Cellular Signalling Vol. 4, No. 3, pp. 321-329, 1992. Printed in Great Britain.

0898-6568/92 $5.00 + 0.00 © 1992 Pergamon Press Lid

PERTUSSIS TOXIN REVERSES PROSTAGLANDIN E 2- AND SOMATOSTATIN-INDUCED INHIBITION OF RAT PARIETAL CELL H+-PRODUCTION JOHANNA SCHMIDTLER,* WALTER ROSENTHAL,t STEFAN OFFERMANNS,I"VOLKER SCHUSDZIARRA,* MEINHARD CLASSEN* and WOLFGANG SCHEPP* *Department of Internal Medicine II, Technical University of Munich, and tDepartment of Pharmacology, Free University of Berlin, Germany

(Received 8 August 1990; and accepted 12 December 1991) Abstract--In enzymatically dispersed enriched rat parietal cells we studied the effect of pertussis toxin on prostaglandin E2 (PGE2)- or somatostatin-induced inhibition of H+-production. Parietal cells were incubated in parallel in the absence (control cells) and presence of pertussis toxin (250 ng/ml; 4 h). [14C]Aminopyrine accumulation by both pertussis toxin-treated and control cells was used as an indirect measure of H+-production after stimulation with either histamine, forskolin or dibutyryl adenosine Y,5'-cyclic monophosphate (dbcAMP) alone and in the presence of PGE 2 (10-9-10 -7 M) or somatostatin (10-9-10 -6 M). PGE 2 inhibited histamine- and forskolin-stimulated [14C]aminopyrineaccumulation but failed to alter the response to dbcAMP. Somatostatin was less effective and less potent than PGE 2 in inhibiting stimulation by histamine or forskolin and reduced the response to dbcAMP. Pertussis toxin completely reversed inhibition by both PGE 2 and somatostatin on histamine- and forskolin-stimulated H+-production but failed to affect inhibition by somatostatin of the response to dbcAMP. After incubation of crude control cell membranes with [32p]NAD+, pertussis toxin catalysed the incorporation of [3rp]adenosine diphosphate (ADP)-ribose into a membrane protein of molecular weight of 41,000, the known molecular weight of the inhibitory subunit of adenylate cyclase (Gi,). Pertussis toxin treatment of parietal cells prior to the preparation of crude membranes almost completely prevented subsequent pertussis toxin-catalysed [32p]ADP ribosylation of the 41,000 molecular weight protein. It is concluded that in rat parietal cells pertussis toxin inactivates Gt~ by ADP-ribosylation thereby preventing inhibition mediated by the inhibitory subunit of adenylate cyclase. PGE 2 exerts its inhibitory effect on rat parietal cell function entirely by activating Gt~. In contrast, somatostatin acts only in part via Gi~, but also through additional pertussis toxin-insensitive mechanisms distal to or independent of the generation of cAMP.

Key words: Prostaglandin E2, somatostatin, rat parietal cell, acid production, GTP-binding protein, pertussis toxin. inhibits the enzyme after being activated by an inhibitory agent. Pertussis toxin has been shown to cause irreversible inactivation of Gia by ADP-ribosylation thereby selectively blocking this inhibitory pathway in a variety of cells [5, 6]. Therefore, pertussis toxin constitutes an excellent pharmacological tool for investigating whether an inhibitory agent reduces histaminestimulated acid production via activation of

INTRODUCTION IT IS generally accepted that the stimulatory effect of histamine on parietal cell function is mediated through adenosine Y,5'-cyclic monophosphate (cAMP) [1-4] which is generated from adenosine 5'-triphosphate (ATP) by the action o f adenylate cyclase. The activity of this enzyme is controlled by two GTP-binding proteins linking the receptor at the cell surface to the catalytic subunit of adenylate cyclase [5, 6]. One of these GTP-binding proteins, G~, activates the catalytic subunit upon hormonal stimulation of the cell, while the other, Gi~,

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Recent studies in isolated canine parietal cells suggested pertussis toxin-sensitive inhibition by prostaglandin E 2 (PGE2) [7] and somatostatin [8] of histamine-stimulated H+-production. 321

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Moreover, pertussis toxin reversed inhibition by somatostatin of histamine-stimulated acid production in the mouse perfused stomach [9]. On the other hand, although the rat is widely used for studies on gastric acid secretion and peptic ulcer, pertussis toxin-sensitive inhibition by somatostatin and/or P G E 2 o f rat parietal cells has only been studied in crude preparations of gastric cells [10, 11]. In this experimental system indirect effects on the parietal cells cannot be excluded. Therefore, in an attempt to reduce substantially any potential indirect effects, we investigated the effect of both P G E 2 and somatostatin on H + - p r o d u c t i o n by enriched rat parietal cells and its sensitivity to pertussis toxin.

