Inhibition of Carbachol Stimulated Acid Secretion by Interleukin 1Β in Rabbit

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782 Gut 2001;48:782–789 Inhibition of carbachol stimulated acid secretion by interleukin 1â in rabbit parietal cells requires protein kinase C Gut: first published as 10.1136/gut.48.6.782 on 1 June 2001. Downloaded from

I L P Beales, J Calam

Abstract secreting enterochromaYn-like (ECL) cell.6 Background—Interleukin 1â (IL-1â)isa Expression of IL-1â in the gastric mucosa is potent inhibitor of gastric acid secretion. increased by inﬂammation, infection, and Regulatory actions at several levels have ulceration7 and it is possible that this local pro- previously been demonstrated, including duction is important in local regulation of acid direct inhibition of parietal cell acid secretion. Increased IL-1â production is seen secretion. Although IL-1â may activate at the edges of gastric ulcers and it may be that several intracellular signalling pathways, inhibition of acid secretion is an important part the mechanisms responsible for inhibition of the repair mechanism by limiting further of carbachol stimulated acid secretion damage.89 have not been determined. Previous studies have shown complex regu- Aims—To investigate the roles of protein lation of parietal cell acid secretion by IL-1â. kinase C (PKC) and the sphingomyelinase Inhibition of histamine stimulated acid secre- signalling pathways in the regulation of tion occurred via a tyrosine kinase and pertus- acid secretion by IL-1â. sis toxin sensitive inhibitory G protein depend- 3 Methods—Rabbit parietal cells were ob- ent pathway reducing cAMP generation. tained by collagenase-EDTA digestion However, the inhibitory actions of IL-1â and centrifugal elutriation. Acid secretion against carbachol stimulated acid secretion stimulated by carbachol and A23187 (to seemed to be independent of these mecha- 3 mimic elevations in intracellular calcium) nisms. A variety of studies have shown that was assessed by 14C aminopyrine uptake in protein kinase C (PKC) has important inhibi- 10 11 response to IL-1â, PKC, and sphingomy- tory roles in parietal cells, and that the elinase manipulation. sphingomyelinase/ceramide signalling pathway Results—IL-1â inhibited carbachol and may mediate important biological eVects of 12 13 A23187 stimulated acid secretion in a dose IL-1â. Therefore, the role of these two http://gut.bmj.com/ dependent manner. The inhibitory actions messenger systems in mediating the inhibitory were completely reversed by each of three actions of IL-1â against carbachol stimulated diVerent PKC inhibitors, staurosporine, acid secretion in cultured rabbit parietal cells H-7, and chelerythrine, as well as by PKC were examined. depletion with high dose phorbol ester pretreatment. IL-1â did not downregulate

parietal cell muscarinic receptor. IL-1â Methods on October 2, 2021 by guest. Protected copyright. signiﬁcantly increased membrane PKC PARIETAL CELL PREPARATION activity. Activation of the Rabbit parietal cells were isolated from New sphingomyelinase/ceramide pathway had Zealand white rabbits and enriched using pre- no eVect on basal or stimulated acid viously described methods.14 Gastric fundic secretion. The inhibitory action of IL-1â mucosa was digested with sequential exposure was independent of protein kinase A and to collagenase (type I 0.175 g/l with type H protein kinase G activity. 0.175 g/l) and EDTA. Parietal cells were Conclusions—IL-1â directly inhibits pari- enriched from the crude suspension using a Department of Gastroenterology, etal cell carbachol stimulated acid secre- Beckman JE 5.0 elutriator rotor using the Royal Postgraduate tion. This action occurs distal to standard elutriation chamber. Parietal cells Medical School, muscarinic receptor activation and eleva- were enriched to >70% homogeneity and Hammersmith tions in intracellular calcium and requires >98% viability, as determined by trypan blue Hospital, Du Cane PKC. exclusion. Road, London (Gut 2001;48:782–789) W12 0NN, UK I L P Beales Keywords: carbachol; gastric acid; interleukin 1 ; J Calam â Abbreviations used in this paper: BSA, bovine serum parietal cell; protein kinase C; sphingomyelinase albumin; CHL, chelerythrine; db-cGMP, guanosine 3', 5'- Correspondence to: cyclic monophosphate; DMEM, Dulbecco’s modiﬁed Dr I L P Beales, Department Eagle’s medium; EBSS, Earle’s balanced salt solution; of Gastroenterology, James Interleukin 1â (IL-1â) is a potent inhibitor of EGTA, ethylene glycol-bis(beta-aminoethyl Paget Hospital, Lowestoft gastric acid secretion.1 The mechanisms medi- ether)-N,N,N',N'-tetra acetic acid; EGF, epidermal Road, Gorleston, Great growth factor; ECL, enterochromaYn-like; HBSS, Yarmouth, Norfolk NR 31 ating this acid inhibitory eVect are complex; the 6LA, UK. cytokine acts centrally to inhibit acid secretion2 Hanks’ balanced salt solution; HEPES, [email protected] N-2-hydroxyethylpiperazine-N'-ethansulphonic acid; but in vitro studies have also shown that IL-1â IL-1â, interleukin 1â; MLCK, myosin light chain kinase; Accepted for publication has inhibitory actions against both the acid PKA, protein kinase A; PKC, protein kinase C; PKG, 19 December 2000 secreting parietal cell3–5 and the histamine protein kinase G; PMA, phorbol-12-myristate-13-acetate.

