INTRACELLULAR SIGNALLING AND REGULATION OF GASTRIC ACID SECRETION

Transcription

1 Quarterly Journal of Experimental Physiology (1989), 74, Printed in Great Britain REVIEW ARTICLE INTRACELLULAR SIGNALLING AND REGULATION OF GASTRIC ACID SECRETION P. J. HANSON AND J. F. HATT Pharmaceutical Sciences Institute, Biology Division, Aston University, Aston Triangle, Birmingham B4 7ET (MANUSCRIPT RECEIVED 17 MARCH 1989, ACCEPTED 27 APRIL 1989) CONTENTS PAGE Introduction 608 Preparations used to investigate intracellular signalling mechanisms 610 Assessment of secretory activity 610 Regulation of adenylate cyclase 611 Activators of adenylate cyclase 611 Histamine 611 Adrenergic agonists 612 Glucagon and related peptides? 612 Opioid peptides? 613 Inhibitors of adenylate cyclase 613 Eicosanoids 613 Somatostatin 613 Adenosine 614 Oxyntomodulin? 615 Actions of cyclic AMP 615 Epidermal growth factor - a regulator of cyclic AMP phosphodiesterase? 615 Polyphosphoinositide metabolism and intracellular calcium 617 Muscarinic cholinergic agonists 617 Gastrin 618 Histamine 619 Gastrotropin (porcine ileal polypeptide) 620 Calcium-activated protein kinases 621 Protein kinase C 621 Involvement in stimulation of acid secretion by gastrin and carbachol 621 Feedback inhibition of the actions of carbachol 622 Effects on the cyclic AMP pathway 622 Targets for intracellular signalling systems 624 Summary and future prospects 627 References 628

2 608 P. J. HANSON AND J. F. HATT K+ f C1- - ffcl-.- K+ Lumen of canaliculus ATP +i ADP r +Piy H+ Conductance ATP -4 ADPA Pump Fig. 1. Model for the secretion of HCI into the secretory canaliculus. INTRODUCTION In recent years there have been tremendous advances in the understanding of the mechanisms by which receptors on the cell surface transmit information into the cell interior. Highlights have been the discovery of a signalling pathway involving protein kinase C (Nishizuka, 1986) and inositol 1,4,5-trisphosphate (Berridge, 1987), the understanding of the role of guanine nucleotide binding proteins (G proteins) in the regulation of adenylate cyclase (Gilman, 1987), and the recognition that there are related families of receptors (see Michell, 1987 for a brief review). The intention of this review is to provide a picture of current understanding of the signal transduction mechanisms in mammalian parietal cells which mediate the effects of activators and inhibitors of acid secretion. Only those agents which may have a direct action on the parietal cell will be considered. The mechanism by which the parietal cell secretes acid has been reviewed by Forte & Wolosin (1987). Acid is secreted across the apical membrane of the parietal cell by a K+-H+-ATPase pump. The pump requires the presence of K+ at the external face of this

3 REGULATION OF GASTRIC ACID SECRETION 609 3Na+ / ATP -ADIP\ +Pi *2K+ Conductance Exchanger ATP -* ADP Pump Fig. 2. The ion transport pathways present in the basolateral membrane of the parietal cell. Pathways involving Ca2` have been excluded. membrane for activity, and the expulsion of H+ across the apical membrane is coupled with the movement of K+ into the cell (Fig. 1). In the resting cell the pump is present in intracellular tubulovesicular structures. There is evidence that these tubulovesicles may fuse with the apical membrane to form secretory canaliculi when the parietal cell is activated. Regulation of the provision of K+ to the extracellular site of the pump is thought to be the mechanism by which K+-H+-ATPase activity is controlled. K+ and Cl- conductances are present in vesicles prepared from the apical membrane of stimulated parietal cells, and enable K+ and Cl- to efflux from the cell. This K+ largely returns via the K+-H+-ATPase so that the Cl- is secreted with H+. The Cl--HCO3- exchanger in the basolateral membrane (Fig. 2) serves to provide Cl- for expulsion across the apical membrane of the parietal cell, and to remove intracellular base generated by the passage of H+ into the secretory canaliculi. Basolateral Na+-H+ exchange may also be a necessary accompaniment of acid secretion (Muallem, Blissard, Cragoe & Sachs, 1988). The abbreviations used routinely in this review are shown in Table 1.

4 610 P. J. HANSON AND J. F. HATT Table 1. List of abbreviations CCK-8 Cholecystokinin octapeptide EGF Epidermal growth factor Gi The heterotrimer, consisting of cx, f and y subunits, which is involved in the inhibition of adenylate cyclase IBMX 3-Isobutyl- I-methylxanthine OAG l-oleoyl-2-acetyl-glycerol PGE2 Prostaglandin E2 TPA 12-o-Tetradecanoylphorbol- 13-acetate PREPARATIONS USED TO INVESTIGATE INTRACELLULAR SIGNALLING MECHANISMS Measurement of acid secretion in vivo is of little use in the investigation of intracellular signalling mechanisms because the effects of agents on acid secretion are the result of a complex interplay of regulatory systems. Gastric mucosa from which the muscle has been stripped can be mounted in chambers in vitro. The advantage of these preparations is that acid secretion can be measured directly, but there are problems in obtaining a response to a full range of secretagogues and inhibitors, and the interpretation of results is made difficult by the complexity of the preparation. In some respects gastric glands are the ideal preparation with which to investigate the regulation of acid secretion. They can be prepared from man (Fellenius, Elander, Wallmark, Haglund, Olbe & Helander, 1979), rabbit (Berglindh & Obrink, 1976) and dog (Berglindh & Hansen, 1984) by treatment of tissue with collagenase. Rat gastric glands are prepared by the incubation of everted stomach sacs with ethylenediaminetetraacetic acid (EDTA) at 4 C (Gespach, Dupont, Bataille & Rosselin, 1980). The parietal cells, which are retained in a functional unit with intercellular connections, make up about 50% of the total cell volume of the gland (Berglindh & Obrink, 1976). Also, there are no problems about access of oxygen, metabolic substrates or regulators of acid secretion. However, the presence of chief cells and other cell types can complicate the interpretation of some types of experiment with gastric glands. By using more vigorous isolation procedures it is possible to obtain parietal cells unattached to other cells. Since these procedures often use Ca2" chelation and/or pronase digestion the possibility that the cells are damaged during isolation must always be considered. Isolated parietal cells can be enriched by centrifugal elutriation or density gradient centrifugation. When these methods are combined virtually pure preparations can be obtained (Chew & Brown, 1986). Highly enriched, or essentially pure, parietal cell preparations are required for many experiments on intracellular signalling mechanisms. Assessment of secretory activity Direct measurements of acid secretion cannot be obtained with gastric glands or isolated parietal cells because the same medium bathes both the apical and basolateral membranes of the parietal cell. Secretory activity has to be assessed indirectly. The most widely used technique is to measure the ratio of the intracellular concentration of ["4C]aminopyrine to that in the medium. Aminopyrine is a weak base with a pka of 5 which can cross membranes when unprotonated, but which becomes trapped in its protonated form in regions of low ph. The distribution of aminopyrine is therefore a function of the ph in the secretory canaliculi and in the incubation medium (Berglindh, Helander & Obrink, 1976),

5 REGULATION OF GASTRIC ACID SECRETION and reflects sequestration of acid in the secretory canaliculi of the parietal cell. Although the aminopyrine accumulation ratio provides only an index, rather than a direct measure, of acid secretion it has proved to be an extremely useful technique in the investigation of intracellular mechanisms regulating acid secretion. Measurements of the changes in oxygen consumption induced by secretagogues are somewhat less convenient to perform than measurements of aminopyrine accumulation but generally seem to give similar results (Chew, Hersey, Sachs & Berglindh, 1980). Interpretation of changes in oxygen consumption may be complicated if agents affect metabolic activity in any non-parietal cells present in the experimental preparation. 611 REGULATION OF ADENYLATE CYCLASE Activators of adenylate cyclase Histamine Histamine stimulated aminopyrine accumulation in rabbit gastric glands (Berglindh et al. 1976) and in parietal cells from rat (Sonnenberg, Berglindh, Lewin, Fischer, Sachs & Blum, 1979), dog (Soll, 1980 a), guinea-pig (Batzri & Dyer, 1981), man (Miederer, Schepp, Dein & Ruoff, 1986) and pig (Mardh, Song, Carlsson & Bjorkman, 1987). The stimulation of oxygen consumption by histamine was also evidence that it increased secretory activity in parietal cells (Berglindh et al. 1976; Soll, 1978). Several investigations (e.g. Dial, Thompson & Rosenfeld, 1981; Leth, Elander, Haglund, Olbe & Fellenius, 1987) have established that the pharmacology of the histamine receptor is of the H2 type (Black, Duncan, Durant, Ganellin & Parsons, 1972). The development of specific histamine H2 receptor antagonists such as cimetidine and ranitidine has led to a revolution in the treatment of peptic ulcer disease. These drugs can heal approximately 80% of duodenal and gastric ulcers in 4-6 weeks, and can also be used as maintenance therapy to prevent relapse (Misiewicz, 1988). Virtually all of the characterization of the histamine H2 receptor on parietal cells has involved measurement of cellular responses such as increased oxygen consumption or aminopyrine accumulation. Receptor binding studies using [3H]histamine have been performed with guinea-pig cells (Batzri, Harmon & Thompson, 1982). The uptake of histamine by the cells from species such as rabbit (Batzri, Thompson & Toles, 1985) complicates the interpretation of receptor binding studies. Cyclic AMP is a second messenger for histamine. The evidence is as follows. Firstly, histamine elevated the cyclic AMP content in rabbit gastric glands (Chew et al. 1980) and in parietal cells from several species (Soll & Wollin, 1979; Batzri & Dyer, 1981; Schepp, Heim & Ruoff, 1983a). Secondly, there was a relationship between the effect of various histamine agonists and antagonists on aminopyrine accumulation and on cyclic AMP content in rabbit gastric glands (Chew et al. 1980). In dog parietal cells cyclic AMP content was related to the secretory response provided that the cyclic AMP content was not elevated above that producing maximal aminopyrine accumulation (Soll & Wollin, 1979). Thirdly, the cyclic AMP phosphodiesterase inhibitor 3-isobutyl- 1 -methylxanthine (IBMX) potentiated the action of histamine on intracellular cyclic AMP content and on aminopyrine accumulation (Soll & Wollin, 1979). Fourthly, histamine stimulated adenylate cyclase in sonicates of isolated parietal cells (Thompson, Chang & Rosenfeld, 1981). Fifthly, the cyclic AMP analogue, dibutyryl cyclic AMP (Berglindh et al. 1976; Sonnenberg et al. 1979; Soll, 1980a), and the activator of adenylate cyclase, forskolin (Hersey, Owirodu & Miller, 1982), both evoke a secretory response similar to that of histamine.