MATERIALS AND METHODS Materials

The following analytical grade reagents were used: Dimethyl [~4C]aminopyrine, specific activity l l4mCi/mmol (Amersham Buchler, Braunschweig, F.R.G.); Quickszint 2000 scintillation cocktail and Biolute S tissue solubilizer (Zinsser Analytic, Frankfurt a.M., F.R.G.); [32p]nicotinamide adenine dinucleotide, specific activity 100-200 Ci/mmol ([32P]NAD÷; ICN, Meckenhein, F.R.G.); bovine serum albumin, Dulbecco's modified Eagle's medium (DMEM), 3-isobutyl- l-methylxanthine (IMX), N-2-hydroxyethylpiperazine-N-ethylsulphonic acid (HEPES), N 6, O2-dibutyryl adenosine 3',5'-cyclic monophosphate sodium salt (dbcAMP), histamine (Serva, Heidelberg, F.R.G.); pertussis toxin, prostaglandin E2, synthetic somatostatin-14, sodiumdodecylsulphate (SDS), acrylamide, N,N'bisacrylamide, ammoniumpersulphate, N,N,N',N'tetramethylethylenediamine (TEMED), phosphorylase B, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, nicotinamide adenine dinueleotide (NAD+), Coomassie brilliant blue (Sigma, Munich, F.R.G.); forskolin (Calbiochem, La Jolla, CA, U.S.A.); pronase E, dithiothreitol, trypan blue, Na2EDTA, and all buffer constituents (Merck, Darmstadt, F.R.G.). Buffers. Medium A consisted of (mmol/1) NaH2PO4 (0.5), Na2HPO 4 (1.0), NaHCO 3 (20), NaC1 (70), KCI (5), glucose (11), Na2EDTA (2), HEPES (50), bovine serum albumin (20 mg/ml). Medium B was of the same composition as medium A, EDTA-free and supplemented with CaCI 2 (1.0) and MgCI 2 (1.5). Medium C differed from B by having a lower albumin concentration (1 mg/ml).

Methods Cell isolation. Stomachs of female Wistar rats (body weight 130-150 g; Charles River, Salzfeid, F.R.G.) were used. Gastric mucosal cells were enzymatically isolated as described [3, 12]. Briefly, after transformation into everted sacs, the stomachs were filled with 2 ml medium A containing pronase E (2.5 mg/ml) and incubated at 37°C in carbogen-gassed pronase-free medium A. After 90 min the stomachs were transferred to medium B and gently stirred for 60 min. The isolated cells were collected by centrifugation (750 rpm, 5 min). The crude cell preparation contained 21 + 3% parietal cells as determined by light microscopy. Cellfractionation. The crude cell preparation was resuspended in medium C containing dithiothreitol (15.5 mg/100 ml) and fractionized by counterflow centrifugation [13] using a Beckman elutriator rotor (JE 6B, Beckman Instruments Inc., Glenrothes, U.K.) run in a J2-21 M/E Beckman centrifuge. Non-parietal cells were washed out at a rotor velocity of 2,000 rpm and a flow rate of 40 ml/minute. The fraction enriched in parietal cells (76 + 4%) was collected at 2,000 rpm and 58 ml/minute as described previously [14] and used for subsequent experiments. Cell viability as determined by trypan blue exclusion exceeded 95%. Pertussis toxin treatment of parietal cells. The enriched parietal cell fraction was resuspended in DMEM (5 x 106 cells/ml, pH 7.4) and divided into two portions in separate incubation flasks. To one flask pertussis toxin (250ng/ml) was added, whereas in the second flask control cells were incubated in the absence of pertussis toxin. Both pertussis toxin-treated and control cells were incubated in parallel at 37°C under continuous gentle stirring. After 4h the phosphodiesterase inhibitor IMX (0.1mM) was added to both flasks and the [14C]aminopyrine experiments were started immediately. [14C]Aminopyrine accumulation. Accumulation of the weak base [~4C]aminopyrine in isolated parietal cells was used as a quantitative index of acid production [15]. Experiments were performed in parallel with both control and pertussis toxin-treated cells as previously described [3, 14]. Briefly, 400 ~l..of the cell suspensions were incubated at 37°C together with [14C]aminopyrine (0.041aCi/tube) and one of the following stimulants: histamine 10 -4 M, forskolin 10 -~ M, dbcAMP 10 -3 M. Each stimulant was tested alone and in the presence of somatostatin (10 -910-rM) or PGE 2 (10-9-10 -7 M). While histamine interacts with H2-receptors, forskolin and dbcAMP are receptor-independent direct stimulants of adenylate cyclase [16] or cAMP-dependent protein-kinase A [17], respectively. After 30min 2001al of the suspension was layered over 10001al of medium C

