Agents Actions37 (1992)

0065-4299/92/020025-05 $1.50+ 0.20/0 9 1992 Birkh/iuser Verlag, Basel

Protein kinase C inhibits stimulation of adenylate cyclase by the histamine H 2 receptor in rat parietal cells J. F. Emly 1 and P. J. Hanson Pharmaceutical SciencesInstitute, Aston University, Aston Triangle, Birmingham B4 7ET, UK


The action of protein kinase C on the stimulation of adenylate cyclase activity by the histamine H 2 receptor was investigated in rat parietal cells. Protein kinase C was activated by preincubating cells with 12-0tetradecanoylphorbol 13-acetate (TPA), and adenylate cyclase activity was measured in sonicated extracts. TPA (100 nM) inhibited adenylate cyclase activity stimulated by histamine (100 nM 500 ~tM). This effect was related to the concentration of TPA. TPA (100 nM) enhanced the stimulation of adenylate cyclase activity by forskolin (100 ~tM) but had no effect on the stimulation by N a F (10 mM). In conclusion, protein kinase C inhibits stimulation of adenylate cyclase by the histamine H 2 receptor. This action could be mediated by changes in the number or affinity of histamine H2 receptors or in the coupling of the receptor to the stimulatory guanine nucleotide regulatory subunit Gs.


Protein kinase C is the name given to a family of enzymes which are involved in many aspects of the regulation of cellular function [1]. One way of investigating the actions of protein kinase C is to use an activator of the enzymes 12-O-tetradecanoylphorbol 13-acetate (TPA). Use of this compound, and another activator, 1-oleoyl-2-acetylglycerol, has suggested that activation of protein kinase C inhibits histamine-stimulated acid secretion in parietal cells from rat, rabbit and guinea pig [2, 3, 4]. In the rat, one site for the inhibitory action of protein kinase C is distal in the secretory pathway to cyclic A M P generation, because the stimulatory effects of dibutyryl cyclic A M P are inhibited by TPA [5]. However, TPA also inhibits the stimuPresent address: WolfsonResearchLaboratories,Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 2TH, UK

latory effect of histamine on the cyclic A M P content of parietal cells [5]. This result suggests, but does not prove [6], that histamine-stimulation of adenylate cyclase might be affected by protein kinase C. The aim of this work was to establish whether stimulation of adenylate cyclase by the histamine H2 receptor was negatively modulated by protein kinase C, and to establish the site at which any effects might occur. Methods and materials

Preparation and incubation of parietal cells A crude suspension of parietal cells was prepared from everted fundic sacs of rat stomach by digestion with pronase and intermittent calcium chelation as described previously [5]. Parietal cells were enriched by centrifugation on a self-generating Percoll gradient [5]. The low-density cell

Agents Actions37 (1992)

26 fraction removed from the top 20% of the gradient contained > 80% parietal cells. This fraction was washed free from Percoll by centrifugation at 200x 9 for 5 min at 15~ and was finally resuspended in Eagle's Minimum Essential Medium containing 20mM N-2-hydroxyethylpiperazineN'-2-ethanesulphonic acid (HEPES), 1 mg/ml of bovine serum albumin and 50 ~tg/ml of gentamicin for 2 h at 37~ with continuous gassing with 95% O2 5% CO2. This preincubation procedure increased the subsequent response to histamine [5].

Incubation of parietal cells with phorbol esters For the final 10 min of the preincubation period TPA, 4~-TPA or the solvent dimethylsulphoxide (final concentration 0.125% v/v) was added to batches of cells. Cells were then washed rapidly by transferring the suspensions to 1.5 ml microfuge tubes, centrifuging for 10 s at 12 000 • g, resuspending the pellet in incubation medium using a plastic pipette tip and centrifuging again. The final resuspension was in 200 ~tl of homogenization buffer (pH 7.8) consisting of 25 mM tris-HC1, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM benzamidine, 10 ~tM leupeptin, 100 U/ml aprotinin and 0.3 mM phenylmethylsulphonyl fluoride. Cells were then sonicated at 4 ~ for 4 x 5 s, with 5 s intervals, by using an MSE Soniprep 150 working at 3-~tm amplitude. TPA was added to intact cells rather than to homogenates for two reasons. Firstly, some isoforms of protein kinase C require the presence of Ca 2 + for the enzyme to be activated by translocation from the cytosol to the membrane in the presence of phorbol esters. If protein kinase C were to be activated in the homogenates adenylate cyclase would therefore have to be assayed in the presence of C a 2 +. This is undesirable because Ca 2+ can inhibit adenylate cyclase [7]. Secondly, comparisons between previously observed effects of TPA on histamine-stimulated cyclic AMP content and the present experiments on adenylate cyclase would be easier to interpret if protein kinase C were activated in intact cells in both cases.