6 612 P. J. HANSON AND J. F. HATT Potentiating interactions were found between histamine and carbachol and between histamine and gastrin (Soll, 1982), but there was no evidence that such interactions involved modification of the rise in cyclic AMP content evoked by histamine (Soll & Wollin, 1979; Batzri & Dyer, 1981). Indeed, the existence of potentiating interactions between carbachol and submaximally effective concentrations of dibutyryl cyclic AMP (Soll, 1982; Pfeiffer, Sauter & Rochlitz, 1987) suggested that these interactions were at sites distal to cyclic AMP production. After stimulation with histamine the cyclic AMP content of parietal cells from dog (Wollin, Soll & Samloff, 1979) and rat (Hatt & Hanson, 1988), rose to a plateau and stayed there. There is thus no evidence for a rapid homologous desensitization of the response to histamine in rat or dog parietal cells. However, in rabbit gastric glands the cyclic AMP response to histamine was more transitory (Chew et al. 1980), and in the human gastric cancer cell line, HGT-1, homologous desensitization of the histamine H2 receptor was evident (Emami & Gespach, 1986). There may be mechanisms for the long-term regulation of histamine receptors on rat parietal cells. Thus in gastric glands isolated from rats fed with a diet of cow's milk for 4 days, histamine stimulated adenylate cyclase activity with an increased efficacy but unchanged potency by comparison with glands obtained from rats fed with a control diet (Gespach, Emami, Chastre, Launay & Rosselin, 1987). Whether cyclic AMP is responsible for the effects of histamine on Ca2+ mobilization, or whether there may be a different second messenger for this action of histamine is discussed later. Adrenergic agonists Hexoprenaline, adrenaline, noradrenaline and isoprenaline increased aminopyrine accumulation in rat parietal cells in a dose-related manner (Ruoff, Wagner, Gunther & Maslinski, 1982; Rosenfeld, 1984). Use of selective agonists and antagonists suggested that a /82-adrenergic receptor was present on rat parietal cells. In pig and human parietal cells adrenaline alone was ineffective but enhanced aminopyrine accumulation in response to histamine (Song, Mardh, Nyren & Loof, 1988). Again the evidence was for the involvement of a /?2-adrenergic receptor. There appears to be no effect of adrenergic agents in dog or rabbit parietal cells (Soll & Berglindh, 1987). Isoprenaline activated adenylate cyclase (Thompson et al. 1981; Ruoff et al. 1982), and increased the cyclic AMP content of an enriched preparation of rat parietal cells (Black, Strada & Thompson, 1988). It is likely that the effects of adrenergic agonists are mediated through stimulation of adenylate cyclase. Glucagon and related peptides? Oxyntomodulin is a peptide isolated from the porcine jejuno-ileum which consists of the entire sequence of pancreatic glucagon with an octapeptide extension at its C-terminal end. Oxyntomodulin increased the cyclic AMP content of rat gastric glands, and was 20 times more potent than pancreatic glucagon in this respect (Bataille, Gespach, Coudray & Rosselin, 1981). The action of pancreatic glucagon in stimulating the cyclic AMP content of gastric glands in the presence of histamine was less than that expected from addition of the separate effects of glucagon and histamine (Gespach, Bataille, Dutrillaux & Rosselin, 1982). However, in rat (Schepp & Ruoff, 1984) and human (Miederer et al. 1986) parietal cells the effects of glucagon and histamine on adenylate cyclase activity were additive. Glucagon did not increase aminopyrine accumulation in rat parietal cells (Schepp & Ruoff, 1984). Either there is a 'glucagon' receptor on rat parietal cells which is coupled to adenylate cyclase, but which does not stimulate acid secretion, or in gastric glands and

7 REGULATION OF GASTRIC ACID SECRETION enriched preparations of parietal cells there is a cell type, other than the parietal cell, which possesses a 'glucagon' receptor linked to stimulation of adenylate cyclase. The problem could be resolved if elutriation were combined with density gradient centrifugation so as to prepare an essentially pure fraction of rat parietal cells. Opioid peptides? D-Ala2-D-leu5-enkephalin and met-enkephalin produced a small enhancement of histamine-stimulated aminopyrine accumulation in parietai cells from guinea-pig and rat (Kromer, Schroder & Netz, 1984; Schepp, Schneider, Schusdziarra & Classen, 1986). The effects of these opioid peptides were blocked by (-)naloxone. No effect of met-enkephalin alone on aminopyrine accumulation was found. Whether these effects are mediated by changes in cellular cyclic AMP content is unknown, but since they were only seen in cells stimulated with submaximal concentrations of histamine (Schepp et al. 1986) this is possible. Inhibitors of adenylate cyclase Eicosanoids Low concentrations of prostaglandins of the prostaglandin E series inhibited histaminestimulated aminopyrine accumulation in parietal cells from dog (Soll, 1980b), and rat (Schepp, Ruoff& Maslinksi, 1983b; Rosenfeld, 1986; Atwell & Hanson, 1988), and in rabbit gastric glands (Levine, Kohen, Schwartzel & Ramsay, 1982). In rat parietal cells prostaglandin E2 (PGE2) inhibited aminopyrine accumulation stimulated by isoprenaline (Rosenfeld, 1986). Prostacyclin and some prostacyclin analogues inhibited histaminestimulated aminopyrine accumulation in dog parietal cells (Soll, 1980b; Soll & Whittle, 1981). Leukotriene C4 inhibited aminopyrine accumulation stimulated by histamine or by dibutyryl cyclic AMP in rabbit gastric glands (Konturek, Bilski, Dembin'ski, Warzecha, Beck & Jendralla, 1987). There was no effect of PGE2 on aminopyrine accumulation stimulated by carbachol, gastrin or dibutyryl cyclic AMP in dog parietal cells (Soll, 1980 b). These results suggest that the site of action of PGE2 is close to adenylate cyclase. PGE2 produced a fall in the histamine-stimulated cyclic AMP content of dog parietal cells (Major & Scholes, 1978; Soll, 1980b), and inhibition of adenylate cyclase by PGE2 in sonicates of rat parietal cells was demonstrated by Schepp et al. (1983a). Pre-incubation of parietal cells with pertussis toxin inhibited the action of PGE2 on histamine-stimulated aminopyrine accumulation (Rosenfeld, 1986; Atwell & Hanson, 1988; Chen, Amirian, Toomey, Sanders & Soll, 1988). In dog parietal cells a prevention by pertussis toxin of the effect of PGE2 on histaminestimulated cyclic AMP content was also found (Chen et al. 1988). Pertussis toxin is known to inactivate Gi by ADP-ribosylating the ac-subunit. A 41 kda protein which can be ADPribosylated by pertussis toxin has been identified in extracts of rabbit (Brown & Chew, 1987) and dog (Chen et al. 1988) parietal cells. It is likely that this protein is the cc-subunit of Gi and that prostaglandin receptors on parietal cells (Tsai, Kessler, Schoenhard, Collins & Bauer, 1987) activate G1 by causing the dissociation of Gia from the heterotrimer. Somatostatin Use of 251I-labelled somatostatin analogues enabled the identification of somatostatin receptors in cell preparations, enriched with parietal cells, and prepared from the rat (Reyl, Silve & Lewin, 1979) and dog (Park, Chiba & Yamada, 1987) stomach. The receptors on the dog cells did not appear to discriminate between somatostatin- 14 and somatostatin-28. Somatostatin inhibited histamine-stimulated aminopyrine accumulation in parietal cells 613