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CELL CULTURE cells were solubilised by incubating in 1% Harvested cells from the parietal cell enriched Triton X-100 and bound and unbound radio- fractions were collected by brief centrifugation, activity was assessed by liquid scintillation pooled, and resuspended in complete culture counting. [3H]Methyl-scopolamine binding in medium (Ham’s F12/Dulbecco’s modified the presence of 100 mM atropine was regarded Gut: first published as 10.1136/gut.48.6.782 on 1 June 2001. Downloaded from Eagle’s medium (DMEM) 50/50 nutrient mix, as non-specific binding and these values were containing 10% heat inactivated fetal calf subtracted from total binding to obtain values serum, 10 mM N-2-hydroxyethylpiperazine- for specific binding. N'-ethansulphonic acid (HEPES), pH 7.4, 100 mg/l gentamicin, 100 mg/l streptomycin, 100 MEASUREMENT OF PKC ACTIVITY mg/l penicillin, 2 mM glutamine, 8 µg/ml PKC activity in parietal cell membranes was hydrocortisone, and 1 µg/ml insulin) and measured using a standardised assay kit cultured by plating 0.5–1.0×106 cells/well onto (Amersham protein kinase C enzyme kit)17 24 well Matrigel coated tissue culture plates based on phosphorylation of a synthetic (Corning). The Matrigel coated plates had peptide substrate by PKC. Isolated parietal been prepared by diluting Matrigel 1:7 with cells (107 cells) in 1 ml of EBSS containing 14 sterile water and uniformly coating the wells. HEPES (10 nmol/l), NaHCO3 (0.22%), and The parietal cell enriched fraction was cultured BSA (0.1%) were incubated for five minutes at ° ° at 37 C in an atmosphere of 5% CO2/95% air 37 C with IL-1â (10 ng/ml), epidermal growth for 40 hours. factor (EGF) (100 nM), or PMA (100 nM). Ice chilled phosphate buVer saline (1 ml) was MEASUREMENT OF ACID SECRETION added to quench the reaction and cells were Intracellular accumulation of 14C aminopyrine immediately centrifuged. Membranes were was used as a measure of functional acid secre- prepared by resuspending cells in 1 ml of soni- tory activity.14 Cultured cells in 24 well plates cate buVer (50 mM Tris HCl, pH 7.5, 5 mM were washed once with 2 ml of Earle’s balanced EDTA, 10 mM ethylene glycol-bis(beta- salt solution (EBSS) containing 0.1% bovine aminoethyl ether)-N,N,N',N'-tetra acetic acid serum albumin (BSA), 10 mM HEPES, pH (EGTA), 0.3% â-mercaptoethanol, 10 mM

7.4, 2 mM glutamine, and 0.22% NaHCO3 to benzamidine, and 25 mg/ml phenylmethyl- remove dead and non-adherent cells. There- sulphonyl ﬂuoride) on ice and sonicated twice after, 1 ml of the above medium was added, for 15 seconds. Sonicates were centrifuged at and 0.1 µCi aminopyrine and the stimulant 1000 g for ﬁve minutes at 4°C. Supernatants substances (carbachol and A23187) were were centrifuged at 12 000 g for 25 minutes at added simultaneously to each well. Cells were 4°C. The membrane containing pellets were incubated for 30 minutes at 37°C in an atmos- resuspended in sonicate buVer and stored at ° phere of 5% CO2/95% air. Incubations were −70 C. Total PKC activity in membranes

terminated by removing the medium from each (30–60 mg protein) was measured in a final http://gut.bmj.com/ well using a vacuum pump and washing twice volume of 100 ml containing 50 mM Tris HCl, with 1 ml of EBSS solution. Cells were lysed 0.05% sodium azide, pH 7.5, 24 mg/ml PMA, with 1 ml of 1% Triton X-100. Aliquots of cell 900 mM peptide, 300 mM dithiothreitol, 150 lysates and incubation media were counted in mM adenosine triphosphate, 45 mM magne- Optiphase Safe (Wallac, Milton Keynes, UK) sium acetate, and 1×106 counts/min of using a Beckman LS 1801 liquid scintillation ã-[32P]adenosine triphosphate (Amersham spe- counter with DPM correction. Dinitrophenol cific activity 1.66 mCi/ml). The reaction (0.1 mM) was added to separate wells to assess mixture was incubated for 15 minutes at 25°C on October 2, 2021 by guest. Protected copyright. non-specific incorporation and values were and stopped by adding 100 ml of stop solution. subtracted from test values.15 IL-1â or guanos- A sample of the final reaction mixture (125 ml) ine 3', 5'- cyclic monophosphate (db-cGMP) was pipetted onto binding paper (2.5×2.5 cm). was added to the wells 15 minutes prior to the Phosphorylated peptide was separated onto initial washing step before adding aminopyrine binding paper. The paper was washed twice and the stimulants. PKC inhibition was with 5% acetic acid and phosphorylation was aVected by preincubating parietal cells for 24 detected by scintillation counting. hours with phorbol-12-myristate-13-acetate (PMA) (500 nM) or PKC inhibitors for 60 CHEMICALS AND DRUGS minutes prior to assessing acid secretion.316 Carbachol, A23187, atropine, staphylococcal sphingomyelinase, and recombinant human RECEPTOR BINDING STUDIES IL-1â were purchased from Sigma (Poole, Muscarinic receptor binding studies were per- UK). Recombinant human EGF, db-cGMP, formed using a previously described method H-8, H-89, and C2-ceramide were from with modifications.17 Briefly, cells on Matrigel Calbiochem (Nottingham, UK). Stau- coated 12 well plates (1×106/well) were incu- rosporine, H-7, and chelerythrine (CHL) were bated with IL-1â (10 ng/ml) for 15 minutes from LC laboratories (Nottingham, UK). and washed three times with Hank’s balanced Triton X-100 and HEPES were from BDH- salt solution (HBSS) containing 0.1% BSA to Merck (Poole, UK).14C-dimethylamine- remove non-adherent cells and incubated at aminopyrine (103 mCi/mmol) and [3H]n- 37°C with [3H]n-methyl-scopolamine (specific methyl-scopolamine (64 Ci/mmol ) were activity 64 Ci/mmol, 100 fmol/sample) in 0.5 obtained from Amersham International (Am- ml of HBSS-0.1% BSA and increasing concen- ersham, UK). Ham’s F12/DMEM (50:50 trations of atropine for 60 minutes. Cells were vol/vol), glutamine, HBSS, basal medium washed twice with ice chilled HBSS. Adherent Eagle’s, and fetal calf serum were obtained

