Biochimica et Biophysica Act|, 1001 (1989) 191-195 Elsevier

191

BBA53020

Stirnulat]on of ]nos]to] phosphate and d]acylglycerol production by RHC 80267, a d]acylglycerol-fipase ]ntfib. tor, rat gastric

parietal eels: effects on hydrogen ion secretion

Andreas Pfeiffer, Reinhard Kopp and Helmut Rochlitz Medizin~sche Klinik 11, Klinikum Grosshadern, Ludwig Maximilians Universtt?it M~nchen, Munich

and Mav-PMn~k Institut /~r Psychiatrie, Abt. Neuropharmakoiogie, Martinsried (F. R. G.)

(Received 27 June 1988) (Revised manuscript received 5 October 1988)

l(ey words: DiacylglyceroI lipase; RHC 80267; lnositol phosphate production; (Rat gastric parietal cell)

RHC 80267, on inhibitor of diacy|g|ycerol ||pase, was used to |nvestigate the ro|e of diacy|g|ycerol in acid secretion by isolated rat gastric parietal ceaYs. Unexpecte¢||y, RHC ~267 stimYated the production of |nosmtom phosphates in [aH]inosito|-pre|abeled cei|s and increased |eve|s of 3ZPo|abeled phosphafidic acid to the same degree as did cafbachoL RHC 80267 increased diacylg|ycero| to a greater extent than d|d carbacho|, and additionally decreased |evels of [3El]arachidon|c acid. This suggests t~at RHC ~QA~267 stimulated phosphoHpase C and inhibited ~iacyig|ycerol ||pase in parietal cells. RHC |nh|bited ]a4C]aminopyrine uptake, a measure off acid secretion, stime|ated by carbacho| or by simdtaneous addition of carbacho| and dibutyryl-cAMPo These data support the model that t~e d|acy|g|ycero|/pr3tein kinase C branch of the phosphoinositide system is |nhihitory to acid secretion.

Introduction

Acid secretion of gastric parietal cells is stimulated by activation of either the cAMP system via histaminergic receptors or by activation of the phos- phatidylinositol system via cholinergic receptors [1,21. The phosphatidylinositol generates at least two second messengers upon activation of phospholipase C: In- ositol 1,4,5-triphosphate and sn-l,2-diacylglycerol [3,4]. The role of these two second messengers in mediating receptor-stimulated acid secretion is not deafly re- solved. There is a good correlation of inositol phosphate and proton production in isolaied parietal cells [5-8]. Regarding diacylglycerol, an i~xli~bition of aminopyrine uptake (an indirect measure of t'!roton production) upon stimulation of protein kinase C by phorbol esters or 1-oleoyl-2-acetylglycerol has been described [8,91. This inhibition was observed after histaminergic and cholinergic stimulation of acid secretion. On the other hand, increases in intracdlalar calcium have bet asso-

Abbreviations: dB-cAMP: dibutyryl cyclic AMP; Hepes, 4-(2-hy- droxyethyl)-l-piperazineethanesuifonic acid.

Correspondence: A. Pfeiffer, Medizinische Klinik II, glinikum GroBhadern, Marchioninistr. 15, D-8000 Munich 70, F.R.G.

elated with proton production and are like]y to result from an action of inositol 1,4,5-triphosphate on release of calcium from intracellular stores [10]. These results seem to indicate that the two limbs of the phosphati- dylinositol system exert opposite effects on acid secre- tion. However, since phorbol esters cause powerful and probably nonselective activation of protein ldnase C, the inhibition may represent a pharmacological phe- nomenon. We therefore searched for a tool to enhance cellular production of diacylglycerol.

Suthedand and Amin [11] developed on inhibitor of diacylglycerol lipase, RHC 80267, which was reported to be specific for this enzyme in dog and rat platelets. This compound also increased cellular, concentrations of diacylglycerol in rodent pancreatic minilobules in response to stimulation by cerulein without significant changes in phospholipids, while levels of arachidonic acid were decreased, as would be expected upon inhibi- tion of diacylglycerol lipase [12]. We therefore em- ployed RHC 80267 to enhance the level of di- acylglycerol and to inb, ibit the level of arachidonic acid in isolated rat gastric parietal cells in order to investi- gate their role in stimulus-response coupfing.

