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. www.gutjnl.com IL-1â inhibition of acid secretion 783 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.