only the parietal cell content is increased. In addition biological data obtainedin these studies indicates that only fraction C displays a significant increase overbasal levels of histamine-stimulated aminopyrine accumulation. Aminopyrineaccumulation is an accepted index of parietal cell activity. Furthermore PGE2 wasfound to be a potent inhibitor of aminopyrine accumulation only in fraction C andhad a variable effect on the small degree of aminopyrine accumulation determined infractions A and B. These arguments would support our conclusions based on a 60 %enrichment in identified parietal cells. Recently our findings have been confirmed by

50


Tsai, Kessler, Schoenhard, Collins & Bauer (1987) using canine mucosal cells with agreater than 80% enrichment of parietal cells.PGE2 binding was also observed to occur in smaller diameter cells although the

degree of binding as well as the ratio of specific: non-specific binding was significantlyless than that detected in the parietal cell fraction. PGE2 exerts a number ofbiological actions on the stomach including inhibition of acid secretion, stimulationof HCO3- and mucus secretion and vasodilation of the gastric vasculature (Miller,1983). Since specific PGE2 binding was detected in each fraction it is possible thatthese biological actions are initiated even by small degrees of PGE2 binding to thecells regulating these actions.

In contrast both the degree of binding and the ratio of specific:non-specificbinding for Iloprost were greatest in a fraction of smaller diameter cells. This fractionof cells contains predominantly mucus and peptic cells as well as cells which areprobably endothelial cells. Exogenous PGE2 exerts actions on the stomach similar toPGE2 (Robert, 1981; Miller, 1983). Furthermore the synthetic analogue Iloprost hasbeen shown to have both antisecretory and cytoprotective properties although itspotency is less than that of PGE2 or native PGU2 (Vischer & Casals-Stenzel, 1982).

Heterogeneous populations of binding sites for PGE2 and Iloprost were observedin unfractionated cells. We have previously observed similar binding characteristicswith high-affinity-low-capacity and low-affinity-high-capacity sites (Tepperman &Soper, 1983; Tepperman et al. 1984). However heterogeneous sites were not observedin every pooled fraction. Two binding sites for [3H]PGE2 were observed in thefraction of large diameter cells (fraction C). Two sites for Iloprost occurred in afraction of smaller cells. In both cases heterogeneous sites were observed in fractionsin which the specific binding of each prostanoid was greatest. In all cases the specificbinding capacities were greater on the enriched fractions than in unfractionatedmaterial suggesting receptor concentration after elutriation. Furthermore, whilesome fractions demonstrate the presence of heterogeneous binding sites both bindingsites may not reside on a single cell. The cell fractions are not pure. Thereforedifferences in binding affinities may represent binding to different cell types in eachfraction.A significantly greater degree of specific compared to non-specific binding was

determined in only one fraction for both PGE2 and Iloprost. The high degree of non-specific binding may interfere with the fitting of these data to either a one-site or two-site binding model. Therefore, data must be interpreted cautiously when the specificbinding of either prostanoid comprised only a small percentage of total binding, i.e.fractions A and B for PGE2 and fractions A and C for Iloprost.PGE2 was a more potent inhibitor of aminopyrine accumulation than was Iloprost.

These results are similar to those of Soll (1980) and Skoglund, Nies & Gebber (1982)using parietal cells isolated from canine stomach. In those studies the IC50 forPGE2 was in the range of 10-7-10-8 M while PGI2 produced a 50% inhibition ofaminopyrine uptake at concentrations greater than 10-6 M. These concentrations aresimilar to those observed in the present study. Furthermore in the present study agood association between [3H]PGE2 binding affinity in parietal cells and the abilityof PGE2 to inhibit histamine-stimulated aminopyrine uptake was demonstratedquantitatively. The Kd in fraction C was found to be 0-3+ 01 x 10" M and