Pertussis toxin and rat parietal cell H+-production and spun down in an Eppendorf table centrifuge. The pellet was dissolved by Biolute S. Scintillation cocktail (4mi) was added after 12h, and after another 4 h radioactivity was determined in a liquid scintillation counter (LS 1801, Beckman, Glenrothes, U.K.). [14C]Aminopyrine accumulation in the presence of dinitrophenol (0.1 mM) represents nonspecific incorporation and was subtracted from the test values. The ratio of [t4C]aminopyrine taken up into the parietal cells to that remaining in the undiluted incubation medium was calculated as described [15]. Preparation of crude membranes. According to previously published protocols [7, 8] cell suspensions of both pertussis toxin-treated (18 h) and -untreated cells were washed once in homogenization buffer containing 5 mM EDTA in 100raM Tris (pH 7.5). Thereafter, cells were homogenized by sonication (sonifier W 250, Branson Ultrasonics B.V., Soest, F.R.G.; 50 W, 3-s sonication periods, 30-s intervals, 10min total; 4°C). Homogenates were centrifuged for 2min at 200g and 4°C. The supernatant was subjected to ultracentrifugation at 20,000g for 30 min. The pellet was resuspended in 25mM Tris containing I mM EDTA. The protein content of the membrane preparation was measured using a commercial kit (Bio-Rad, Munich, F.R.G.) and the suspension diluted to a concentration of 2-4 mg protein/ml. Membrane suspensions were stored at - 7 0 ° C until ADP-ribosylation. In vitro [32P]ADP-ribosylation. ADP-ribosylation of membrane proteins was performed as previously described [18]. Pertussis toxin (0.1 mg/ml) was preactivated in the presence of DTT (20 mM) and ATP (1 mM) at 37°C for 30 min. The final assay mixture contained 25 mM Tris-HC1 (pH 7.5), I mM EDTA, 5 mM ATP, 1 mM GTP, 10 mM thymidine, 20 lag/ml DNase, 1 ~tM NAD +, 1.7 lag/ml preactivated pertussis toxin and 1.8 laCi [32p]NAD+ in a total volume of 60 lal/tube. The reaction was started by addition of membrane protein (100~tg/tube). Membranes of pertussis toxin-treated and -untreated ceils were assayed in duplicate. Membranes prepared from the hamster clonal insulinoma cell line HIT-TI5 were used as an internal standard since in these cells pertussis toxin is known to catalyse specifically ADP ribosylati~n of a 41,000 molecular weight membrane protein [19]. Incubations were performed at 30°C for 30 min. The reaction was stopped using cold Tris buffer (50 mM). SDS-polyacrylamide gel electrophoresis was performed according to Laemmli [20]. Proteins were dissolved in Laemmli buffer and loaded on a 10% separating gel (stacking gel 6%). Electrophoresis was performed at a constant current of 20 rnA. Gels were stained with Coomassie blue (1%). Following destaining in 10% acetic acid, gels were dried and exposed to a Kodak X-OMAT AR CELLS 4:3-F