MaterfaJs and Methods

Enncked rat gastric parietal cells were prepared as described by Lewin et al. [13] with modifications as

0005-2760/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division~

192

detailed previously [7]. After isolation, parietal cells were enriched to a content of 70~ cell number by isopycnic centrifugation in 27~ percoll. The cells were incubated in buffer C (concentrations in raM): NaCI, 70; KCI, 5; Hepes, 50 (pH 7.4); sodium bicarbonate, 20; sodium phosphate, 1.5; bo':~ae serum albumin, 0.1%.

Determination of inositol phosphate The isolated cells were incul:~ated with 10 pCi/ml per

5.106 cells n!vo-inositol (15 Ci/mmol specific activity) for 120 rain at 36 °C in oxyge~,~ated buffer C to achieve labeling of the membrane lipi~,~. After washing, the cells were incubated in a volume of 0.8 ml in buffer C confining 10 mM LiCl at 1 106 celis/ml. Test com- pounds were added to the tubes in a total volume of 8 ~tl. The incubations were stop!3ed by adding an ice-cold mix of 2 ml methanol and 1 ml chloroform. The further extraction was performed as described [6,7] and phosphoinositides were as~yed according to Downes and Michell [14] by use of Dowex l-X8 1.ml column chromatography. The elution and recovery of inositol phospholipids were controlled using [~4C]inositol 1- phosphate as a standard [7].

Determination of phospholipids Isolated cells were labeled with 20-50 /~Ci/ml

[32p]orthophosphate in buffer C containing 0.15 instead of 1.5 mM "sodium phosphate for 60 min. Cells were then washed and incubated with test compounds in a final volume of 0.8 ml. Phospholipids were extracted as described in Ref. 12, After drying of the chloroform phase under nitrogen, the lipids were resuspended in 50 ~tl of a 2:1 mix chloroform/methanol, and 15 ~tl were analyzed by thin-layer chromatography on silica plates (Merck, Darmstadt, F.R.G.), according to Ref. 15. Phospholipids were stained with iodine and identified by comigration with standards and by autoradiography. Radioactivity in the spots was quantified in a fl-counter after scraping.

Determination of neutral lipids Isolated parietal cells were incubated in buffer C

containing 0.1% fatty acid-free bovine serum albumin for 60 rain at 36eC with 1 ~Ci/ml [SH]arachidonic acid (Amersham 225 Ci/mmol specific activity) which had previously been vortexed fcr 10 rain in 10 vol. 50 mM sodium carbonate. Uptake of arachidonic acid was 70~ of the total label added. After washing twice by centri- fugation and resuspension in fresh buffer, the cells were incubated with test compounds as described above. Incubations were ~topped by adding 3 ml ice-cold methanol/chloroform (2:1) contaimng 0.005~ buty- lated hydroxytoluene as antioxidant. After vortexing and standing on ice for at least 30 rain, another ml of chloroform and of medium was added and the two phases were separated by centrift,gation. The chloro-

form phase was saved, the upper phase washed with another ml of chloroform and the combined lower phases were evaporated under nitrogen. The lipids were analyzed according to Dixon and Hokin [12]. 1,2-Di- acylglycerol and arachidonic acid were identified by comigration with standards, scraped and ~uantified by liquid scintillation spectrophotometry with 4 ml of scin- tillation fluid (Szintigel, Roth, Darmstadt, F.R.G.). All values reported were expressed relative to control values run in parallel.

[14 C]A minopyrine The accumulation of [~4C]aminopyrine was used as

an indirect measure of acid production by the celJs [16]. 2 ml of cell suspension were incubated with 0.05 ~tCi/ml [14C]aminopyrine and the appropriate drugs at 36 °C for 75 rain in a horizontally positioned Eppendorf tube of 2.2 ml col and shaken at 180 cycles/rain. Incubations were terminated by placing 0.5 ml cell suspension on 1 ml cold buffer C, followed by centrifugation for 20 s in an Eppendorf table centrifuge. The cell pellet was surface-washed with I ml cold buffer, dissolved in 100 ~tl 0,5 M NaOH and countec~ in 4 ml scintillation fluid. Radioactivity in the supernatant wa~ also determined (100/~1 in 4 ml). The uptake was calculated according to Berglindh and co-workers [16] using the aminopyrine uptake ratio = pellet cpm/ (2 ~tl rag). (mg dry weight). (medium cpm per ~tl). The unstimulated aminopyrine uptake ratio was 0.91 + 0.40 and maximal uptake was 80 :t: 7 in the experiments shown ( n - 7). Nonspecific trapping was corrected by running parallel tubes con- talning 10 mM NaSCN, which blocks aminopyrine up- take, and subtracting this value.