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from Gibco (Paisley, UK). Basement mem- A * * brane Matrigel was from Universal Biologicals 160 * (London, UK.). All other reagents were from Sigma. 120 † Gut: first published as 10.1136/gut.48.6.782 on 1 June 2001. Downloaded from 80 STATISTICAL ANALYSIS 40

All data are presented as mean (SEM) of 3–7 basal) (% above

separate cell preparations. Data were compared Aminopyrine uptake 0 using one way analysis of variance and the Stu- + Carbachol 100 µM dent’s t test to determine signiﬁcance. A p value + IL-1β 10 ng/ml of <0.05 was regarded as signiﬁcant. +SP +H-7 +CHL

Results 120 B * EFFECT OF IL-1â ON STIMULATED ACID 100 ** SECRETION 80 Carbachol eVectively stimulated acid secretion † in cultured rabbit parietal cells; 100 mM 60 increased acid secretion by 155 (10)%. The 40 calcium ionophore A23187 (1 mM), used to 20 (% above basal) (% above

mimic post-receptor signalling (elevations in Aminopyrine uptake 0 2++ [Ca ]i) initiated by carbachol, was also an + A23187 1 µM eVective stimulant of acid secretion, increasing acid secretion by 80 (15)%. IL-1â inhibited the + IL-1β 10 ng/ml stimulatory activities of both carbachol and +SP +H-7 +CHL A23187. Maximal inhibition of acid secretion was seen with 10 ng/ml IL-1 (carbachol by 36 Figure 2 EVect of protein kinase C inhibitors on â interleukin 1â (IL-1â) inhibition of carbachol (A) and (4)%, A23187 by 38 (4)%) and this concentra- A23187 (B) stimulated parietal cell acid secretion. Parietal tion was used for further studies (fig 1). cells were incubated with staurosporine 100 nM (SP), H-7 100 nM (H-7), or chelerythrine 10 mM (CHL) for 60 minutes prior to exposure to IL-1â 10 ng/ml for 15 minutes. EFFECT OF PKC MODULATION ON IL-1â Results are expressed as per cent aminopyrine uptake above INHIBITION OF ACID SECRETION unstimulated basal over 30 minutes (mean (SEM), n=7). The three diVerent PKC inhibitors had no †p<0.05 v stimulant alone, *p<0.05 v stimulant+IL-1â. eVect on basal or stimulated acid secretion. All three agents (staurosporine (100 nM), H-7 (100 nM), and CHL (10 mM)) abolished the inhibitory eVects of IL-1 against carbachol â http://gut.bmj.com/ 200 A and A23187 stimulated acid secretion (fig 2). CHL reversed inhibition of carbachol stimu- 150 lated acid secretion by IL-1â in a dose depend- * * ent manner (fig 3). 100 Pretreatment with high dose PMA (500 nM for 24 hours) to deplete cellular active PKC 50 itself caused a small decrease in stimulated acid (% above basal) (% above on October 2, 2021 by guest. Protected copyright.

Aminopyrine uptake secretion but completely abolished the inhibi- 0 tory eVects of IL-1â against carbachol and + Carbachol 100 µM A23187 stimulated acid secretion (ﬁg 4). + IL-1β (ng/ml) 0.1 1.0 10 100 200 100 B * * 160 * * 120

* * 80 50 40 (% above basal) (% above

Aminopyrine uptake 0

(% above basal) (% above + Carbachol 100 µM Aminopyrine uptake 0 + IL-1β 10ng/ml + A23187 1 µM + CHL (_log M) β + IL-1 (ng/ml) 1098765 0.1 1.0 10 100 Figure 3 Dose response relationship of the protein kinase Figure 1 EVect of increasing concentrations of interleukin C inhibitor chelerythrine (CHL) against interleukin 1â 1â (IL-1â) on parietal cell acid secretion stimulated by (IL-1â) inhibition of carbachol stimulated acid secretion. carbachol (A) and the calcium ionophore A23187 (B). Parietal cells were incubated with increasing concentrations Parietal cells were incubated with IL-1â for 15 minutes of CHL for 60 minutes prior to exposure to IL-1â (10 before stimulation of acid secretion. Results are expressed as ng/ml) for 15 minutes. Acid secretion was then stimulated per cent aminopyrine uptake above unstimulated basal over by carbachol 100 µM for 30 minutes. Results are expressed 30 minutes (mean (SEM), n=4). *p<0.05 compared with as per cent above unstimulated basal (mean (SEM), n=3). appropriate agonist stimulated control. *p<0.05 v carbachol+IL-1â.

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200 A Agonist alone Agonist + CHL 150 30 * *

* Gut: first published as 10.1136/gut.48.6.782 on 1 June 2001. Downloaded from 100 cells 6

50 20 * (% above basal) (% above

Aminopyrine uptake 0 µ + Carbachol 100 M 10 + IL-1β + IL-1β † † † † PMA pretreatment

PKC activity/pmol/10 0 β 100 B Control PMA EGF IL-1

80 Figure 6 EVect of phorbol ester (PMA 100 nM), * epidermal growth factor (EGF 100 nM), interleukin 1â 60 (IL-1â) (10 ng/ml), and chelerythrine (CHL 10 mM) on protein kinase C (PKC) activity in parietal cell 40 membranes. Parietal cells were stimulated with agonists for ﬁve minutes before membrane PKC activity was assessed. 20 Parietal cells were exposed to CHL for 60 minutes prior to (% above basal) (% above adding other agonists. Results are expressed as pmol/106