8 614 P. J. HANSON AND J. F. HATT from guinea-pig (Batzri, 1981), rat (Schepp, et al. 1983b; Atwell & Hanson, 1988), rabbit (Chew, 1983) and dog (Park et al. 1987). Somatostatin inhibited the stimulatory effect of histamine on the cyclic AMP content of guinea-pig and dog parietal cells (Batzri, 1981; Park et al. 1987) and of gastric glands from rat and guinea-pig (Gespach et al. 1980; Gespach, Hui Bon Hoa & Rosselin, 1983). Somatostatin inhibited histamine-stimulation of adenylate cyclase assayed in sonicates of rat parietal cells (Schepp et al a). Inhibition of prostaglandin biosynthesis by indomethacin did not diminish the effect of somatostatin in rabbit gastric glands (Nylander, Bergqvist & Obrink, 1985). It is therefore unlikely that prostaglandins mediate the inhibitory effect of somatostatin. Pre-incubation with pertussis toxin abolished the inhibitory effect of somatostatin on histamine-stimulated aminopyrine accumulation (Park et al. 1987; Atwell & Hanson, 1988) and cyclic AMP content (Park et al. 1987). The most reasonable interpretation of all of the above data is that somatostatin receptors on parietal cells are coupled through Gi to inhibition of adenylate cyclase. Some observations suggest, however, that there may be a second site of action for somatostatin. Thus, in dog parietal cells (Park et al. 1987), but not rabbit gastric glands (Chew, 1983; Nylander et al. 1985), somatostatin inhibited secretory activity induced by dibutyryl cyclic AMP. Pre-incubation with pertussis toxin did not affect this inhibitory action of somatostatin against aminopyrine accumulation induced by dibutyryl cyclic AMP (Park et al. 1987). Cytosolic binding sites for somatostatin were detected after subcellular fractionation of homogenates prepared from rat gastric mucosa (Reyl & Lewin, 1982), or from cell preparations enriched with rabbit parietal cells (Arilla, Lopez-Ruiz, Gonzalez- Guijarro & Prieto, 1985). Finally, somatostatin is a potent activator of phosphoprotein phosphatases in cytosolic extracts from rat fundic mucosa (Reyl & Lewin, 1982). It might be worth investigating whether somatostatin could cause the specific dephosphorylation of any32p-labelled proteins in dog parietal cells Adenosine stimulated with histamine. In dog and guinea-pig parietal cells adenosine analogues inhibited secretory activity induced by histamine but not that induced by carbachol or by dibutyryl AMP (Gerber, Nies & Payne, 1985; Heldsinger, Vinik & Fox, 1986). The relative potency of the adenosine analogues suggested an action at anal receptor (see Daly, 1985, for review of adenosine receptors). The action of adenosine in dog parietal cells was probably not mediated by prostaglandins (Gerber et al. 1985). L-N6-Phenylisopropyl adenosine (100 nm) caused a slight, but significant, reduction of the cyclic AMP content of dog parietal cells stimulated with histamine and a cyclic AMP phosphodiesterase inhibitor. Higher concentrations of L- N8-phenylisopropyl adenosine did not affect cyclic AMP content, but had a greater inhibitory effect on aminopyrine accumulation than 100 nm-l-n-phenylisopropyl adenosine (Gerber et al. 1985). The possibility that high concentrations of the adenosine analogue might be stimulating adenylate cyclase in another cell type in the preparation was proposed but not tested. In conclusion, the possibility that adenosine Al receptors couple to Gi in dog parietal cells and thereby inhibit adenylate cyclase remains to be established. An alternative possibility that adenosine might down-regulate histamine receptors should perhaps be investigated. Endogenous adenosine modulated the secretory response of dog parietal cells to histamine (Gerber & Payne, 1988). What could be the physiological significance of this effect? Adenosine production is increased in cells which suffer a drop in ATP and a rise in

9 REGULATION OF GASTRIC ACID SECRETION AMP (Arch & Newsholme, 1978). Adenosine could therefore act as a feedback regulator to prevent overstimulation of the parietal cell and depletion of ATP by too much K+-H+- ATPase activity. Alternatively, adenosine might reduce secretory activity in haemorrhagic shock when its production would be increased by local anoxia. Arguments for the fundamental importance of adenosine as a direct regulator of parietal cell function are not strengthened by results which show that although adenosine analogues can inhibit acid secretion in the rat in vivo (Scarpignato, Tramacere, Zappia & Del Soldato, 1987), there is no direct action of adenosine agonists on rat parietal cells in vitro (Puurunen, Ruoff & Schwabe, 1987; Atwell & Hanson, 1988). Oxyntomodulin? Oxyntomodulin did not inhibit histamine-stimulated aminopyrine accumulation in isolated rat parietal cells (Gehl, Jeppesen, Poulsen & Holst, 1988). In vivo oxyntomodulin was a more effective inhibitor of pentagastrin-stimulated acid secretion than that induced by histamine (Dubrasquet, Audousset-Puech, Martinez & Bataille, 1986). The rat parietal cells did not exhibit a secretory response to gastrin. If this lack of response were due to damage to the putative gastrin receptor, then the possibility of a direct inhibitory effect of oxyntomodulin against gastrin in the rat parietal cell cannot be completely ruled out. Actions of cyclic AMP There is good evidence from a variety of techniques that histamine activates cyclic AMPdependent protein kinase in stomach cells (Mangeat, Marchis-Mouren, Cheret & Lewin, 1980; Jackson & Sachs, 1982; Mangeat, Gespach, Marchis-Mouren & Rosselin, 1982; Chew, 1985a). Both types I and II cyclic AMP-dependent protein kinase were present in an enriched, but not pure, rabbit parietal cell fraction (Chew, 1985a), but- histamine appeared only to activate the cytosolic type I kinase. Potential targets for cyclic AMPdependent protein kinase are discussed later. 615 EPIDERMAL GROWTH FACTOR-A REGULATOR OF CYCLIC AMP PHOSPHODIESTERASE? This section considers the inhibitory action of epidermal growth factor (EGF) on gastric acid secretion, and examines the hypothesis that the mechanism of action of this agent might involve an increase in cyclic AMP phosphodiesterase activity in the parietal cell. Epidermal growth factor is a single chain polypeptide of fifty-three amino acid residues and Mr of 6045 (mouse EGF). It is present in saliva and is resistant to degradation by pepsin in the stomach. As well as having a short-term inhibitory effect on acid secretion, in the long term it promotes proliferation of the gastric mucosa and numerous other cell types (see Gregory, 1985 for review). Epidermal growth factor receptors were detected on gastric glands from guinea-pigs (Forgue-Lafitte, Kobari, Gespach, Chamblier & Rosselin, 1984) and parietal cells from dog (Chen, Amirian & Soll, 1984) by binding experiments using "2II-labelled EGF as a probe. Use of an EGF receptor antibody coupled with immuno-electron microscopy showed EGF receptors to be present on the basolateral membranes of human gastric parietal cells (Mori, Morishita, Sakai, Kurimoto, Okamoto, Kawamoto & Kuroki, 1987). The absence of receptors from the apical membrane of these cells argues against a role for EGF from the gastric lumen in the regulation of parietal cell activity. Epidermal growth factor inhibited aminopyrine accumulation stimulated by histamine in 24 EPH 74

10 616 P. J. HANSON AND J. F. HATT rat parietal cells but was ineffective if secretory activity was stimulated by carbachol or by dibutyryl cyclic AMP or if 01l mm-ibmx was present with histamine (Shaw, Hatt, Anderson & Hanson, 1987). Similar results were reported in an abstract by Chen et al. (1984) using dog parietal cells. Epidermal growth factor inhibited the stimulation by histamine of the cyclic AMP content of a rat stomach cell population containing greater than 80% parietal cells (Hatt & Hanson, 1988). The half-maximally effective concentration of EGF for inhibition of cyclic AMP content was 3 9 nm and the same measurement for inhibition of aminopyrine accumulation was 30 nm. The addition of 0Ml mm-ibmx blocked the effect of EGF on cyclic AMP content. This action of IBMX could not be ascribed to its enhancement of the response to histamine. These results suggest that EGF inhibits histamine-stimulated acid secretion by decreasing parietal cell cyclic AMP content. 'In rabbit gastric glands EGF inhibited aminopyrine accumulation stimulated by both histamine and by dibutyryl cyclic AMP (Dembin'ski, Drozdowicz, Gregory, Konturek & Warzecha, 1986). These results were interpreted as suggesting a site for EGF action distal to the production and hydrolysis of cyclic AMP. However, the inhibitory action of EGF against dibutyryl cyclic AMP-stimulated secretion appeared to be competitive, but its action against histamine-stimulated secretion was non-competitive. Furthermore close inspection of the data suggests that the half-maximally effective concentration of EGF required for inhibition of aminopyrine accumulation stimulated by dibutyryl cyclic AMP was possibly 10-fold higher than that required to inhibit histamine-stimulated secretion. A somewhat similar finding was mentioned by Berglindh (1984). The action of EGF in inhibiting acid secretion by sheets of guinea-pig gastric mucosa was slower when the tissue had been stimulated with dibutyryl cyclic AMP than with histamine (Finke, Rutten, Murphy & Silen, 1985). One interpretation of these findings is that EGF acts at separate sites to inhibit secretion induced by histamine and by dibutyryl cyclic AMP. A potent inhibitor of histamine-stimulated cyclic AMP production in parietal cells is PGE2. Furthermore EGF was shown to increase prostaglandin production by the isolated perfused rat stomach (Chiba, Hirata, Taminato, Kadowaki, Matsukura & Fujita, 1982). It is therefore necessary to consider whether prostaglandins might mediate the inhibitory action of EGF. The following results do not support this idea. Firstly, the action of EGF on aminopyrine accumulation in rat parietal cells was not prevented by the specific cyclooxygenase inhibitor flurbiprofen, or by the cyclo-oxygenase and lipoxygenase inhibitor nordihydroguaiaretic acid (Shaw et al. 1987). Secondly, the action of PGE2, unlike that of EGF, was not prevented by IBMX (Atwell & Hanson, 1988). Thirdly, the concentration of PGE2 in the medium surrounding enriched parietal cell preparations, and containing 200 nm-egf, was 84 pm which is substantially below the concentration of PGE2 required for half-maximal inhibition of histamine-stimulated aminopyrine accumulation (24 nm) (Hatt & Hanson, 1988). The simplest explanation for the prevention of the inhibitory action of EGF by IBMX is that EGF was decreasing parietal cell cyclic AMP content by increasing cyclic AMP phosphodiesterase activity. IBMX would inhibit the phosphodiesterase on which EGF acted, and consequently there would be no stimulation of cyclic AMP break-down. This suggestion is supported indirectly by the absence of an effect of EGF on histaminestimulated adenylate cyclase in homogenates of parietal cells (J. F. Hatt & P. J. Hanson unpublished work). However, direct proof of the idea has not yet been obtained.