Assay of adenylate cyclase The assay was performed according to [8]. 20 ~tl of homogenate was added to 105 pA of incubation medium such that the final composition of the assay mixture (pH 7.8) was: 25 mM tris-HC1, 1 mM

EDTA, 1 mM dithiothreitol, 10 mM MgC12, 1 mM 3-isobutyl-l-methylxanthine (IBMX), 0.1 mM ATP, 16 mM creatine phosphate, 10 U/ml of creatine kinase and 1 mg/ml of BSA. The mixture was incubated for 10 rain at 30~ The reaction was stopped by the addition of an equal volume of icecold ethanol and the transfer of the tubes to ice.

Assay of cyclic AMP Ethanolic extracts were evaporated in a vacuum oven at 40 ~ dissolved in 0.05 M sodium acetate buffer (pH 6.2) and were analysed for cyclic AMP by using an acetylated [lzsI] radioimmunoassay procedure (New England Nuclear kit).

Expression of results Variation in responsiveness between batches of parietal cells is a common problem [5] and, as here, data are usually presented in normalised form to remove this source of variation. Statistical analyses have been performed on the untransformed data using procedures such analysis of variance or paired t-tests to separate out the effects of phorbol esters from that of different batches of cells.

Animals and reagents Male Wistar rats (200-300 g body weight) were fed on Heygates breeding diet supplied by Pilsbury (Birmingham, UK). Creatine phosphate and creatine kinase were obtained from Boehringer Mannheim, Lewes, UK. Protease inhibitors and TPA were obtained from Sigma, Poole, UK, and the 4~-phorbol derivative, 4~-TPA, was from Scientific Marketing Associates, London N1, UK. Bovine serum albumin was purchased from ICN Biomedicals, High Wycombe, UK. Results

Formation of cyclic AMP was linearly related to time and was proportional to the amount of homogenate added to the assay system. Basal adenylate cyclase activity, and the stimulation by 0.5 mM histamine were broadly in agreement with other workers [9, 10]. There was no effect of preincubation with 100 nM TPA on basal adenylate cyclase activity which was (pmol/10 min per 10 6 cells, mean _+SEM from five batches of cells): 1.4_+0.46 and 1.6


Agents Actions 37 (1992)

+0.45 in the absence and presence of TPA, respectively. Preincubation with TPA caused a significant inhibition of the stimulatory effect of histamine at all concentrations tested between 100 nM and 500 taM (Fig. 1). The inhibition by TPA of the stimulation of adenylate cyclase by 500 taM histamine was related to the concentration of TPA and was significant at 10 and 100 n M T P A (Fig. 2). Preincubation with 100 n M T P A enhanced the stimulation of adenylate cyclase activity by forskolin but had no effect

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on the stimulatory action of sodium fluoride (Table 1). Preincubation with 4ct-TPA had no effect on the stimulatory effects of histamine, forskolin or sodium fluoride on adenylate cyclase activity (Table 1) Preincubation of parietal cells with pertussis toxin (100ng/ml) for 2h, did not abolish effects of T P A on histamine- or forskolin-stimulated adenylate cyclase activity. In two such experiments the results of measurements of adenylate cyclase were: histamine-stimulation with T P A preincubation, 43 and 52% of control; forskolin-








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log {TPA concn. (M)}

log {histamine concn. (M)} Figure I Effect of preincubation of intact parietal cells with 100 nM TPA for 10 min on the subsequent stimulation of homogenate adenylate cyclase activity by histamine. Results are means+-SEM from five batches of cells and have been normalized to the stimulation obtained with 0.5 mM histamine in the absence of TPA which was 4.9 +- 1.6 pmol/106 cells per 10 min (O) control; (11) 100 nM TPA. * P

Protein kinase C inhibits stimulation of adenylate cyclase by the histamine H2 receptor in rat parietal cells.

The action of protein kinase C on the stimulation of adenylate cyclase activity by the histamine H2 receptor was investigated in rat parietal cells. P...
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