Aminopyrine uptake 0 µ cells (mean (SEM), n=4). *p<0.05 compared with basal, + A23187 1 M †p<0.05 compared with agonist stimulated PKC activity in the absence of CHL. + IL-1β + IL-1β PMA pretreatment PMA (by 29 (7)%) and EGF (by 40 (4)%) Figure 4 EVect of protein kinase C depletion on interleukin 1â (IL-1â) inhibition of carbachol (A) and were eVective in inhibiting carbachol stimu- A23187 (B) stimulated parietal cell acid secretion. Parietal lated acid secretion (fig 7). CHL (10 µM) cells were incubated with the active phorbol ester, abolished basal and agonist stimulated mem- phorbol-12-myristate-13-acetate (PMA) 500 nM for 24 hours before acid secretion with or without IL-1â (10 brane PKC activity (fig 6) and reversed the ng/ml) was assessed. Results are expressed as per cent acid inhibitory action of IL-1â as well as PMA aminopyrine uptake above identically treated unstimulated and EGF (fig 7). controls (mean (SEM), n=4). *p<0.05 compared with agonist stimulated acid secretion in the absence of IL-1â.

3 EFFECT OF MANIPULATION OF PROTEIN KINASES A EFFECT OF IL-1â ON [ H]METHYL-SCOPOLAMINE ANDGONIL-1âINHIBITION OF ACID SECRETION BINDING TO PARIETAL CELLS Addition of the cell permeable cGMP analogue

Pretreatment of parietal cells with IL-1 in an http://gut.bmj.com/ â db-cGMP (100 µM) inhibited carbachol acid inhibitory concentration (10 ng/ml) did stimulated acid secretion by 25 (4)%. This was not alter binding to the muscarinic receptor (fig completely ameliorated by pretreatment with 5). H-8 (0.1 µM) (fig 8). This concentration of H-8 had no eVect on the inhibitory action of EFFECT OF IL-1â ON PARIETAL CELL MEMBRANE IL-1â (fig 8). The protein kinase A (PKA) PKC ACTIVITY inhibitor H-89 (10 nM) had no eVect on basal PKC activity in parietal cell membranes was or carbachol stimulated acid secretion. Simi- on October 2, 2021 by guest. Protected copyright. increased by acid inhibitory concentrations of larly, H-89 did not alter the inhibitory actions IL-1â. PKC activity was increased by 50 (6)%. of IL-1 or db-cGMP (fig 8). This eVect was less than that seen with the two â positive controls, the phorbol ester PMA 100 nM and EGF 100 nM (fig 6).17 200 † † 160 120 _ IL-1β * 120 β * 100 + IL-1 80

80 40 (% above basal) (% above

Aminopyrine uptake 0 60 + Carbachol 100 µM + EGF + PMA 40 +CHL +CHL 20 Figure 7 EVect of protein kinase C activation with phorbol ester (PMA) and epidermal growth factor (EGF) on carbachol stimulated acid secretion. Parietal cells were H] scopolamine binding (%maximum) 0 3

[ C 11 10 9 8 7 6 incubated with PMA or EGF (both 100 nM) for 15 Atropine (_log M) minutes before stimulation of acid secretion. Chelerythrine (CHL) 10 mM was added 60 minutes prior to protein Figure 5 EVect of interleukin 1â (IL-1â) (10 ng/ml) on kinase C activators. Results are expressed as per cent [3H]methyl-scopolamine binding to parietal cells. Parietal aminopyrine uptake above unstimulated basal over 30 cells were pretreated with IL-1â for 15 minutes before 60 minutes (mean (SEM), n=3). *p<0.05 compared with minutes incubation with [3H]methyl-scopolamine and carbachol stimulated control, †p<0.05 compared with eVect increasing concentrations of atropine (mean (SEM), n=3). of EGF or PMA in the absence of CHL.

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200 A EFFECT OF ACTIVATION OF THE † SPHINGOMYELINASE/CERAMIDE PATHWAY 160 Exogenous sphingomyelinase and the cell wall 120 * permeable C2 ceramide had no eVect on basal, or A23187 or carbachol stimulated acid secre- Gut: first published as 10.1136/gut.48.6.782 on 1 June 2001. Downloaded from 80 tion. Similarly, no enhancement or reversal of 40 the inhibitory eVects of IL-1â were apparent (% above basal) (% above with either agent (ﬁg 9). Biological activity of Aminopyrine uptake 0 µ C2 ceramide and sphingomyelinase was con- + Carbachol 100 M ﬁrmed by the ability to induce interleukin 8 + IL-1β 10 ng/ml production in cultured AGS cells16 18 +CHL +H-8 +H-89 TOXICITY Morphology and toxicity were assessed in par- 200 B † allel experiments with all agents used. No 160 alteration in parietal cell morphology, or * 120 impairment of cell viability, as assessed by trypan blue exclusion or lactate dehydrogenase 80 release,19 was seen under any experimental 40 condition, either following the 30 minute (% above basal) (% above stimulation period or after washing the cells Aminopyrine uptake 0 and continuing culture in complete culture + Carbachol 100 µM medium for a further 24 hours. + db-cGMP 100 µM Discussion +CHL +H-8 +H-89 This study demonstrates that IL-1â directly Figure 8 EVect of protein kinase inhibitors on modulation inhibits acid secretion by isolated cultured of acid secretion in parietal cells. The protein kinase C parietal cells. The mechanism is dependent on (chelerythrine, CHL 10 µM), protein kinase G (H-8 0.1 PKC and the inhibitory action is at a point dis- µM), and protein kinase A (H-89 10 nM) inhibitors were added 60 minutes prior to exposure of parietal cells to either tal to the generation of increased intracellular interleukin 1â (IL-1â) (A) or guanosine 3', 5'- cyclic calcium as a second messenger. IL-1â eVec- monophosphate (db-cGMP) (B) and stimulation of acid tively inhibited both carbachol and A23187 secretion with carbachol (mean (SEM), n=4). *p<0.05 compared with carbachol stimulated acid secretion, induced acid secretion showing that the inhibi- †p<0.05 compared with eVects of IL-1â or db-cGMP. tory action against carbachol stimulated acid secretion is unlikely to be mediated by inhibit- 2+ ing rises in [Ca ]i. These data are consistent

with the ﬁnding that IL-1â did not inhibit http://gut.bmj.com/ muscarinic receptor binding at concentrations 300 A which were eVective at inhibiting acid secre- 250 tion, and thus the inhibitory action of IL-1â 200 does not appear to involve direct inhibition of either receptor binding or early signal trans- 150 duction. 100 The inhibitory actions of IL-1â were blocked on October 2, 2021 by guest. Protected copyright. 50 by manoeuvres which inhibited PKC. Three