51


0-72 + 035 x 10-9 M for high- and low-affinity sites respectively, while the computer-estimated Ki for biological action (aminopyrine accumulation) was calculated to be0-4 + 0-2 x 10-9 M. The concentration of PGE2 which saturates 50% of the bindingsites was not different from the concentration required to inhibit the biologicalresponse by 50%. Therefore biological action was correlated with binding to low-affinity receptors primarily with some possibility of spare receptors. The high degreeof specific binding of PGE2 to the parietal cell-enriched fraction as well as the goodassociation between binding and biological activity of PGE2 in this group of cellssuggests that PGE2 binds at true receptor sites.The low degree of specific [3H]Iloprost binding to the parietal cell-enriched

fraction demonstrated only high-affinity sites (Kd 0-22+ 001 x 10-10 M) while theKi for Iloprost inhibition of aminopyrine uptake was of the order of 0 4 x 10-8 M.Under these circumstances at an Iloprost concentration of 10-8 M most if not all ofthe saturable binding sites would be occupied but the biological response is only half-maximal. Therefore binding of Iloprost to its sites on the parietal cell does not appearto mediate inhibition of acid secretion. This is especially apparent as specific Iloprostbinding comprised only a small fraction of total binding of this analogue to thesecells. The action of Iloprost on parietal cell activity may be as a result of binding toPGE2 receptors. This possibility has been confirmed by the demonstration thatIloprost displaces [3H]PGE2 binding in each of the three enriched cell fractions.Similar conclusions have been reached by Seidler, Beinborn & Sewing (1987) usingIloprost and parietal cells isolated from rabbit gastric mucosa. Alternatively thesedata may indicate that Iloprost is not the most appropriate PGI2 analogue to use inthese studies. Soll & Whittle (1981) have demonstrated that 16-phenoxy analoguesof PGI2 were equipotent to PGE2 especially as related to inhibition of histamine-stimulated aminopyrine accumulation. Therefore the data in the present study maynot truly represent differences between PGE2 and PGI2.

In the present study both PGE2 and Iloprost produced some degree of protectionof the isolated cells against ethanol-induced damage. Therefore protection of oxyntictissue may be mediated partially via a direct effect on the mucosal cell. PGE2 hasbeen previously reported to protect isolated gastric glands from the damagingactions of ethanol (Tarnawski, Brzozowski, Hollander, Krause & Gregely, 1986).Likewise Terano, Mach, Stachura, Tarnawski & Ivey (1984) and Ternao, Ota, Mach,Hiraishi, Stachura, Tarnawski & Ivey (1987) have reported protection againstaspirin- and taurocholate-induced damage of mucous cells in culture. Muller-Lissner,Fimmel, Sonnenberg, Peskar, Fischer & Blum (1981) have observed PGE2-mediatedprotection of isolated mucosal cells in response to sodium taurocholate. However inthe present study neither PGE2 nor Iloprost produced a clear dose-related reductionin Trypan Blue staining. Therefore a quantitative relation between binding andprotection cannot be made on the basis of these data.We have used the Trypan Blue dye exclusion test to assess cellular viability in

response to ethanol in the presence and absence of prostanoids. The Trypan Blue testhas been used as an index of gastric mucosal cellular viability in response toulcerogens and prostaglandins (Terano et al. 1987). However in the present study itmay not provide the sensitivity necessary to firmly establish the quantitativeassociation between prostanoid binding and prostanoid-mediated cellular protection.

52


Both prostanoids produced dose-related increases in cellular cyclic AMP levels inmost of the fractions examined. The most potent stimulatory effects were observed inthe smaller diameter, non-parietal cell fractions. This confirms the findings of Wollin,Soll & Samloff, (1979). PGE2 weakly stimulated cyclic AMP in the parietal cell-enriched fraction while Iloprost was ineffective. While it is unlikely that thesestimulatory actions on cyclic AMP are relevant to parietal cell function, the actionsof prostaglandin on other mucosal cells may be mediated via stimulation of cellularcyclic AMP. However the EC50 values for cyclic AMP stimulation in these cells wereat least two orders of magnitude greater than those which produce an occupation of50% of the maximal number of binding sites for either prostanoid, even in fractionsin which there was a high degree of specific binding. The reasons for this poorassociation are unclear. The cell fractions while enriched for some cell types are stillrelatively heterogeneous. Interactions between cell types might account for this poorcorrelation. Also our assay may not have detected small but significant changes incyclic AMP formation in response to the lower range of prostaglandin concentrations.Furthermore, the transduction of the stimulatory signal to the target compartmentmay not only require cyclic AMP as a messenger but other messengers as well.Therefore measurement of other intracellular mediators and their interaction withcyclic AMP may be necessary to correlate the role of cyclic AMP with prostanoidbinding. Alternatively the significance of prostanoid stimulation of cyclic AMP inthese cellular fractions is questionable. Confirming this is the demonstration thatprostaglandin-mediated protection has been shown to be independent of an increasein cellular cyclic AMP (Simon, Muller & Kather, 1981; Terano et al. 1987).