RHC 80267, 1,6-di(-O-(carbamoyl)cyclohexanone oxime)hexane, was dissolved in ethanol and directly added to the incubation buffer at a final concentration of 0.5% ethanol. All other incubations also contained 0.5~ ethanol, which did not significantly affect inositol phosphate production or aminopyrine accumulation.

Materials All reagents were of analytical grade and were

purchased from sources previously identified [7]. Lipid standards were from Serva, Heidelberg, or from Sigma, TaufldrcLen, F.R.G. RHC 80267 was a kind gift from Dr. Charles A. Sutherland, Revlon Health Care Group, New York.

Data are presented as means ± S.E. from n indepen- dent experiments performed in triplicate. Statistical analysis was by analysis of variance or by Student's t-test for independent samples (Fig. 2).

Results

RHC 80267 is a known inhibitor of diacylglycerol iipase. In initial experiments, we monitored its effect on

inositot phospho!ipid hydrolysis by first labeling cellu- lax li#ds with [~H]inositol and then measuring the RHC 80267-induced accumulation of [aH]inositol mor:ophosphate in the presence of 10 mM lithium. I.i*.hiu~ was added to inhibit inositol phosphate de- phosp~ ~rylation [17]. RHC a0267 caused a dose-depen- dent i~:,croase in inositol monophosphate accumulation which ~indicates activation of phospholipase C (Fig. 1). The f~gure also shows the response to a maximally effective dose of carbachol (0.1 mM), which was com- parable to the maximal response elicited by RHC 80267. In a second set of four independent experiments, the incre~se in inositol monophosphate above basal levels caused by carbachol was 239 ± 25%, and that by 1000 #M LHC 80267 was 206 ± 28%, measured 10 rain after additon of the drugs. When both compounds were added together, there was only a slight further enhance- ment of inosi~ol monophosphate accumulation, indicat- ing that the effects of RHC 80267 and carbachol were not additive (Fig. 1). However, whereas atropine (1 #M) completely suppressed t h e response to carbachol, i t did not affect the ~.esponse to RHC 80267 (data not shown).

To assess whether RHC 80267 caused breakdown of phosphatidylinositol 4,5-bisphosphate, we also de- termined the production of polyphosphoinositides elut- ing at 1 M concentrations of ammonium formate from the ion exchange columns. Carbachol (0.1 mM) caused an increase by 98 + 19 @m/million cells (n = 8, P < 0.01) and RHC 80267 (0.1 raM) caused on increase by 63 + 31 dpm/rmI5¢n cells (n = 8, P < 0.05) over con- trol values, determined 10 rain after application of the compounds. This increase is comparable to that ob- served earlier [7], and suggests that breakdown of poly- phosphoinositides does occur.

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Fig. 1. Dose-response curve for stimulation of inositoi phospha*:e production by RHC 80267. Incubations (n = 3) were performed f~r 30 rain at 36 o C in the presence of 1O mM I,iCl. Open circles show the effect of RHC 80267. Closed citrics show the effect o¢ 0.1 mM

carbachol either alone or in the presence of 200 pM RHC 80267.

193

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Fig. 2. Effect of RHC 80267 (100 t~M), dB-cAMP (100 #M) and of ezrbachol (100 #M), either alone or combined, on levels of di- acylglycerol. Numbers in the bars indicate numbers of independent experiments. Basal levels were 1369+ 146 cpm/mill ion cells (n = 6).

*P < 0.05 compared to dB-cAMP or carbachoi alone.