Aminopyrine uptake 0 diVerent inhibitors all eVectively blocked the (% basal unstimulated) Basal + Carbachol 100 µM action of IL-1â. Depletion of cellular PKC with high dose phorbol ester was similarly + SMase + C2 ceramide eVective, confirming the essential nature of this (u/ml) (nM) _ 5 _ 4 _ 3 pathway in the inhibitory actions of IL-1â. 10 10 10 1 10 100 IL-1â increased the activity of PKC in parietal 250 B cell membranes. Increased membrane associated activity represents translocation of inac- 200 tive cytosolic enzyme to the cytoplasmic leaflet 150 of the plasma membrane and subsequent activation early in receptor mediated signalling 100 pathways.17 This increase in kinase activity fur- 50 ther supports the role of PKC in mediating the inhibitory eVects of IL-1â. Aminopyrine uptake 0 (% basal unstimulated) The PKC inhibitor CHL abolished both Basal + A23187 1 µM IL-1â inhibition of acid secretion and IL-1â + SMase + C2 ceramide stimulation of membrane PKC activity. CHL (u/ml) (nM) also abolished the increase in membrane asso- _ _ _ 10 5 10 4 10 3 1 10 100 ciated PKC activity and the acid inhibitory actions of the two positive controls (PMA and Figure 9 EVect of activation of the sphingomyelinase signalling pathway on carbachol (A) and A23187 (B) EGF). This strongly suggests that increased stimulated acid secretion. Parietal cells were incubated with PKC activity is essential for inhibition of acid increasing concentrations of sphingomyelinase (SMase) or secretion. C2 ceramide for 15 minutes prior to assessing acid secretion. Results are expressed as per cent unstimulated While these kinase inhibitors are not abso- basal aminopyrine uptake (mean (SEM), n=4). lutely specific for PKC,17 the corresponding

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actions of the three diVerent agents, combined the regulation of acid secretion in parietal cells. with data on PKC depletion by a PKC specific Some acute stimulatory action has been agent as well as increased PKC activity levels demonstrated but the majority of studies following IL-1â treatment, strongly implicate suggest PKC has an inhibitory role which may PKC in the pathway mediating the eVects of be mediated by receptor downregulation and at Gut: first published as 10.1136/gut.48.6.782 on 1 June 2001. Downloaded from IL-1â. more distal points in the acid secretion One potential criticism of all studies requir- pathway.11 17 38 This current study further ing kinase inhibitors is the lack of specificity supports the important role of PKC in mediat- against target kinases. In addition to the actions ing the eVects of a variety of extracellular utilised within the study, all of the agents have inhibitors of acid secretion. the ability to block the activity of other A variety of PKC isoforms have been intracellular enzymes, such as PKA (cAMP demonstrated in parietal cells although the activated kinase), protein kinase G (PKG) specific role of any of these remains to be clari- (cGMP activated kinase), myosin light chain fied and data on the distribution and function kinase (MLCK), and calmodulin kinase. The of each are scanty and often conflicting. Mura- concentrations used were carefully chosen to matsu et al demonstrated alpha, gamma, and enhance the specificity of the actions and con- zeta mRNA by northern blotting in guinea pig trol experiments performed to assess the eVects parietal cells but did not provide data on the of the other kinases. Staurosporine and H-7 are distribution or activity.39 In canine parietal cells potent but relatively non-specific PKC inhibi- EGF increased membrane associated PKC 2+ tors. The Ki value for H-7 against PKC is 6 µM activity and the translocation of the Ca compared with MLCK 97 µM, PKA 3.0 µM, dependent á and â1 isoforms to the cell mem- and PKG 5.8 µM.20–23 Staurosporine inhibits brane.17 It has been suggested that the so-called 2+ PKC with an IC50 of 0.7 nM compared with “atypical” Ca independent isoforms may have 7.0 nM for PKA and 8.5 nM for PKG.22 24–27 important roles in regulating inhibitory path- CHL is a less potent but a more specific PKC ways in parietal cells. Chew et al demonstrated

inhibitor, the IC50 for PKC is 660 nM with immunoblotting that rabbit parietal cells compared with >0.1 mM for PKA, tyrosine express abundant levels of the epsilon, mu, kinase, and calmodulin kinase.28 29 CHL, at iota, lambda, and zeta protein isoforms but concentrations used in this study, selectively relative paucity of alpha and beta 1 and beta inhibits PKC compared with MLCK.30 MLCK 2.10 McKenna et al showed that PKC-á and calmodulin kinase are essential parts of the mediated the inhibitory eVect of phorbol ester

signalling pathway leading to acid secretion on histamine H2 receptor activation of ade- subsequent on activation of the parietal cell.31 32 nylate cyclase in HGT-1 cells.40 Activation of Stimulation of these pathways appears to PKC isoforms by IL-1â may be important in increase acid secretion and thus they are parietal cell regulation and recent work has

unlikely to be activated by IL-1â, which clearly identiﬁed important roles for PKC-lambda/ http://gut.bmj.com/ inhibits acid secretion. The consistency of iota,41 epsilon, and delta42 in NIH 3T3 cells and results with the three PKC inhibitors at the PKC-zeta in Schwann cells13 in mediating the concentrations used would seem to conﬁrm eVects of IL-1â. As yet the PKC isoforms this. involved in IL-1â signal transduction within The potential role of the cyclic nucleotide parietal cells remain to be elucidated. IL-1â, dependent kinases was studied with H-8 and EGF, and PMA inhibited acid secretion by an

H-89. H-8 is a potent inhibitor of PKG (Ki 480 equivalent amount, despite a greater increase nM) and signiﬁcantly less potent against PKA in membrane PKC activity with EGF and on October 2, 2021 by guest. Protected copyright.