In conclusion we have shown that PGE2 and Iloprost demonstrate high degrees ofspecific binding to different cellular types of rabbit oxyntic mucosa. Binding ofPGE2 to parietal cell receptors appears to physiologically mediate a decrease in H+secretion (as determined by aminopyrine uptake). The PGI2 analogue Iloprost bindsto receptors on smaller diameter cells. While Iloprost binding may be associated withthe ability to protect the cell against ethanol-induced damage, a quantitativerelationship could not be demonstrated in the present study.

We thank Ms Kim Clarke for help in preparation of the manuscript. A portion of this work waspresented at the 87th annual meetfng of the American Gastroenterological Association, SanFrancisco, 1986. This study was supported by a grant from the Medical Research Council ofCanada (MT-6426) to Dr B. L. Tepperman.

REFERENCES

BEINBORN, M., NETZ, S., STAAR, U. & SEWING, F.-FR. (1986). Localization and characterization ofprostaglandin E2 binding sites in the porcine fundic mucosa. Gastroenterology 90, 1343.

BRADFORD, M. M. (1976). A rapid and sensitive method for quantification of microgram quantitiesof protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.

COOK, H. C. (1982). Periodic acid-Schiff-toluidine blue aurantia; a stain for the gland cells of thestomach. Stain Technology 37, 317-319.

DELEAN, A., MUNSON, P. J. & RODBARD, D. (1978). Simultaneous analysis of families of sigmoidalcurves: application to bioassay, radioligand assay and physiological dose-response curves.American Journal of Physiology 235, E97-102.

KIERNAN, J. A. (1981). Histological and Histochemical Methods: Theory and Practice. Toronto:Pergamon Press.

53


MCPHERSON, G. A. (1983). A practical computer-based approach to the analysis of radioligandbinding experiments. Computer Programs in Biomedical Science 17, 107-114.

MILLER, T. A. (1983). Protective effects of prostaglandins against gastric mucosal damage: currentknowledge and proposed mechanisms. American Journal of Physiology 245, G601-623.

MULLER-LISSNER, S. A., FimMEL, C., SONNENBERG, A., PESKAR, B., FisCHER, J. A. & BLUM, A. L.

(1981). The effect of prostaglandins in isolated rat gastric cells. Scandinavian Journal ofGastroenterology 16, 229-232.

MuNsoN, P. J. & RODBARD, D. (1980). LIGAND: a versatile computerized approach for thecharacterization of ligand binding systems. Analytical Biochemistry 107, 220-239.

PAUL, J. (1975). In Cell and Tissue Culture, 5th edn, pp. 367-369. New York: ChurchillLivingston.

PHILLIPS, H. J. (1973). Dye exclusion tests for cell viability. In Tissue Culture Methods andApplications, ed. KRAUSE, P. F. & PATTERSON, M. K., pp. 406-408. New York: AcademicPress.

ROBERT, A. (1981). Prostaglandins and the gastrointestinal tract. In Physiology of theGastrointestinal Tract, ed. JOHNSON, L. R., pp. 1407-1434. New York: Raven Press.

SCATCHARD, G. (1949). The attractions of proteins for small molecules and ions. Annals of the NewYork Academy of Sciences 51, 660-72.

SEIDLER, U., BEINBORN, M. & SEWING, K.-FR. (1987). The inhibitory effect of prostaglandins onacid secretion is mediated by the PGE2-receptor.Gastroenterology 92, 1631.

SHEPPARD, M. S., SPENCE, J. W. & KRAICER, J. (1979). Release of growth hormone from purified

somatotrophs: role of adenosine 3'5' monophosphate and guanosine 3'5' monophosphate.Endocrinology 105, 261-266.