11 REGULATION OF GASTRIC ACID SECRETION 617 POLYPHOSPHOINOSITIDE METABOLISM AND INTRACELLULAR CALCIUM Muscarinic cholinergic agonists The tritiated muscarinic cholinergic antagonist, quinuclidinyl benzilate, can be used in binding studies to probe for the presence of muscarinic cholinergic receptors. Use of this agent enabled the identification of a single population of bindingsites on rat parietal cells. The pharmacological specificity of these sites was appropriate to muscarinic cholinergic receptors (Ecknauer, Thompson, Johnson & Rosenfeld, 1980). Subsequent work (Ecknauer, Dial, Thompson, Johnson & Rosenfeld, 1981) linked this receptor with the stimulation of aminopyrine accumulation by carbachol. Recent work has focused on the muscarinic receptor subtype present on the parietal cell. The receptor subtype can be identified by comparing the relative potencies of the antagonists pirenzepine and atropine. Pirenzepine was a much less potent antagonist of carbachol-stimulated aminopyrine accumulation than was atropine (Rosenfeld, 1983). Pirenzepine was also much less potent than atropine in inhibiting the binding of N- methylscopolamine to parietal cells, or in inhibiting the effect of carbachol on the accumulation of inositol phosphates in parietal cells (Pfeiffer, Rochlitz, Herz & Paumgartner, 1988). These results all suggest that an M2 muscarinic cholinergic receptor is present on parietal cells. Interestingly, pirenzepine is used as an antisecretory agent to facilitate the healing of peptic ulcer because it has fewer side-effects than muscarinic receptor antagonists which are not subtype specific. The results mentioned above make it unlikely that the antisecretory effects of pirenzepine are exerted directly on the parietal cell. Rather, a presynaptic muscarinic receptor could be involved in the mechanism by which pirenzepine inhibits acid secretion. Activation of the M2 muscarinic receptor has been linked, in a number of studies, with the activation of a phospholipase C and the break-down of phosphatidylinositol 4,5- bisphosphate to form 1,2-sn-diacylglycerol (henceforth referred to as diacylglycerol) and inositol polyphosphates. Thus, carbachol increased the inositol trisphosphate content of parietal cells from rabbit (Chew & Brown, 1986), rat (Puurunen & Schwabe, 1987; Pfeiffer et al. 1988) and dog (Chiba, Fisher, Park, Seguin, Agranoff & Yamada, 1988). Chiba et al. (1988) showed that the production of total inositol phosphates was associated with a decline in phosphatidylinositol 4,5-bisphosphate content. Furthermore, the similarity between the concentrations of carbachol required for half-maximal effect on stimulation of aminopyrine accumulation, production of inositol phosphates and for decreasing phosphatidylinositol content was such as to suggest that these effects were interrelated. As mentioned above carbachol might also be expected to increase the diacylglycerol content of parietal cells. Such an effect was found in rat parietal cells (Pfeiffer et al. 1987). Diacylglycerol activates an enzyme called protein kinase C by effecting its association with the cell membrane. Since dog parietal cells incubated with carbachol exhibited an increased association of protein kinase C with a membrane fraction (Park et al. 1987), it also seems likely that carbachol increases diacylglycerol in dog parietal cells. The possible roles of protein kinase C are dealt with later. Cholinergic stimulation of acid secretion is strongly dependent on the presence of extracellular Ca2+ (Berglindh, Sachs & Takeguchi, 1980; Soll, 1981), and is associated with an influx of Ca2+ into the parietal cell (Soll, 1981) or gastric glands (Muallem & Sachs, 1985). Muallem & Sachs (1984), using dog parietal cells loaded with the fluorescent Ca2+ indicator, Quin-2, suggested that the increase in intracellular Ca2+ induced by carbachol was entirely dependent upon the entry of extracellular Ca2+ into the cell. Later experiments 24-2

12 618 P. J. HANSON AND J. F. HATT using Fura-2 in rabbit parietal cells (Chew & Brown, 1986), and microspectrofluorimetric measurements from single parietal cells in Fura-2-loaded rabbit gastric glands (Negulescu & Machen, 1988a, b) have provided a different picture. These experiments with Fura-2 showed that carbachol induced an initial rapid rise in cellular Ca2", which was due to the mobilization of intracellular stores. This initial effect was independent of extracellular Ca2". The intracellular Ca2l then fell but was maintained above basal concentrations provided that extracellular Ca2" was present. The implication of these results is that the release of Ca2" from intracellular stores was followed by a stimulation of Ca2" influx across the plasma membrane. The putative membrane Ca2l channel was insensitive to the Ca2" channel blockers nicardipine and nifedipine (Chew & Brown, 1986; Negulescu & Machen, 1988 a), but was blocked by La3" (Negulescu & Machen, 1988b). The initial spike may not have been detectable in cells loaded with Quin-2 (Muallem & Sachs, 1984) because it is necessary to load a much higher concentration of this indicator than Fura-2 to get an adequate signal. At such concentrations Quin-2 may have buffered Ca2" transients. In many types of cell, including parietal cells (Tsunoda, Takeda, Asaka, Nakagaki & Sasaki, 1988), inositol 1,4,5- trisphosphate causes mobilization of Ca2" from intracellular stores, and inositol 1,4,5- trisphosphate may therefore be responsible for the initial rise in intracellular Ca2". Whether inositol polyphosphates are responsible for inducing the subsequent Ca2" influx across the cell membrane is presently uncertain (Berridge, 1987). Stimulation of rabbit gastric glands with carbachol for more than 3 min depleted the intracellular Ca2" stores. Thus no further response to carbachol was detectable provided that reloading from the extracellular medium was prevented by the absence of Ca2l from this medium, or by the presence of La3+ (Negulescu & Machen, 1988b). A probable explanation is that during the 3 min stimulation with carbachol intracellular Ca2" was extruded from the cell by a calmodulin-dependent Ca2+-ATPase pump (Muallem & Sachs, 1985). The nature of the leak which allows the refilling of intracellular Ca2" stores from the medium is uncertain. This leak was not prevented by the presence of atropine, but it was blocked by La3" (Negulescu & Machen, 1988 b). If stimulation was terminated at the peak of the initial spike of intracellular Ca2" then significant reloading of intracellular Ca2+ stores could occur from the cytosol (Negulescu & Machen, 1988b). In summary, activation of the M2 muscarinic cholinergic receptor on the parietal cell causes the break-down of phosphatidylinositol 4,5-bisphosphate to yield inositol trisphosphate and diacylglycerol. The inositol trisphosphate causes mobilization of intracellular Ca2+. The mechanism effecting the subsequent increase in Ca2" influx into the cell is presently unclear. Gastrin There is good evidence for a gastrin receptor on dog parietal cells. Specific binding of I251-labelled-[Leu`5]gastrin correlated with the proportion of parietal cells in cell fractions prepared by elutriation and density gradient centrifugation from dog stomach (Soll, Amirian, Thomas, Reedy & Elashoff, 1984). The gastrin receptor exhibited equal recognition of gastrin and cholecystokinin octapeptide (CCK-8) (Soll et al. 1984). Gastrin-stimulated oxygen consumption (Soll, 1978) and aminopyrine accumulation (Soll, 1980a) in dog parietal cells was unaffected by the pharmacological blockade of histamine H2 or muscarinic cholinergic receptors. Large effects of gastrin on secretory activity in rabbit gastric glands were only seen in the presence of dithiothreitol or IBMX (Chew & Hersey, 1982). These effects in rabbit gastric glands were partly due to stimulation

13 REGULATION OF GASTRIC ACID SECRETION of histamine release by gastrin (Chew & Hersey, 1982). Indeed Berglindh (1984) questioned the existence of a direct receptor-mediated effect of gastrin on rabbit parietal cells. However, Chew & Brown (1986) found effects of gastrin and CCK-8 on intracellular Ca2" in highly purified rabbit parietal cells (see below). Such effects are difficult to explain if gastrin receptors were absent, for there were virtually no other cells to act as a source of histamine, and any histamine released from another cell type would have been substantially diluted in the incubation medium. Stimulation of aminopyrine accumulation by gastrin has also been demonstrated in guinea-pig parietal cells (Tsunoda, 1987), but we are not aware of any reports of consistent stimulation of aminopyrine accumulation by gastrin in rat parietal cells. Gastrin probably activates a phospholipase C because it increased formation of inositol trisphosphate in parietal cells from dog (Chiba et al. 1988). The dependence on the concentration of gastrin of aminopyrine accumulation and inositol phosphate production in dog parietal cells suggested that changes in inositol phosphate production might be linked with the effect of gastrin on the secretory response (Chiba et al. 1988). Induction of an initial rise in intracellular Ca2" by gastrin/cck-8, if it involved mobilization of intracellular stores by inositol 1,4,5-trisphosphate, might be expected to be largely independent of extracellular Ca2". Such an independence from extracellular Ca2" was demonstrated by Chew & Brown (1986) and by Tsunoda et al. (1988). Prior incubation with carbachol virtually abolished the subsequent effects of CCK-8 on intracellular Ca2" in rabbit parietal cells, while previous incubation with CCK-8 attenuated the subsequent response to carbachol (Chew & Brown, 1986). Gastrin/CCK-8 and carbachol therefore appear to mobilize the same intracellular pool of Ca2". Tsunoda & Matsumiya (1987) found that gastrin-induced mobilization of intracellular Ca2" caused a depolarization of the guinea-pig parietal cell membrane, which was possibly the result of an enhanced chloride efflux. If gastrin activates a phosphoinositide-specific phospholipase C then it would generate diacylglycerol as well as inositol phosphates. Indirect evidence that diacylglycerol is produced by gastrin is its induction of an increase in the protein kinase C activity associated with a membrane fraction from dog parietal cells (Park et al. 1987). A sustained effect of gastrin on aminopyrine accumulation was dependent on extracellular Ca2" (Berglindh et al. 1980; Soll, 1981; Tsunoda, 1987). It is likely therefore that a gastrin-stimulated influx of Ca2" across the plasma membrane (Tsunoda, 1987) is necessary for the maintenance of the secretory response. In conclusion, carbachol and gastrin/cck-8 probably activate the same intracellular signalling system. They both induce inositol trisphosphate production, they both cause a change in the subcellular distribution of protein kinase C (Park et al. 1987) and they seem to mobilize the same intracellular pool of Ca2". Histamine Until recently it was thought that the actions of histamine were mediated by cyclic AMP and those of muscarinic cholinergic agonists and gastrin by Ca2". Then Chew (1986), using rabbit gastric glands, and Chew & Brown (1986) using rabbit parietal cells, showed that histamine elevated intracellular Ca2" as detected by using the fluorescent indicators Quin- 2 and Fura-2 respectively. This finding was confirmed by microspectrofluorimetric measurements on single parietal cells in rabbit gastric glands (Negulescu & Machen, 1988 a). The pattern of response to histamine was qualitatively the same as that with carbachol. That is, there was an initial spike of intracellular Ca2", which was independent of extracellular Ca2". Afterwards intracellular Ca2" declined to control levels if extracellular 619