(Ki 1.2µM), MLCK (68 µM), and PKC (15 PMA. This may imply that speciﬁc isoforms µM).33 34 Selectively for PKG is usual at the are responsible for inhibition of acid secretion. concentration used here of 100 nM.34 Activa- The apparent lack of direct relationship tion of PKG with db-cGMP inhibited carba- between measured PKC activity and inhibition chol stimulated acid secretion, as has been of acid secretion may be due to total PKC reported previously.35 36 This eVect was blocked enzyme activity being assessed and that iso- by H-8. No eVect of H-8 against IL-1â inhibi- forms are activated by PMA and EGF which tion of acid secretion was demonstrated, are not immediately related to regulation of suggesting that PKG is not involved in IL-1â acid secretion. mediated acid inhibition. H-89 is a potent PKA Further studies will be performed using

inhibitor (Ki 48 nM); it has been used more isozyme speciﬁc inhibitors and immuno- eVectively to selectively distinguish the eVects blotting to attempt to determine the exact sig- of PKA.17 37 In this study inhibition of PKA did niﬁcance of the diVerent PKC isoforms, cellu- not alter the inhibitory action of IL-1â. Thus lar location, and translocation in the regulation within the constraints of studies utilizing kinase of parietal cell function. inhibitors, by using carefully chosen concentra- A variety of signal transduction pathways tions and combinations of inhibitors, this study mediating the many biological eVects of IL-1â strongly implicates PKC as an essential part of have been described. Many actions of IL-1â are the IL-1â activated signalling pathway. dependent on G protein coupled stimulation of DiVerent PKC isoforms have been detected cAMP formation and activation of PKA.43 in parietal cells. It has been demonstrated pre- Increased levels of cAMP stimulate acid secre- viously that EGF inhibits carbachol stimulated tion in parietal cells44 and our previous studies acid secretion via a PKC dependent mech- did not demonstrate any role for G protein anism, also acting distally to generation of coupled pathways mediating the inhibitory ++ 17 increased [Ca ]i. PKC has a complex role in actions of IL-1â against carbachol stimulated