The effect of RHC 80267 on levels of diacylglycerol was monitored in cells prelabeled with [3H]arachidonic acid. Incubation with 0.1 mM RHC 80267 caused a small but reproducible increase in diacylglycerol above the baseline, which amounted to 20 ± 5.4% after 1 rain, 25 _+ 4.0% after 10 nfin and 62 ± 9.3% after 30 rnin (n = 3-5 separate experiments). After 30 rain, the in- crease in diacylglycerol by 0.1 mM carbachol was 17 ± 9% above the baseline, which was significantly different from the effect of RHC 80267 (DF = 4, T = 3.25, P < 0.05). The effect of RHC 80267 was not significantly altered in the presence of 0.1 mM dibutyryl cAMP (dB-cAMP), whereas ~he effect of carbachol was en- hanced significantly (Fig. 2).

Since RHC has been reported to specifically inhibit diacylglycerol lipase, which generates vachidonic acid by cleavage of diacylglycerol, we also measured levels of arachidonic acid. The medium contained 0.17o fatty acid-free albumin, which is known to extract and equi- librate with the cellularly produced arachidon~c acid [12]. In the presence of RHC, levels of arachidonic acid were decreased to approx. 50% of baseline levels (Fig. 3). Carbachol (0.1 mM) did not affect arachidonic acid, since levels measured after 1 or 30 rrdn of incubation were similar to control values (Fig. 3). The effect of RHC 80267 was not altered in the presence of either carbachol or dB-cAMP (Fig. 3).

Stimulation of inositol phospholipid turnover is usu- ally accompanied by a rapid increase in phosphatidic acid, which is partly generated by phosphorylation of the diacylglycerol generated by activation of phos- pholipase C. The increase in [32 P]phosphatidic acid was monitored as an independent index of phospholipid turnover. RHC 80267 and carbachol both caused rapid inc,oeases in phosphaddic acid which lasted for at least 30 Kiln (Fig. 4).

The upte.ke of [~4C]aminopyrine by parietal cells was measured as an int',ex of acid secretion and was ex-

194

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t ~'~ 40 eE ~1 OCAROACHOL 0.1 mM

@lIMe 80 |07 0.1 mM 2 0 zgHC CO2@? O.'l mM* CAflIIACHOL 0.1 mM

[]RHC eo|61r o.1 mM* dO OAMP 0.1 IIM

' , i .,'o e+b " " 8o role (t)

Fis. 3. Time-course of the effect of RHC 80267 alone or in the presence of cadmchol (100 tiM) or dB-cAMP (100 FM), and of carbachol alone, on levels of [~H]arachidonic acid. Basal levels of [3 H]arachidonic acid were 6356 :J: 621 cpm/million cells ( . ,- 4). After prelabeling the cells with arachidonic acid, incubations were per- formed for the times indicated in the presence of the aSen~ specified on the figure. Incubations were stopped and lipids ext,'~ted as

described under Materials and Methods,

pressed ~s the aminopyrine ratio, i.e,, the concentration of aminopyrine in the cells over the concentration pre- sent in the extracellular medium [16]. RHC 80267 did not alter the basal ~nopyr ine ratio of 0.91 4. 0.40 ( n - 4 ) at doses between 0.1-100/~M. Carbachol (0.1 raM) increased &minopyrine uptake from 0.91 to an aminopyrine ratio of 6.6 + 0.9 after 75 rain. This re. sponse was inhibited dose-dependently in the presence of 1-100/~M RHC 80267 to an aminopyrine ratio of 3.1 4. 0.8 at 100 ~M (n ffi 6, P < 0.01). The synergistic stimulation by earbachol and dB-cAMP resulted in an aminepyfine-ratio of 80 4. 7, which was inhibited dose- dependendy in the pre.senc~,', of 1-100/AM RHC to a minimal aminopyrine ratio of 45 4. II at IO0/xM IU-IC 80267 (n -" 7, P < 0.025),

O 1SO "Xl !l,so 0 3

1 10 ~0 3 0 min (t)

Fig+ 4. Tmte-course of the effect of carbachol and of RHC 80267 on levels of phosphatidic acid. Basal cpm in the [32P]phosphatidic acid spot were 1125, 1994 and 3602 cpm/milfion cells in the experhnents

sl~'.wn.

Discussion

We report a stimulation of inositol phosphate pro- duction by the diacylglycerol lipase inhibitor RHC 80267 in isolated parietal cells. The accumulation of inositol phosphate in the presence of LiCl indicates that RHC 80267 activates a cleavage of phosphatidylinositol or o~" phosphatidylinositol polyphosphates by phospholipase C. The increase in inositol phosphate accumulation by RHC 80267 was maximally 885 of the response stimu- lated by carbachol. The simultaneous addition of both compounds caused no further substantial increase in inositol phosphate production. RHC 80267 thus caused a near maximal stimulation of inositol phosphate breakdown and the effects of both agents were not additive. The effects of RHC 80267 were not blocked by atropine, suggesting that muscarinic receptors were not activated.