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film (Eastman Kodak, Rochester, NY). The following molecular weight markers were used: phosphorylase B (molecular weight 94,000), BSA (67,000), ovalbumin (43,000), carbonic anhydrase (30,000) and soybean trypsin inhibitor (20,000). Calculations and statistics. [~4C]Aminopyrine accumulation obtained in the presence of either histamine (10 -4 M), forskolin (10 -5 M) or dbcAMP (10 -3 M) alone represents maximal stimulation with the respective agent (100%). Accumulation achieved after addition of either somatostatin or PGE2 is indicated in per cent of maximal stimulation. It did not seem to be reasonable to investigate the effects of PGE2 and somatostatin at concentrations above 10 -7 and 10 -6 M, respectively. Thus, inhibition achieved with these concentrations was considered maximal and was the basis for calculation of apparent IC50 values. Data are expressed as means _ 1 S.E.M. from five to seven independent experiments each performed in quadruplicate. Statistical significance of differences was calculated by analysis of variance for multiple determinations. Stimulated [J4C]aminopyrine accumulation was compared to that in the presence of PGE2 or somatostatin. Values of P < 0.05 were considered significant.

RESULTS

1. Effect of PGE 2 on [14C]aminopyrine accumulation in the presence and in the absence of pertussis toxin In the absence o f pertussis toxin, P G E 2 dosedependently reduced [t4C]aminopyrine accumulation stimulated by 10 -4 M histamine. Parietal cell response to histamine (100%) was inhibited to 78.4 + 5.7% by l0 - g M P G E 2 ( P < 0.01; Fig. l, left). A t a concentration o f 10 -7 M, P G E 2 reduced aminopyrine accumulation to 31.2 + 4 . 5 % . (P < 0.01). The ICs0 was 5.6-1- 1.5 x l0 -9 M. P G E 2 was equally effective in inhibiting forskolin-stimulated aminopyrine accumulation. Indeed, parietal cell response to l0 -5 M forskolin was maximally reduced to 39.1 + 7.9% (P < 0.01; Fig. 2, left). The corresponding ICs0 was 2.8 + 0.9 x l0 -9 M. In contrast, P G E 2 had no effect on d b c A M P stimulated aminopyrine accumulation (not shown). Treatment with pertussis toxin did not alter basal or stimulated [t4C]aminopyrine accumulation (Table 1). Pertussis toxin completely

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reversed the inhibitory effect o f P G E 2 on both histamine-stimulated (Fig. 1, fight) and forskolin-stimulated aminopyrine accumulation (Fig. 2, right). Even in the presence o f the maximally effective concentration o f PGE2 acid production reached 98.0 + 3.2% and 1 0 1 . 3 + 3 . 2 % , respectively, o f the response to histamine or forskolin alone.

2. Effect of somatostatin on [l'C]aminopyrine accumulation in the presence and absence of pertussis toxin C o m p a r e d to P G E 2, somatostatin turned out to be less effective and also slightly less potent in inhibiting aminopyrine accumulation. Following stimulation with 10 -4 M histamine (100%), acid production was reduced to 57.0 + 3.4% by 10 -6 M somatostatin (P < 0.01; Fig. 3, left). The half maximal effect was achieved at a concentration o f 30.0 + 7.9 x 10 -9 M somatostatin. Somatostatin inhibited aminopyrine accumulation induced by 10 -5 M forskolin (100%) to 63.5 ___ 4.1% (P < 0.01; Fig. 4, left)

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FIG. 5. Effect of somatostatin on dbeAMP-stimuiated [~4C]aminopyrineaccumulation by isolated rat parietal cells in the absence (left) or presence (right) of pertussis toxin. 10-3 M dbcAMP alone = 100% (open symbols); n = 5; *P < 0.05, ** P < 0.01 vs. dbcAMP alone.

with an IC50 of 9.8 + 3.0 x 10 -9 M somatostatin. In contrast to PGE 2, somatostatin significantly reduced the parietal cell response to 10-3M dbcAMP ( = 100%) (Fig. 5, left). Aminopyrine accumulation was reduced to 85.0+5.7% and 66.6 + 5.3% (P < 0.01) by 10 -9 and 10 -6 somatostatin, respectively. Pertussis toxin completely restored the response to 10 -4 M histamine: in the presence of the maximally effective somatostatin concentration acid production reached 97.5 + 8.2% of the response to histamine alone (Fig. 3, right). Similarly, somatostatin failed to inhibit the response to forskolin after preincubation with pertussis toxin (Fig. 4, right). Pertussis toxin slightly attenuated the effect of 10 -9 M somatostatin on dbcAMP-stimulated H+-production (89.6 + 6.4; Fig. 5, right). However, the inhibitory effect of higher somatostatin concentrations on the response to dbcAMP was insensitive to pertussis toxin (Fig. 5, right).