14 620 P. J. HANSON AND J. F. HATT Ca2" was absent, or declined to a plateau significantly above control concentrations if extracellular Ca2" was present (Negulescu & Machen, 1988a). The spike response to histamine was, however, quantitatively less than that to carbachol. An effect of histamine on intracellular Ca2" was also evident in pig parietal cells (Mardh et al. 1987). The effect of histamine was mediated through an H2 receptor because it was prevented by H2 receptor antagonists (Chew, 1986; Negulescu & Machen, 1988a), while H1 receptor antagonists had no effect (Negulescu & Machen, 1988 a). Prior stimulation with carbachol virtually abolished the subsequent response of intracellular Ca2+ to histamine, while preincubation with histamine attenuated the carbachol response (Chew & Brown, 1986; Negulescu & Machen, 1988a). Therefore histamine and carbachol released Ca2+ from the same intracellular pool. A reasonable hypothesis might be that the histamine H2 receptor was not only coupled to adenylate cyclase but also to a polyphosphoinositide-specific phospholipase C. However, histamine did not stimulate production of inositol trisphosphate in parietal cells from rabbit (Chew & Brown, 1986), rat (Puurunen & Schwabe, 1987) or dog (Chiba et al. 1988). Furthermore histamine, unlike carbachol and pentagastrin, did not induce an increase in the membrane-associated activity of protein kinase C (Park et al. 1987). Such an action of histamine would have been expected had it caused the break-down of polyphosphoinositides to diacylglycerol and inositol phosphates. An alternative possibility is that cyclic AMP might either directly or indirectly induce elevation of intracellular Ca2+. Forskolin activates the catalytic subunit of adenylate cyclase and is a useful tool to elevate cyclic AMP without activating the histamine receptor. Forskolin increased intracellular Ca2+ in rabbit gastric glands (Chew, 1986) and in pig parietal cells (Mardh et al. 1987). These findings with forskolin support a role for cyclic AMP in mobilizing Ca2+, but the inability of dibutyryl cyclic AMP to increase intracellular Ca2+ (Mardh et al. 1987; Negulescu & Machen, 1988a), unless cells were stimulated with histamine a short time previously (Negulescu & Machen, 1988 a), is at present not readily explained. Finally, the sustained increase in intracellular Ca21 may not be essential for the secretory response to histamine. Removal of extracellular Ca2' abolished this sustained elevation of intracellular Ca2+ (Negulescu & Machen, 1988 a) but did not affect the histaminestimulated secretory activity in rabbit gastric glands (Berglindh et al. 1980; Chew, 1985b). In conclusion, histamine increases intracellular Ca2+ in parietal cells in addition to elevating cyclic AMP. It seems possible that changes in Ca2+ may be secondary to changes in cyclic AMP. Gastrotropin (porcine ileal polypeptide) There is a polypeptide which can be isolated from the distal region of the ileum of the dog and pig, and which can stimulate gastric acid secretion both in vivo and by isolated parietal cells in vitro (Defize, Wider, Walz & Hunt, 1988). Half-maximally effective concentrations of this peptide for stimulation of aminopyrine accumulation by isolated parietal cells were close to basal circulating levels of the peptide in adult pigs. Porcine gastrotropin has a Mr of and contains 127 amino acid residues. The amino acid sequence exhibits 35 % homology with the amino acid sequence of rat liver fatty acid binding protein (Walz, Wider, Snow, Dass & Desiderio, 1988). Porcine ileal polypeptide increased cytoplasmic Ca2+ in Fura-2-loaded guinea-pig parietal cells (Tsunoda & Wider, 1987). It seems likely that this peptide will prove to act via its own receptor to induce phosphatidylinositol 4,5-bisphosphate break-down in a similar manner to carbachol and gastrin.

15 REGULATION OF GASTRIC ACID SECRETION 621 Calcium-activated protein kinases A Ca2+-calmodulin-activated protein kinase was present in a cytosolic extract derived from a homogenate of a cell preparation containing 79% rabbit parietal cells (Oddsdottir, Modlin, Zucker, Zdon & Goldenring, 1987). The kinase activity was detected by the phosphorylation of an endogenous 100 kda protein. It is presently unclear whether this kinase might be involved in mediating the effects of rises in Ca2" in the parietal cell. Gastric mucosal membranes enriched in K+-H+-ATPase were found to possess endogenous Ca2+-calmodulin-dependent protein kinase activity which phosphorylated an 88 kda protein (Shaltz, Bools & Reimann, 1981). Since the starting material was gastric mucosa, a definite association of the substrate and kinase with the parietal cell cannot be made. PROTEIN KINASE C Protein kinase C was first identified in extracts of brain as an enzyme which was dependent on Ca2" and phospholipid, particularly phosphatidylserine, for activity. 1,2-sn-Diacylglycerols reduced the Ca2" requirement of the enzyme to concentrations close to those found in the cytosol of resting cells (Nishizuka, 1986). More recently it has emerged that there are multiple forms of protein kinase C which appear to differ in their tissue expression and in their sensitivity to Ca2" and diacylglycerol (Nishizuka, 1988). The tumour-promoting phorbol ester, 12-o-tetradecanoylphorbol- 13-acetate (TPA), can substitute for diacylglycerol as an activator of protein kinase C, and has proved a valuable tool to investigate the function of the enzyme. In intact cells activation of protein kinase C seems to involve its translocation from the cytosol to the cell membrane where it can come into contact with diacylglycerol and phosphatidylserine. The functions of protein kinase C in the regulation of cellular activity seem to be several. Activation of protein kinase C can synergize with elevation of intracellular Ca2" to enhance the response effected by receptors which activate phosphatidylinositol 4,5-bisphosphate break-down to inositol phosphates and diacylglycerol. Protein kinase C can exert feedback inhibition of such receptors, and finally it can be involved in 'cross-talk' with receptors coupled to adenylate cyclase. Whether these actions occur in parietal cells will now be discussed. Involvement in stimulation of acid secretion by gastrin and carbachol Pentagastrin, carbachol and TPA increased the association of protein kinase C with membranes in dog parietal cells, and therefore can be presumed to activate it (Park et al. 1987). Protein kinase C has also been identified in rabbit (Chew, 1985 a), rat (Anderson & Hanson, 1985) and guinea-pig (Beil, Mannschedel & Sewing, 1987) parietal cells. Evidence that such an activation could stimulate acid secretion is as follows. Firstly, TPA stimulated basal aminopyrine accumulation in rabbit parietal cells (Brown & Chew, 1987). Such an effect was not found in rat parietal cells. An explanation for the discrepancy is suggested by Hatt & Hanson (1989). Secondly, TPA enhanced the response of rabbit gastric glands to submaximal concentrations of dibutyryl cyclic AMP (Brown & Chew, 1987). This result is compatible with the finding, mentioned earlier, that carbachol can enhance the secretory response to dibutyryl cyclic AMP. Thirdly, TPA further increased aminopyrine accumulation in rat parietal cells in which secretory activity had been stimulated by the presence of 100 mm-k' in the incubation medium. The half-maximally effective concentration of TPA was 1 1 nm (Hatt & Hanson, 1989). This effect did not involve changes in cellular cyclic AMP, and was mimicked by 1-oleoyl-2-acetyl-glycerol (OAG) which also activates protein kinase C when added to intact cells.

16 622 P. J. HANSON AND J. F. HATT There is thus evidence that activation of protein kinase C in rat and rabbit parietal cells can, under rather particular circumstances, enhance secretory activity. Investigation of the effects of Ca2l on the stimulatory effects of TPA have been hampered by the apparent inability of Ca2" ionophores to raise intracellular Ca2" without exerting non-specific effects on secretory activity in parietal cells (G. P. Shaw, J. F. Hatt & P. J. Hanson, unpublished work; see also Chew, 1985b; Muallem, Fimmel, Pandol & Sachs, 1986). Feedback inhibition of the actions of carbachol TPA inhibited aminopyrine accumulation induced by carbachol in rat parietal cells (Anderson & Hanson, 1984), and in rabbit gastric glands (Muallem et al. 1986; Brown & Chew, 1987). TPA also reduced the stimulation of inositol trisphosphate production induced by carbachol in a cell fraction containing 60-70% rat parietal cells (Puurunen, Lohse & Schwabe, 1987). Protein kinase C may exert negative feedback modulation of the muscarinic cholinergic receptor in parietal cells thereby moderating the response to carbachol. The exact site at which protein kinase C exerts this effect remains to be established. Effects on the cyclic AMP pathway TPA inhibited histamine-stimulated aminopyrine accumulation in both crude and enriched preparations of rat parietal cells, with half-maximal effect occurring at 2-7 nm- TPA (Anderson & Hanson, 1984). No effects were found with a phorbol ester which did not activate protein kinase C. The relative potency of phorbol esters in inhibiting histamine-stimulated aminopyrine accumulation paralleled their ability to activate isolated protein kinase C (Anderson & Hanson, 1985). OAG also showed a dose-dependent inhibition of histamine-stimulated aminopyrine accumulation. Similar results have been obtained by Brown & Chew (1987) using rabbit gastric glands and parietal cells. TPA inhibited histamine-stimulated acid secretion in vivo (Shaw & Hanson, 1986), and in rat intact gastric mucosa in vitro (Pearce, Baird, Williamson & Evans, 1981). These results made it likely that inhibition of aminopyrine accumulation by TPA in vitro genuinely represented an inhibition of acid secretion, and not simply an increased efflux of acid from intracellular secretory canaliculi. Beil et al. (1987) showed that inhibition of the K+-H+-ATPase purified from guinea-pig parietal cells was half-maximal at a TPA concentration of 6 5 /SM. This value was nearly 1000-fold higher than the half-maximally effective concentration of TPA for inhibition of histamine-stimulated aminopyrine accumulation in guinea-pig parietal cells. Furthermore no experiments were performed with phorbol esters, which do not activate protein kinase C, so as to establish the structural specificity of the inhibitory effect of TPA on the K+-H+- ATPase. High extracellular K+ is thought to activate acid secretion by acting at a site very close to the K+-H+-ATPase. Therefore, if TPA were to inhibit histamine-stimulated aminopyrine accumulation by inhibition of the proton pump then it should also inhibit aminopyrine accumulation induced by 100 mm-k+. As mentioned previously, low concentrations of TPA stimulated aminopyrine accumulation in rat parietal cells incubated with 100 mm-k+. Higher concentrations of TPA inhibited this stimulatory effect, but at 100 nm-tpa aminopyrine accumulation was the same as that in cells incubated with 100 mm-k' alone, and substantially above that found in cells incubated in a medium containing 4 5 mm-k' and no secretagogues (Hatt & Hanson, 1989). It is extremely unlikely that the inhibitory effects of low nanomolar concentrations of TPA on histamine-stimulated aminopyrine accumulation are exerted by inhibition of the proton pump. Similar