RHC 80267 ~ d carbachol both caused similar in- creases in phosphatidic acid. This lipid is mainly de- rived from phosphorylation of diacylglycerol and may be regarded as an indirect marker of phospholipid de- gradation resulting in the production of diacylglycerol. The responses were apparen~: within 1 rain after ad- dition of both compounds demonstrating a rapid effect of both, ¢arbachol and RHC, on phospholipase C. Based on these data it is, however, not possible to decide whether RHC 80267 activates phospholipase C by a direct reaction or via some intermediary metabolic process in parietal cells.

RHC 80267 has originally been described as a specific inhibitor of diacylglycerol lipase [11] in canine and rat platelets, which only marginally inhibited rat platelet phosphofipase A 2 at a concentration of 100 pM and did not inhibit a soluble rat platelet phospholipase C at a concentration of 300 pM. This finding has been con- firmed by Dixon and Hokin [12], who showed a selec- tive increase in diacylglycerol levels and a decrease in arachidonic acid without changes in monoacylglycerol, triacylglycerol or phosphofipids in pancreatic minilob- ules. Reports on the effect of RHC 80267 in intact human platelets have been contradictory. While Bishop and Bell [19] and Chau and Tai [20] observed inhibition of diacylglycerol lipase in intact platelets, Bross and co-workers [21] reported that this drug was without effect. Ogiesby and German [22] observed inhibition of diacylglycerol lipase, cyclooxygenase and of phos- phatidylchofine hydrolysis at a concentration of 250 +~M RHC 80267 in intact human platelets. The differences between results obtained in rat and human platelets may relate to species-specific differences. However, as our results show, RHC 80267 also exerts some effects unrelated to inhibition of diacylglycerol lipase in rat cell preparations. Although RHC 80267 did not affect the activity of p~osphofipase C in rat platelet supernatants [11], it may directly activate the membrane-associated

enzyme in rat parietal cells. Another possibility is that RHC 80267 activated phosphohpase C by disturbing the lipid bilayer or through other indirect means. The precise mechanism by which phospholipase C is activated remains to be elucidated.

In the present experiments, we observed an increase in levels of diaeylglycerol and a decrease in arachidonic acid consistent with inhibition of diacylglycerol lipase by RHC 80267. In the same experiments, we observed no change in levels of arachidonic acid upon incubation with carbachol.

The accumulation of aminopyrine by parietal cells was measured as an indirect index of acid secretion [16]. RHC 80267 did not stimulate amJnopyrine accumula- tion when added alone. RHC 80267 reproducibly inhibited responses to carbaehol and to simultaneous stimulation by carbachol and riB-cAMP. It has been shown that agents activating protein kinase C inhibit acid secretion [8,9]. The inhibitory effect of RHC 80267 on aminopyfine accumulation, therefore, may be explained by an activation of protein ldnase C due to the increased levels of diacylglycerol. The net effect of RHC 80267 on acid secretion is likely to result from the balance of inositol 1,4,5-triphosphate and diacylglycerol formation, with the former being stimulatory :and the latter being inhibitory to acid secretion. Since RHC 80267 did not show a reproducible stimulatory effect on aminopyrine accumulation, the enhanced activation of protein kinase C may have been sufficient to counter the stimulatory effect of inositol 1,4,5-tr/phosphate for- mation.

Arachidonic acid and its metabolites were recently proposed to act ~s second messengers in signal trans- duction [23,24]. The inhibitory effects of RHC 80267, particularly on carbachol-stimulated aminopyri~e accu- mulation, may therefore relate to the decrease in arachidonic acid caused by the compound. The pleio- tropic effects of RHC 80267, however, suggest caution in the interpretation of results obtained with this com- pound.

Acknowledgements

This work was supported by the Deutsche For- schungsgemeinschaft, Pf 164/3-4. The authors are in-

195

debted to Prof. A. Herz and to Prof. G. Pam ngartner for their support and encouragement.

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