94,000 molecular weight as determined from appropriate molecular weight markers (Fig. 6). Incubation of crude membranes from control cells in the presence of pertussis toxin and [32p]NAD+ was followed by incorporation of [32p]ADP-ribose into a single membrane protein of approximately 41,000 molecular weight (n = 5 independent experiments; Fig. 7, lane 1). Gel-electrophoretically, the [32p]ADP-ribosylated protein comigrated with the pertussis toxin substrate in HIT-T15 cells (Fig. 7, lane 4) which has been characterized as representing Gi~ [19]. On the other hand, preincubation with pertussis toxin of parietal cells prior to the preparation of membranes substantially reduced subsequent [32p]ADP-ribosylation of the 41,000 molecular weight membrane protein (Fig. 7, lane 3), suggesting that pertussis toxin had catalysed ADP-ribosylation of this protein during preincubation of intact cells. However, cell treatment with pertussis toxin did not totally prevent [32p]ADP-ribosylation of the 41,000 molecular weight protein. These data are similar to findings in canine parietal cells [7] and indicate that under the conditions tested ADP-ribosylation in the intact cells was marked but not complete.

3. In vitro [32 P]ADP-ribosylation Gel electrophoresis resulted in accurate separation of proteins ranging from 20,000 to

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DISCUSSION In the present study PGE 2 exhibited potent inhibitory effects on both histamine-stimulated and forskolin-stimulated acid production by isolated enriched rat parietal cells. Pretreatment of parietal cells with pertussis toxin abolished these PGE 2 effects. PGE 2 did not interfere with dbcAMP-stimulated aminopyrine accumulation. Compared to PGE 2, somatostatin appeared to be less effective and also less potent in inhibiting histamine- and forskolin-stimulated acid production. In contrast to PGE 2 somatostatin significantly inhibited dbcAMPstimulated aminopyrine accumulation. Pertussis toxin completely abolished the effect of somatostatin on histamine- and forskolin-stimulated acid production, but did not alter the effect of somatostatin (10-8-10 -6 M) on dbcAMPstimulated [~4C]aminopyrine accumulation. Regarding the inhibitory effect of PGE 2 and somatostatin on histamine-stimulated acid production, the present study is in line with previous investigations using fundic glands or isolated parietal cells [4, 7, 8, 10, 11]. These data suggest a direct effect of both agents on the parietal cell. Accordingly, parietal cell receptors for PGE2 and somatostatin have been characterized [8, 21]. The direct inhibitory effect of both agents on the parietal cell is mediated by inhibition of adenylate cyclase and subsequent reduction of cAMP production since both PGE 2 and somatostatin inhibit cAMP production as well as [~4C]aminopydne accumulation following stimulation by histamine and forskolin [4, 7, 8]. In line with previous data from enriched canine parietal cells [7, 8] the present study demonstrates that pertussis toxin abolishes the inhibitory effect of PGE2 and somatostatin on histamine-stimulated [t4C]aminopyrine accumulation also in rat parietal cells. Furthermore, we observed that in rat, as in canine [7], parietal cells pertussis toxin ADP-ribosylates a membrane protein of molecular weight 41,000, the apparent molecular weight of G~. Taken together our data also suggest that in rat parietal cells PGE 2 and somatostatin inhibit