SIMON, B., MULLER, P. & KATHER, H. (1981). Molecular aspects of prostaglandin action in humangastric mucosa. In Stomach Diseases - Current Status, ed. MAERKE, Y. W. F., MOER, E. M. J. &PELCKMANS, P. A. R., pp. 22-28. Amsterdam: Exerpta Medica.

SKOGLUND, M. L., NIES, A. S. & GEBBER, J. G. (1982). Inhibition of acid secretion in isolated canineparietal cells by prostaglandins. Journal of Pharmacology and Experimental Therapeutics 220,371-374.

SOLL, A. H. (1978). The actions of secretagogues on oxygen uptake by isolated mammalian parietalcells. Journal of Clinical Investigation 61, 370-380.

SOLL, A. H. (1980). Specific inhibition by prostaglandins E2 and 12 of histamine stimulated14C-aminopyrine accumulation and cyclic adenosine monophosphate generation by isolated canine

parietal cells. Journal of Clinical Investigation 65, 1222-1229.SOLL, A. H. & WHITTLE, B. J. R. (1981). Interaction between prostaglandins and cyclic AMP in the

gastric mucosa. Prostaglandins 21, suppl., 39-45.SOLL, A. H. & WOLLIN, A. (1979). Histamine and cyclic AMP in isolated canine parietal cells.American Journal of Physiology 237, E444-450.

TARNAWSKI, A., BRzOZOWSKI, T., HOLLANDER, D., KRAUSE, W. J. & GREGELY, J. (1986).

Prostaglandin protection of isolated gastric glands against ethanol-induced injury. Evidence for

cytoprotection in vitro. Gastroenterology 90, 1658.TEPPERMAN, B. L. & SOPER, B. D. (1981). Prostaglandin E2 binding sites in cAMP production in

porcine fundic mucosa. American Journal of Physiology 241, G313-320.TEPPERMAN, B. L. & SOPER, B. D. (1983). Subcellular distribution of 3H-prostaglandin E2 binding

sites in porcine gastric mucosa. Prostaglandins 25, 425-441.TEPPERMAN, B. L., SOPER, B. D. & EMERY, S. K. (1984). Specific binding of a stable prostacyclin

analogue (Iloprost) to rat oxyntic mucosa. Prostaglandins 28, 477-484.TERANO, A., MACH, T., STACHURA, J., TARNAWSKI, A. & IVEY, K. J. (1984). Effect of 16, 16-

dimethyl prostaglandinE2 aspirin induced damage to rat gastric epithelial cells in tissue culture.Gut 25, 19-25.

TERANO, A., OTA, S., MACH, T., HIRAISHI, H., STACHURA, J., TARNAWSKI, A. & IVEY, K. J. (1987).Prostaglandin protects against taurocholate-induced damage to rat gastric mucosal cell culture.Gastroenterology 92, 669-677.

TSAI, B. S., KESSLER, L. K., SCHOENHARD, G., COLLINS, P. W. & BAUER, R. F. (1987). E-typeprostaglandin binding sites in isolated canine parietal cells: Elucidation with [3H]misoprostolfree acid. Zeitschrift fur Gastroenterologie 25, 201-206.

54


VISCHER, P. & CASALS-STENZEL, J. (1982). Pharmacological properties of Iloprost, a stableprostacyclin analogue in the gastrointestinal tract. Prostaglandins Leulkiotrienes and Medicine 9,517-529.

WOLLIN, A. (1984). Carbonic anhydrase activity and aminopyrine uptake in isolated gastricmucosal cells. American Journal of Physiology 247, G213-219.

WOLLIN, A., SOLL, A. H. & SAMLOFF, I. M. (1979). Actions of histamine, secretion and PGE2 oncyclic AMP production by isolated canine fundic mucosal cells. American Journal of Phy8iology237, E437-443.

Woo, S. K., ROZKOWSKI, A. P., WATERBURY, L. D. & GARAY, G. L. (1986). Gastric mucosabinding studies with enprostil: A potent anti-ulcer prostaglandin. Prostaglandins 32, 243-249.

Documents

enriched in parietal cells. [3H]Iloprost binding occurred