17 REGULATION OF GASTRIC ACID SECRETION <100 * 80 CUE 60- E E 40 - CU 20 C log [histamine] (M) Fig. 3. Effect of pre-incubation of intact parietal cells in the presence (A) or absence (AL) of 100 nm-tpa for 10 min on histamine-stimulated adenylate cyclase activity assayed subsequently in cell sonicates. Results are means + S.E. of the mean from five experiments and are expressed as a percentage of the stimulation of adenylate cyclase activity in control cell homogenates by 0-5 mm-histamine, which was pmol cyclic AMP formed/ 10 min per 106 cells. Adenylate cyclase activity in the absence of histamine was (picomoles cyclic AMP formed/ 10 min per 106 cells) and for cells incubated respectively in the absence and presence of TPA. *, P < 0-05; **, P < 0-01; ***, P < for the effect of pre-incubation with TPA by paired t test. Some symbols have been omitted to allow visualization of the standard error bars. arguments can be used to refute the possibility that inhibitory effects are mediated by a protonophoric action of TPA causing the dissipation of H+ accumulated inside secretory canaliculi. In rat parietal cells TPA inhibited dibutyryl cyclic AMP-stimulated aminopyrine accumulation (Anderson & Hanson, 1984), with a potency similar to that for inhibition of histamine-stimulated aminopyrine accumulation (Hatt & Hanson, 1989). A similar effect of TPA was found in guinea-pig parietal cells (Beil et al. 1987). However, as mentioned above, in rabbit gastric glands TPA enhanced the response to a submaximally effective concentration of dibutyryl cyclic AMP (Brown & Chew, 1987). This difference is not due to a component of the secretory response in rat parietal cells being a consequence of interaction between endogenous histamine and dibutyryl cyclic AMP. Thus 10 /tmcimetidine, which should inhibit any contribution of histamine to secretory activity had no effect on aminopyrine accumulation stimulated by 1 mm-dibutyryl cyclic AMP in rat parietal cells (Hatt & Hanson, 1989). In conclusion, there seems to be an inhibitory site for the action of protein kinase C in rat parietal cells which is located distal to the production and hydrolysis of cyclic AMP but proximal to the K+-H+-ATPase pump. This site would appear to be absent from rabbit parietal cells. In rat parietal cells there seems to be a second inhibitory site close to the histaminestimulated adenylate cyclase. Thus TPA inhibited the histamine-induced elevation of the cyclic AMP content of parietal cells with a half-maximally effective concentration of 3 nm (Hatt & Hanson, 1989). Pre-incubation of intact cells with TPA also inhibited histaminestimulated adenylate cyclase activity assayed subsequently in cell homogenates (Fig. 3). Activation of protein kinase C in parietal cells by TPA can induce a strong inhibitory

18 624 P. J. HANSON AND J. F. HATT Table 2. Profiles for the action of inhibitors of the histamine pathway of acid secretion in isolated rat parietal cells Inhibition of dibutyryl Sensitivity of action cyclic AMP- to pre-incubation with Activity in Inhibitor stimulated secretion pertussis toxin presence of IBMX TPA Yes Little effect Yes Somatostatin * Major effect Yes PGE2 No Major effect Yes EGF No Major effect No Data from: Rosenfeld (1986); Shaw, Hatt, Anderson & Hanson (1987); Atwell & Hanson (1988); Hatt & Hanson (1989). * To our knowledge not known in rat, variable in other species - see this review. effect on histamine-stimulated aminopyrine accumulation. This pathway could be a means by which the muscarinic cholinergic receptor exerts negative modulation of the response to histamine in parietal cells. However, TPA, by producing a massive and sustained activation of protein kinase C, may make this interaction appear more prominent than it is in cells incubated with physiological secretagogues. Thus, as mentioned earlier, the net effect of carbachol is not to inhibit the secretory response to histamine, but generally to enhance it. An alternative explanation for the above findings with TPA and histamine-stimulated aminopyrine accumulation is that protein kinase C mediates the inhibitory mechanism of action of an exogenous inhibitor of acid secretion. If this inhibitor were to activate a phospholipase C which acted on a phospholipid other than phosphatidylinositol 4,5- bisphosphate then it could still produce diacylglycerol and activation of protein kinase C without mobilization of cell Ca2. Comparison of the inhibitory actions of somatostatin, PGE2 and EGF with that of TPA (Table 2) suggests that it is unlikely that their actions are mediated by the activation of protein kinase C. Inhibitory actions of substance P and of thyrotropin-releasing hormone against aminopyrine accumulation stimulated by histamine or dibutyrylcyclic AMP in rat parietal cells have recently been reported in two abstracts (Schepp, Tatge, Schusdziarra & Classen, 1988 a, b). The possibility that these agents could be activating protein kinase C remains to be evaluated. In conclusion, there is evidence for both stimulatory and inhibitory actions of protein kinase C in rat parietal cells. Whether these two sets of actions might be mediated by different isoforms of the enzyme is an intriguing possibility worthy of future consideration. TARGETS FOR INTRACELLULAR SIGNALLING SYSTEMS There must be links between the signal transduction systems and the machinery of acid secretion. Identification of likely targets for the signal transduction machinery and investigation of the substrate proteins for parietal cell protein kinases are complementary approaches towards establishing such links. The activity of the K+-H+-ATPase is increased by an 80 kda cytosolic protein, but whether this protein is a target for the signal transduction machinery has not been established (Bandopadhyay, Das, Wright, Nandi, Bhattacharyyay & Ray, 1987). An increased Cl- and K+ flux across the apical membrane (Fig. 1) is necessary to provide K+ at the external site of the K+-H+-ATPase. A labile, ATP-induced Cl- flux was found by

19 Table 3. REGULATION OF GASTRIC ACID SECRETION Proteins phosphorylated in response to activation of acid secretion in preparations of the rabbit gastric mucosa 625 Secretion Phosphorylated Preparation induced by protein (kda) Cell fraction (1) Gastric glands F 92 Unknown (2) Gastric glands H/F 120,94,80 Apical membrane H/IBMX (3) Parietal cells (98% enrichment) H 40, g pellet (4) Parietal cells (80% enrichment) H (Ca"+-depleted) 148,130,47, g supernatant 130,51, g pellet (5) Parietal cells (80% enrichment) H g supernatant Abbreviations: H, histamine; F, forskolin; IBMX, 3-isobutyl-1-methylxanthine. Sources: (1) Modlin, Schafer, Tyshkov, Ballantyne, Fratesi, Roberts & Zdon (1986); (2) Urushidani, Hanzel & Forte (1987); (3) Chew & Brown (1987); (4) Malinowska, Sachs & Cuppoletti (1988); (5) Oddsdottir, Goldenring, Adrian, Zdon, Zucker & Modlin (1988). Soumarmon, Abastado, Bonfils & Lewin (1980) in a crude preparation of pig or rabbit gastric microsomes. The effect of ATP was prevented by cyclic AMP-dependent protein kinase inhibitor protein, and could therefore suggest modulation of Cl- flux by cyclic AMPdependent protein kinase. This possibility does not appear to have been confirmed or followed up. Acid secretion is dependent upon the effective removal of OH- from the parietal cell. A potential control site is therefore carbonic anhydrase (Fig. 2). However, experiments with isolated rabbit parietal cells do not support a major regulatory role for this enzyme (Wollin, 1984). The basolateral Na+-H+ exchanger (Fig. 2) in rabbit parietal cells was activated when the cells were stimulated with histamine or forskolin (Muallem et al. 1988). This stimulation occurred even when the K+-H+-ATPase pump was inhibited by SCH28080 which is a competitive inhibitor for the external K+ site on the pump. Changes in exchanger activity were therefore not secondary to activation of the proton pump. The implication is that the basolateral Na+-H+ exchanger is a target for the signal transduction system. Results of the investigation of targets for histamine-activated protein phosphorylation in rabbit parietal cells are summarized in Table 3. These data were obtained by pre-incubating preparations with [32P]orthophosphate so that intracellular ATP and proteins became radioactively labelled. Incubation in the presence or absence of secretagogues was then performed. To assess the labelling of intracellular proteins cells were broken open, and in some cases subcellular fractionation was performed. Finally phosphorylation was measured by autoradiography after separation of proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis, sometimes coupled with isoelectric focusing. The most disappointing feature of this summary (Table 3) is the lack of agreement between the various investigations in respect of the molecular weight of phosphorylated proteins and/or in their subcellular location. One cause of variation in the molecular weight of phosphorylated bands may be proteolysis during homogenization prior to subcellular fractionation. Of those listed in Table 3 only Chew & Brown (1987) included a full range of protease inhibitors in their homogenization buffer. Another problem with this approach is that it is often difficult to identify the phosphorylated proteins or to be certain that they are directly involved in the secretory

20 626 P. J. HANSON AND J. F. HATT 205 vi Ca PS + + TPA + + Fig. 4. Autoradiogram showing effects of adding Ca'+, phosphatidylserine and TPA on phosphorylation of endogenous proteins in a 100:000 g supernatant derived from a cell fraction enriched with parietal cells. The concentration of phosphatidylserine (PS) was 20 jig/ml and that of TPA 20 ng/ml. The free-ca` concentration was calculated to be 100 p.m. The values on the left are in kilodaltons and denote the position of molecular weight markers. process. Urushidani et al. (1987) found that the phosphorylation of three proteins (120, 94, 80 kda) present in an 18 % Ficoll fraction was increased by stimulation with histamine plus forskolin or IBMX. This fraction was highly enriched in the K+-H+-ATPase and was thought to be constituted of apical membranes from parietal cells. The location of these phosphorylated proteins makes it likely that they could be involved in the secretory response. The 130 kda and the 47 kda proteins seen by Malinowska et a!. (1988) could be vinculin and an actin-binding protein respectively (Cuppoletti & Malinowska, 1988). Since histamine mobilizes Ca'+, some of the phosphorylations listed in Table 3 could have been mediated by Ca"~-dependent kinases. Exceptions are where Ca"~-depleted cells were used (Malinowska et al. 1988) or where additional experiments were performed with Ca`~ ionophores to clarify the position (Chew & Brown, 1987).