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histamine-stimulated acid production via activation of Gi,. Although in rat, as in canine [7], parietal cells pertussis toxin failed to induce complete ADP-ribosylation of Gi,, the high degree of ADP-ribosylation is apparently sufficient to fully reverse inhibition of [taC]aminopyrine accumulation by PGE 2 and somatostatin. In contrast to the present data, reduction but not complete abolition of the effect of PGE 2 and somatostatin on histamine-stimulated acid production was demonstrated in previous studies using isolated rat gastric cells [10, 11]. Yet, in these studies crude cell preparations containing only 15-20% parietal cells were used. In these crude preparations pertussis toxin-insensitive inhibitors might be released from non-parietal cells and might overcome the pertussis toxin effect. In our enriched preparation the percentage of non-parietal cells is markedly reduced, thus making release of endogenous inhibitors at least much more unlikely. This might allow pertussis toxin to reverse completely inhibition by PGE 2 and somatostatin in the cell system tested here. Forskolin has been shown to stimulate directly adenylate cyclase [16]. Moreover, the diterpene derivative stimulates the catalytic sub-unit of the enzyme in the absence of Gs~ [22]. PGE 2 and somatostatin act on specific receptors on the cell surface, i.e. proximal to the catalytic subunit. Accordingly, one would have to expect that these inhibitors do not affect the response to forskolin. Yet, this hypothesis is not supported by the present data and by previous studies which show reduction of forskolin-stimulated H+-production by PGE 2 [7] and somatostatin [8]. These and the present data rather suggest that the action of forskolin on adenylate cyclase is at least in part dependent on regulatory GTP-binding proteins. Further evidence for this hypothesis comes from the observation that the enzymatic activity obtained with the combination of forskolin, Gs~, and the catalytic subunit exceeds that observed with forskolin and the catalytic subunit alone [23]. In line with this hypothesis pertussis toxin, by blocking the inhibitory limb,

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FIG. 7. Autoradiography of the identical gel as in Fig. 6. Effect of pertussis toxin on ADP ribosylation in crude membranes from enriched rat parietal (lanes 1, 2; 3) and from HIT-TI5 cells (lane 4). Lane numbers correspond to the pertussis toxin treatment of intact cells indicated in the legend to Fig. 6. For use in membranes pertussis toxin was preactivat~d in the presence of DTT and ATP. Crude membranes (100 ~tg protein/60 ~tl) were incubated in duplicate for 30 min at 30°C in the absence (lane 2) or presence of 1.7 ~tg/ml preactivated pertussis toxin (lanes 1 and 3) and 1.8 I~Ci [32p]NAD+. After the ribosylation reaction 100 ~tg of membrane protein was subjected tO [~Jl:lS-polyaeryl~de gel (10%) electrophoresis with subsequent autoradiography. One experiment representativ©~i0f a total ~ , = 5 similar experiments is shown.

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Pertussis toxin and rat parietal cell H+-production restores the activity of forskolin in the presence of PGE2 and somatostatin. In contrast to P G E 2 somatostatin inhibited H +-production following stimulation with dbcAMP in our study as well as in canine parietal cells [8]; this inhibition was insensitive to pertussis toxin in both cell preparations. Apparently, somatostatin reduces H÷-production only in part by inhibition of adenylate cyclase but also through additional mechanisms distal to the generation of cAMP. Similar observations have been made regarding the somatostatin effect in a pituitary tumour cell line [24]. As in parietal cells these distal mechanisms should be activated via H2-receptor mediated stimulation of adenylate cyclase, it is astonishing that in the present and in a previous study [8] pertussis toxin completely reversed the effect of somatostatin on the response to histamine. Presently we cannot explain these findings. On the other hand, pertussis toxininsensitive inhibition by somatostatin of the response to dbcAMP is in line with the observation that somatostatin is more effective in reducing the response to histamine than that to dbcAMP. In canine parietal cells somatostatin has been shown to reduce H÷-production in response to gastrin [8]. It is not possible to investigate this effect in isolated rat parietal cells since @ey do not respond to gastrin [25, 26]. ,~ In conclusion, the present data from rat parietal cells suggest that, similar to canine parietal cells, PGE 2 inhibits histamine-stimulated acid production entirely via activating Gi, while somatostatin acts by additional pertussis toxin-insensitive'mechanisms. Acknowledgements--This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Sche 229/224) to :W.S. The skilful technical assistance of CATaR~NTATGEand KERSTINDEn~ is gratefully acknowledged.

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Pertussis toxin reverses prostaglandin E2- and somatostatin-induced inhibition of rat parietal cell H(+)-production.

In enzymatically dispersed enriched rat parietal cells we studied the effect of pertussis toxin on prostaglandin E2 (PGE2)- or somatostatin-induced in...
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