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acid secretion.3 The lack of eVect of H-89, used isoforms and their precise roles will be at a concentration specific for the inhibition of determined in the future. PKA,37 is also strongly suggestive of the inhibitory actions of IL-1â not being mediated via This work was supported by the Medical Research Council in the adenylate cyclase/cAMP/PKA pathway. the form of a Research Training Fellowship for ILPB. This work Gut: first published as 10.1136/gut.48.6.782 on 1 June 2001. Downloaded from was presented and published in abstract form at the 6th UEGW In other systems the biological actions of (Birmingham 1997; Gut 1997;41(suppl 3): A95) and the British IL-1â subsequent to receptor binding appear Society of Gastroenterology (Harrogate 1998; Gut to be mediated by activation of sphingomyeli- 1998;42(suppl 1): F98). nases in the cell membrane and liberation of 12 1 Wallace JL, Cucala M, Mugridge K, et al. Secretagogue- ceramide second messenger. Exogenous specific eVects of interleukin-1 on gastric acid secretion. sphingomyelinase and cell permeable ceramide Am J Physiol 1991;261:G559–64. 2 Saperas ES, Yang H, Rivier C, et al. Central action of analogues have been used to explore the recombinant interleukin-1 to inhibit acid secretion in rats. biological role of this system.18 No eVect of Gastroenterology 1990;99:1599–606. 3 Beales I, Calam J. Interleukin-1â and tumour necrosis biologically active ceramide or sphingomyeli- factor-á inhibit acid secretion in cultured rabbit parietal nase on acid secretion was demonstrated. Thus cells by multiple pathways. Gut 1998;42:227–34. 4 Nompleggi DJ, Beinborn M, Roy A, et al. The eVect of the current data do not support a role for this recombinant cytokines on [14C]-aminopyrine accumula- pathway in mediating control of parietal cell tion by isolated canine parietal cells. J Pharmacol Exp Ther 1994;270:440–5. acid secretion by IL-1â. 5 Schepp W, Dehne K, Herrmuth H, et al. Identification and Previously, we have shown that tumour functional importance of IL-1 receptors on rat parietal cells. Am J Physiol 1998;275:G1094–105. necrosis factor á inhibited carbachol stimu- 6 Prinz C, Neumayer N, Mahr S, et al. Functional impairment lated secretion via a tyrosine kinase pathway.3 of rat enterochromaYn-like cells by interleukin 1 beta. Gastroenterology 1997;112:364–75. Although some of the varied biological eVects 7 Yamaoka Y, Kita M, Kodama T, et al. Induction of various of IL-1â are mediated by tyrosine kinases,16 cytokines and development of severe mucosal inflamma- tion by cagA gene positive Helicobacter pylori strains. Gut previous studies demonstrated that the inhibi- 1997;41:442–51. 8 Tache Y, Saperas E. Potent inhibition of gastric acid tory action against carbachol stimulated acid secretion and ulcer formation by centrally and peripherally secretion was independent of tyrosine kinase administered interleukin-1. AnnNYAcadSci1992;664: activity.3 353–68. 9 Wahlstrom KJ, Knudsen K, Lee JI, et al. Sequential changes Several of the agents used within this study in IL-1 alpha and TNF-alpha expression during healing of experimental gastric ulcers. Gastroenterology 1995;108: have the ability to induce toxicity or apoptosis A251. in various cellular models.45 46 A portion of the 10 Chew CS, Zhou CJ, Parente JA Jr. Ca2+-independent protein kinase C isoforms may modulate parietal cell HCl inhibitory action of IL-1â against ECL cells is secretion. Am J Physiol 1997;272:G246–56. due to decreased cell viability.6 We failed to 11 Nandi J, Bosche MC, Levine RA. EVects of a phorbol ester and isoquinoline sulfonamides on rabbit parietal cell func- demonstrate any reduction in parietal cell tion. J Pharmacol Exp Ther 1996;279:97–105. viability or alteration in morphology, even at 24 12 Saklatvala J. Intracellular signalling mechanisms of interleukin 1 and tumour necrosis factor: possible targets for hours after exposure to test agents. Thus it therapy. Br Med Bull 1995;51:402–18. seems unlikely that any alterations in acid 13 Carlson CD, Hart RP.Activation of acidic sphingomyelinase and protein kinase C zeta is required for IL-1 induction of http://gut.bmj.com/ secretion occurred as a secondary phenomena LIF mRNA in a Schwann cell line. Glia 1996;18:49–58. to toxicity or apoptosis. 14 Beales I, Calam J. EVect of Nalpha-methyl-histamine on acid secretion in isolated cultured rabbit parietal cells: IL-1 is a potent inhibitor of acid secretion implications for Helicobacter pylori associated gastritis and when administered parenterally47 or intracister- gastric physiology. Gut 1997;40:14–9. 2 15 Wang L, Lucey MR, Fras AM, et al. Epidermal growth fac- nally ; at least some of these inhibitory actions tor and transforming growth factor-alpha directly inhibit are mediated by direct inhibition of parietal3 parietal cell function through a similar mechanism. J Phar- 6 macol Exp Ther 1993;265:308–13. and ECL cell function. Recent studies have 16 Beales I, Calam J. Stimulation of IL-8 production in human both confirmed an inhibitory action of IL-1â gastric epithelial cells by Helicobacter pylori, IL-1â and on October 2, 2021 by guest. Protected copyright. TNF-á requires tyrosine kinase activity, but not protein and demonstrated the presence of IL-1â kinase C. Cytokine 1997;9:514–20. receptor and corresponding mRNA in highly 17 Wang L, Wilson EJ, Osbourn J, et al. Epidermal growth fac- 5 tor inhibits carbachol-stimulated canine parietal cell enriched populations of rabbit parietal cells. function via protein kinase C. Gastroenterology 1996;110: IL-1 may be an important pathophysiological 469–77. 18 Masamune A, Shimosegawa T, Masamune O, et al. Helico- regulator of acid secretion. Inhibition of acid bacter pylori-dependent ceramide production may mediate secretion is seen in systemic sepsis and disease. increased interleukin 8 expression in human gastric cancer cell lines. Gastroenterology 1999;116:1330–41. Profound hypochlorhydria may be seen in 19 Beales I, Blaser MJ, Srinivasan S, et al. EVect of acute and chronic Helicobacter pylori Helicobacter pylori products and recombinant cytokines 48–50 on gastrin release from cultured canine G cells. Gastroenter- infection, where it is associated with in- ology 1997;113:465–71. creased expression of IL-1â.749Cytokine over- 20 Boulis NM, Davis M. Blockade of the spinal excitatory eVect of cAMP on the startle reflex by intrathecal adminis- expression and acid secretion return towards tration of the isoquinoline sulfonamide H-8: comparison to normal with eradication of the infection.49 51 the protein kinase C inhibitor H-7. Brain Res 1990;525: 198–204. Low levels of IL-1â may be expressed in 21 Quick J, Ware JA, Driedger PE. The structure and biologi- normal healthy gastric mucosa,7 suggesting cal activities of the widely used protein kinase inhibitor, H7, diVer depending on the commercial source. Biochem that the cytokine may have a physiological role Biophys Res Commun 1992;187:657–63. 22 Schachtele C, Seifert R, Osswald H. Stimulus-dependent in the control of acid secretion along with the inhibition of platelet aggregation by the protein kinase C other luminal, paracrine, endocrine, and neu- inhibitors polymyxin B, H-7 and staurosporine. Biochem rocrine factors. Biophys Res Commun 1988;151:542–7. 23 Takahashi I, Kobayashi E, Nakano H, et al. Potent selective In conclusion, this study demonstrates that inhibition of 7-O-methyl UCN-01 against protein kinase C. J Pharmacol Exp Ther 1990;255:1218–21. IL-1â inhibits carbachol stimulated acid secre- 24 Tischler AS, Ruzicka LA, Perlman RL. Mimicry and inhibition in cultured parietal cells. This eVect tion of nerve growth factor eVects: interactions of occurs distal to second messenger generation staurosporine, forskolin, and K252a in PC12 cells and normal rat chromaYn cells in vitro. J Neurochem 1990;55: and appears to involve a PKC dependent path- 1159–65. 25 Tamaoki T, Nomoto H, Takahashi I, et al. Staurosporine, a way but is independent of PKA, PKG, and potent inhibitor of phospholipid/Ca++ dependent protein ceramide/sphingomyelinase. The exact PKC kinase. Biochem Biophys Res Commun 1986;135:397–402.