21 REGULATION OF GASTRIC ACID SECRETION As a firststep towards establishing the substrates for protein kinase C in parietal cells we have investigated the effects of TPA, Ca2" and phosphatidylserine on protein phosphorylation in g supernatants derived from homogenates of rat gastric mucosal cells enriched to 75 % with parietal cells (G. P. Shaw & P. J. Hanson, unpublished work). TPA and phosphatidylserine stimulated the phosphorylation of a protein of Mr (n = 6) (Fig. 4). Despite the inhibition by Ca2" of the phosphorylation induced by TPA and phospholipid, there is evidence that the 89 kda protein is a substrate for protein kinase C. Thus the phosphorylation was blocked by10 /IM-polymyxin B, at which concentration it may be a relatively specific inhibitor of protein kinase C (Wooten & Wrenn, 1984). Also, pre-incubation of intact cells with TPA to cause the removal of protein kinase C from the cytosol prevented the subsequent phosphorylation of an 89 kda band in g supernatants in the presence of phospholipid and TPA. The 89 kda band was more prominent in homogenates derived from preparations enriched in parietal cells. A protein with a widespread tissue distribution and a molecular weight in the range kda, 'the 87 kda protein', has been proposed as a major substrate for protein kinase C (Albert, Walaas, Wang & Greengard, 1986). It is possible that this protein is present in parietal cells. The mechanism of the inhibition of phosphorylation by Ca2` deserves further investigation. Changes in protein phosphorylation can be effected by the regulation of protein phosphatase activity, as well as by regulation of protein kinases. A histone phosphate phosphatase was found in rat heavy gastric membranes enriched with K+-H+-ATPase (Im, Blakeman, Bleasdale & Davis, 1987). These membranes were prepared by differential and density gradient centrifugation from the gastric mucosa of rats which hadbeen injected I h previously with carbachol to stimulate acid secretion. An endogenous protein kinase was present in the vesicles because incubating them with ATP caused several proteins to be phosphorylated. Stimulation of the above phosphatase by 2 mm-mg2t caused the dephosphorylation of endogenous phosphoproteins. Most interestingly this dephosphorylation was associated with a loss of the ability of extravesicular Kt to stimulate H+ accumulation by the vesicles. This effect seemed specific because H+ accumulation could be restored by the ionophore, valinomycin. The implication is that the activity of the K+ conductance in the apical membrane is controlled by phosphorylation/dephosphorylation. 627 SUMMARY AND FUTURE PROSPECTS Muscarinic cholinergic agonists and gastrin stimulate secretion by inducing the breakdown of phosphatidylinositol 4,5-bisphosphate, which leads to the generation of inositol trisphosphate, the release of Ca2` from intracellular stores and probably its influx across the cell membrane The secretagogue, histamine, increases the cyclic AMP content of parietal cells but also increases intracellular Ca2+. The mechanism by which histamine raises intracellular Ca2t probably does not involve inositol trisphosphate. The inhibitors PGE2, somatostatin and probably adenosine all act against the stimulation of adenylate cyclase by histamine. EGF may stimulate the break-down of cyclic AMP by phosphodiesterases, but this remains to be proven. Future work is likely to be most profitable in the following areas. Firstly, it seems unlikely that all of the direct modulators of parietal cell acid secretion have been discovered. For example, the inhibitory actions of pancreastatin are only just being characterized (Lewis, Zdon, Adrian & Modlin, 1988), and unlike the other well-established inhibitors discussed above, it inhibits secretion induced by both histamine and carbachol. Also, there are still gaps in the understanding of the signals generated by inhibitors like somatostatin and EGF.

22 628 P. J. HANSON AND J. F. HATT Secondly, identification of the specific roles and targets for calcium-calmodulin-activated protein kinases and members of the protein kinase C family is still a largely unexplored area. Thirdly, work with vesicles derived from the apical membrane of stimulated parietal cells should provide further understanding of the regulation of K+ and Cl- conductances. The purification of the proteins functioning as these conductances, the generation of monoclonal antibodies to them, and the cloning of their cdna would represent major advances. REFERENCES ALBERT, K. A.. WALAAS, S. I., WANG, J. K.-T. & GREENGARD, P. (1986). Widespread occurrence of '87 kda', a major specific substrate for protein kinase C. Proceedings of the National Academy of Sciences of the USA 83, ANDERSON, N. G. & HANSON, P. J. (1984). Inhibition of gastric acid secretion by a phorbol ester: effect of 12-o-tetradecanoylphorbol 13-acetate on aminopyrine accumulation by rat parietal cells. Biochemical and Biophysical Research Communications 121, ANDERSON, N. G. & HANSON, P. J. (1985). Involvement of calcium-sensitive phospholipid-dependent protein kinase in control of acid secretion by isolated rat parietal cells. Biochemical Journal 232, ARCH, J. R. S. & NEWSHOLME, E. A. (1978). The control of metabolism and the hormonal role of adenosine. In Essays in Biochemistry, vol. 14, ed. CAMPBELL, P. N. & ALDRIDGE, W. N., pp London, New York, San Francisco: Academic Press. ARILLA, E., LoPEz-Ruiz, M. P., GONZALEz-GuIJARRO, L. & PRIETO, J. C. (1985). Somatostatin binding sites in cytosolic fractions of parietal and non-parietal cells from rabbit fundic mucosa. Bioscience Reports 5, ATWELL, M. M. & HANSON, P. J. (1988). Effect of pertussis toxin on the inhibition of secretory activity by prostaglandin E.,, somatostatin, epidermal growth factor and 12-o-tetradecanoylphorbol 13-acetate in parietal cells from rat stomach. Biochimica et biophysica acta 971, BANDOPADHYAY, S., DAS, P. K., WRIGHT, M. V., NANDI, J., BHATTACHARYYAY, D. & RAY, T. K. (1987). Characteristics of a pure endogenous activator of the gastric H+,K+-ATPase system. Evaluation of the role as a possible intracellular regulator. Journal of Biological Chemistry 262, BATAILLE, D., GESPACH, C., COUDRAY, A. M. & ROSSELIN, G. (1981). 'Enteroglucagon': a specific effect on gastric glands isolated from the rat fundus. Evidence for an 'oxyntomodulin' action. Bioscience Reports 1, BATZRI, S. (1981). Direct action of somatostatin on dispersed mucosal cells from guinea-pig stomach. Biochimica et biophysica acta 677, BATZRI, S. & DYER, J. (1981). Aminopyrine uptake by guinea-pig gastric mucosal cells: mediation by cyclic AMP and interaction among secretagogues. Biochimica et biophysica acta 675, BATZRI, S., HARMON, J. W. & THOMPSON, W. F. (1982). Interaction of histamine with gastric mucosal cells. Effect of histamine agonists on binding and biological response. Molecular Pharmacology 22, BATZRI, S., THOMPSON, W. F. & TOLES, R. (1985). Distortion of H,-antagonist equilibrium constants by uptake in rabbit gastric mucosal cells. Pharmacology 30, BEIL, W., MANNSCHEDEL, W. & SEWING, K.-FR. (1987). Protein kinase C and parietal cell function. Biochemical and Biophysical Research Communications 149, BERGLINDH, T. (1984). The mammalian gastric parietal cell in vitro. Annual Reciev of Phy'siology 46, BERGLINDH, T. & HANSEN, D. (1984). Characterization of isolated gastric glands from the dog. Federation Proceedings 43, 996. BERGLINDH, T., HELANDER, H. F. & OBRINK, K. J. (1976). Effects of secretagogues on oxygen consumption, aminopyrine accumulation and morphology in isolated gastric glands. Acta physiologica scandinavica 97, BERGLINDH, T. & OBRINK, K. J. (1976). A method for preparing isolated glands from the rabbit gastric mucosa. Acta physiologica scandinavica 96,