www.gutjnl.com IL-1â inhibition of acid secretion 789

26 Tamaoki T. Use and specificity of staurosporine, UCN-01, 39 Muramatsu S, Tani N, Miwa T, et al. Protein kinase C gene and calphostin C as protein kinase inhibitors. Methods expression in dispersed guinea-pig gastric parietal cells. Enzymol 1991;201:340–7. Digestion 1998;59:40–6. 27 Yanagihara N, Tachikawa E, Izumi F, et al. Staurosporine: 40 McKenna P, Williams JM, Hanson PJ. Protein kinase C 2+ an eVective inhibitor for Ca /calmodulin-dependent pro- alpha is the isoform responsible for inhibition of histamine tein kinase II. J Neurochem 1991;56:294–8. H2 receptor mediated stimulation of adenylate cyclase in 28 Herbert JM, Augereau JM, Gleye J, et al. Chelerythrine is a the human gastric cancer cell line HGT-1. Gut: first published as 10.1136/gut.48.6.782 on 1 June 2001. Downloaded from potent and specific inhibitor of protein kinase C. Biochem Soc Biochem 1993;21:192S. Biophys Res Commun 1990;172:993–9. Trans 29 Barg J, Belcheva MM, Coscia CJ. Evidence for the implica- 41 Bonizzi G, Piette J, Schoonbroodt S, et al. Role of the pro- tion of phosphoinositol signal transduction in mu-opioid tein kinase C lambda/iota isoform in nuclear factor-kappaB inhibition of DNA synthesis. J Neurochem 1992;59:1145– activation by interleukin-1beta or tumor necrosis factor- 52. alpha: cell type specificities. Biochem Pharmacol 1999;57: 30 Venema RC, Raynor RL, Noland TA Jr, et al. Role of protein 713–20. kinase C in the phosphorylation of cardiac myosin light 42 Varley CL, Brown BL, Groome N, et al. Interleukin-1beta- chain 2. Biochem J 1993;294:401–6. induced expression of protein kinase C (PKC)-delta and 31 Mamiya N, Goldenring JR, Tsunoda Y, et al. Inhibition of epsilon in NIH 3T3 cells. Cytokine 1997;9:577–81. acid secretion in gastric parietal cells by the Ca2+/ 43 Brooks JW, Mizel SB. Interleukin-1 and signal transduction. calmodulin-dependent protein kinase II inhibitor KN-93. Eur Cytokine Netw 1994;5:547–61. Biochem Biophys Res Commun 1993;195:608–15. 44 Park J, Chiba T, Yamada T. Mechanisms for direct 32 Sugita Y, Nagao T, Urushidani T. Nonspecific eVects of the inhibition of canine gastric parietal cells by somatostatin. J pharmacological probes commonly used to analyze signal Biol Chem 1987;262:14190–6. transduction in rabbit parietal cells. Eur J Pharmacol 1999; 45 Jarvis WD, Turner AJ, Povirk LF, Induction of 365:77–89. et al. 33 Hagiwara M, Inagaki M, Hidaka H. Specific binding of a apoptotic DNA fragmentation and cell death in HL-60 novel compound, N-[2-(methylamino)ethyl]-5-isoquin- human promyelocytic leukemia cells by pharmacological olinesulfonamide (H-8) to the active site of cAMP- inhibitors of protein kinase C. Cancer Res 1994;54:1707– dependent protein kinase. Mol Pharmacol 1987;31:523–8. 14. 34 Hidaka H, Inagaki M, Kawamoto S, et al. Isoquinolinesul- 46 Bruno S, Ardelt B, Skierski JS, et al. DiVerent eVects of fonamides, novel and potent inhibitors of cyclic nucleotide staurosporine, an inhibitor of protein kinases, on the cell dependent protein kinase and protein kinase C. Biochemis- cycle and chromatin structure of normal and leukemic try 1984;23:5036–41. lymphocytes. Cancer Res 1992;52:470–3. 35 Heim HK, RuoV HJ. Cyclic GMP and acid production in 47 Saperas E, Cominelli F, Tache Y. Potent inhibition of gastric isolated gastric cells. Naunyn Schmiedebergs Arch Pharmacol acid secretion by intravenous interleukin-1 beta and -1 1985;330:147–54. alpha in rats. Peptides 1992;13:221–6. 36 Brown JF, Hanson PJ, Whittle BJ. The nitric oxide donor, 48 Beales I. H. pylori-associated hypochlorhydria. Gastroenter- S-nitroso-N-acety-penicillamine inhibits secretory activity ology 1998;114:618–20. in rat isolated parietal cells. Biochem Biophys Res Commun 49 Yasunaga Y, Shinomura Y, Kanayama S, et al. Mucosal 1993;195:1354–9. interleukin-1 beta production and acid secretion in 37 Chijiwa T, Mishima A, Hagiwara M, et al. Inhibition of enlarged fold gastritis. 1997; : forskolin-induced neurite outgrowth and protein phospho- Aliment Pharmacol Ther 11 rylation by a newly synthesized selective inhibitor of cyclic 801–9. AMP-dependent protein kinase, N-[2-(p-bromocinn- 50 Konturek PC, Brzozowski T, Konturek SJ, et al. Mouse amylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of model of Helicobacter pylori infection: studies of gastric PC12D pheochromocytoma cells. J Biol Chem 1990;265: function and ulcer healing. Aliment Pharmacol Ther 5267–72. 1999;13:333–46. 38 Chiba T, Fisher SK, AgranoV BW, et al. Autoregulation of 51 el Omar EM, Oien K, el Nujumi A, et al. Helicobacter pylori muscarinic and gastrin receptors on gastric parietal cells. infection and chronic gastric acid hyposecretion. Gastroen- Am J Physiol 1989;256:G356–63. terology 1997;113:15–24. http://gut.bmj.com/ 1st Asia Pacific Forum on Quality Improvement in Health Care Three day conference Wednesday 19 to Friday 21 September 2001 Sydney, Australia on October 2, 2021 by guest. Protected copyright. We are delighted to announce this forthcoming conference in Sydney. Authors are invited to submit papers (call for papers closes on Friday 6 April), and delegate enquiries are welcome. The themes of the Forum are: x Improving patient safety x Leadership for improvement x Consumers driving change x Building capacity for change: measurement, education and human resources x The context: incentives and barriers for change x Improving health systems x The evidence and scientific basis for quality improvement. 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