23 REGULATION OF GASTRIC ACID SECRETION BERGLINDH, T., SACHS, G. & TAKEGUCHI, N. (1980). Ca2l-dependent secretagogue stimulation in isolated rabbit gastric glands. American Journal of Physiology 239, G BERRIDGE, M. J. (1987). Inositol trisphosphate and diacylglycerol; two interacting second messengers. Annual Reviewt of Biochemistry 56, BLACK, E. W., STRADA, S. J. & THOMPSON, W. J. (1988). Relationships of secretagogue-induced camp accumulation and acid secretion in elutriated rat gastric parietal cells. Journal of Pharmacological Methods 20, BLACK, J. W., DUNCAN, W. A. M., DURANT, C. J., GANELLIN, C. R. & PARSONS,E. M. (1972). Definition and antagonism of histamineh12-receptors. Nature 236, BROWN, M. R. & CHEW, C. S. (1987). Multiple effects of phorbol ester on secretory activity in rabbit gastric glands and parietal cells. Canadian Journal of Physiology and Pharmacology 65, CHEN, M. C., AMIRIAN, D. A. & SOLL, A. H. (1984). Epidermal growth factor (EGF) binding and inhibitory effect on acid secretion on isolated canine parietal cells. Federation Proceedings 43, CHEN, M. C. Y., AMIRIAN, D. A., TOOMEY, M., SANDERS, M. J. & SOLL, A. H. (1988). Prostanoid inhibition of canine parietal cells: mediation by the inhibitory guanosine triphosphate-binding protein of adenylate cyclase. Gastroenterology 94, CHEW, C. S. (1983). Inhibitory action of somatostatin on isolated gastric glands and parietal cells. American Journal of Physiology 245, G CHEW, C. S. (1985a). Parietal cell protein kinases. Selective activation of type I cyclic AMPdependent protein kinase by histamine. Journal of Biological Chemistry 260, CHEW, C. S. (1985b). Differential effects of extracellular calcium removal and nonspecific effects of Ca2l antagonists on acid secretory activity in isolated gastric glands. Biochimica et biophysica acta 846, CHEW, C. S. (1986). Cholecystokinin, carbachol, gastrin, histamine and forskolin increase [Ca2"], in gastric glands. American Journal of Physiology 250, G CHEW, C. S. & BROWN, M. R. (1986). Release of intracellular Ca2+ and elevation of inositol triphosphate by secretagogues in parietal and chief cells isolated from rabbit gastric mucosa. Biochimica et biophysica acta 888, CHEW, C. S. & BROWN, M. R. (1987). Histamine increases phosphorylation of 27- and 40 kda parietal cell proteins. American Journal of Physiology 253, G CHEW, C. S. & HERSEY, S. J. (1982). Gastrin stimulation of isolated gastric glands. American Journal of Physiology 242, G CHEW, C. S., HERSEY, S. J., SACHS, G. & BERGLINDH, T. (1980). Histamine responsiveness of isolated gastric glands. American Journal of Physiology 238, G CHIBA, T., FISHER, S. K., PARK, J., SEGUIN, E. B., AGRANOFF, B. W. & YAMADA, T. (1988). Carbamoylcholine and gastrin induce inositol lipid turnover in canine gastric parietal cells. American Journal of Physiology 255, G CHIBA, T., HIRATA, Y., TAMINATO, T., KADOWAKI, S., MATSUKURA, S. & FUJITA, T. (1982). Epidermal growth factor stimulates prostaglandin E release from isolated perfused rat stomach. Biochemical and Biophysical Research Communications 105, CUPPOLETTI, J. & MALINOWSKA, D. H. (1988). Cytoskeletal determinants of control of gastric acid secretion. Progress in Clinical and Biological Research 258, DALY, J. W. (1985). Adenosine receptors. In Advances in Cyclic Nucleotide and Protein Phosphorylation Research, vol. 19, ed. COOPER, D. M. F. & SEAMON, K. B., pp New York: Raven Press. DEFIZE, J., WIDER, M. D., WALZ, D. & HUNT, R. H. (1988). Isolation and partial characterization of gastrotropin from canine ileum: further studies of the parietal and chief cell response. Endocrinology 123, DEMBIN4SKI, A., DROZDOWICZ, D., GREGORY, H., KONTUREK, S. J. & WARZECHA, Z. (1986). Inhibition of acid formation by epidermal growth factor in the isolated rabbit gastric glands. Journal of Physiology 378, DIAL, E., THOMPSON, W. J. & ROSENFIELD, G. C. (1981). Isolated parietal cells: histamine response and pharmacology. Journal of Pharmacology and Experimental Therapeutics 219, DUBRASQUET, J. M., AUDOUSSET-PUECH, M.-P., MARTINEZ, J. & BATAILLE, D. (1986). Somatostatin enhances the inhibitory effect of oxyntomodulin and its C-terminal octapeptide on acid secretion. Peptides 7, suppi. 1,

24 630 P. J. HANSON AND J. F. HATT ECKNAUER, R., DIAL, E., THOMPSON, W. J., JOHNSON, L. R. & ROSENFELD, G. C. (1981). Isolated rat gastric parietal cells: cholinergic response and pharmacology. Life Sciences 28, ECKNAUER, R., THOMPSON, W. J., JOHNSON, L. R. & ROSENFELD, G. C. (1980). Isolated parietal cells: [:'H]QNB binding to putative cholinergic receptors. American Journal of Physiology 239, G EMAMI, S. & GESPACH, C. (1986). Desensitization by histamine of H 2-receptor activity in HGT-1 human cancerous gastric cells. Agents and Actions 18, FELLENIUS, E., ELANDER, B., WALLMARK, B., HAGLUND, U., OLBE, L. & HELANDER, H. (1979). Studies on acid secretory mechanism and drug action in isolated gastric glands from man. In Hormone Receptors in Digestion and Nutrition, ed. ROSSELIN, G., FROMAGEOT, P. & BONFILS, S., pp Amsterdam: Elsevier/North Holland Biomedical Press. FINKE, U., RUTTEN, M., MURPHY, R. A. & SILEN, W. (1985). Effects of epidermal growth factor on acid secretion from guinea-pig gastric mucosa: in vitro analysis. Gastroenterology 88, FORGUE-LAFITTE, M.-E., KOBARI, L., GESPACH, C., CHAMBLIER, M.-C. & ROSSELIN, G. (1984). Characterization and repartition of epidermal growth factor-urogastrone receptors in gastric glands isolated from young and adult guinea-pigs. Biochimica et biophysica acta 798, FORTE, J. G. & WOLOSIN, J. M. (1987). HCI secretion by the gastric oxyntic cell. In Physiology of the Gastrointestinal Tract, 2nd edn, ed. JOHNSON, L. R., pp New York: Raven Press. GEHL, J., JEPPESEN, J. L., POULSEN, S. S. & HOLST, J. J. (1988). The gastric acid secretagogue gastrinreleasing peptide and the inhibitor oxyntomodulin do not exert their effect directly on the parietal cell in the rat. Digestion 40, GERBER, J. G., NIES, A. S. & PAYNE, N. A. (1985). Adenosine receptors on canine parietal cells modulate gastric acid secretion to histamine. Journal of Pharmacology and Experimental Therapeutics 233, GERBER, J. G. & PAYNE, N. A. (1988). Endogenous adenosine modulates gastric acid secretion to histamine in canine parietal cells. Journal of Pharmacology and Experimental Therapeutics 244, GESPACH, C., BATAILLE, D., DUTRILLAUX, M.-C. & ROSSELIN, G. (1982). The interaction of glucagon, gastric inhibitory peptide and somatostatin with cyclic AMP production systems present in rat gastric glands. Biochimica et biophysica acta 720, GESPACH, C., DUPONT, C., BATAILLE, D. & ROSSELIN, G. (1980). Selective inhibition by somatostatin of cyclic AMP production in rat gastric glands. Demonstration of a direct effect on parietal cell function. FEBS letters 114, GESPACH, C., EMAMI, S., CHASTRE, E., LAUNAY, J.-M. & ROSSELIN, G. (1987). Up- and downregulation of membrane receptors as possible mechanisms related to the antiulcer actions of milk in rat gastric mucosa. Bioscience Reports 7, GESPACH, C., HUI BON HOA, D. & ROSSELIN, G. (1983). Regulation by vasoactive intestinal peptide, histamine, somatostatin-14 and -28 of cyclic adenosine monophosphate levels in gastric glands isolated from the guinea-pig fundus or antrum. Endocrinology 112, GILMAN, G. (1987). G proteins: Transducers of receptor-generated signals. Annual Review of Biochemistry 56, GREGORY, H. (1985). In vivo aspects of urogastrone-epidermal growth factor. Journal of Cell Science Supplement 3, HATT, J. F. & HANSON, P. J. (1988). Inhibition of gastric acid secretion by epidermal growth factor. Effects on cyclic AMP and on prostaglandin production in rat isolated parietal cells. Biochenmical Journal 255, HATT, J. F. & HANSON, P. J. (1989). Sites of action of protein kinase C on secretory activity in rat parietal cells. American Journal of Physiology 256, G HELDSINGER, A. A., VINIK, A. I. & Fox, I. H. (1986). Inhibition of guinea-pig oxyntic cell function by adenosine and prostaglandins. Journal of Pharmacology and Experimental Therapeutics 237, HERSEY, S. J., OWIRODU, A. & MILLER, M. (1982). Forskolin stimulation of acid and pepsinogen secretion by gastric glands. Biochimica et biophysica acta 755, IM, W. B., BLAKEMAN, D. P., BLEASDALE, J. E. & DAVIS, J. P. (1987). A protein phosphatase associated with rat heavy gastric membranes enriched with (H+-K+)-ATPase influences membrane K+ transport activity. Journal of Biological Chemistry 262, JACKSON, R. J. & SACHS, G. (1982). Identification of gastric cyclic AMP binding proteins. Biochimica et biophysica acta 717,

25 REGULATION OF GASTRIC ACID SECRETION 631 KONTUREK, S. J., BILSKI, J., DEMBIN~SKI, A., WARZECHA, A., BECK, G. & JENDRALLA, H. (1987). Effects of leukotrienes on gastric acid and alkaline secretions. Gastroenterology 92, KROMER, W., SCHRODER, P. & NETZ, S. (1984). Stereospecific inhibition by naloxone of histaminestimulated acid secretion in isolated guinea-pig parietal cells. Pharmacology 29, LETH, R., ELANDER, B., HAGLUND, U., OLBE, L. & FELLENIUS, E. (1987). Histamine H2-receptor of human and rabbit parietal cells. American Journalof Physiology 253, G LEVINE, R. A., KOHEN, K. R., SCHWVARTZEL, E. H. & RAMSAY, C. E. (1982). Prostaglandin E2- histamine interactions on camp, cgmp and acid production in isolated fundic glands. American Journal of Physiology 242, G LEWIS, J. J., ZDON, M. J., ADRIAN, T. E. & MODLIN, I. M. (1988). Pancreastatin: a novel peptide inhibitor of parietal cell secretion. Surgery 104, MAJOR, J. S. & SCHOLES, P. (1978). The localization of a histamine H2-receptor adenylate cyclase system in canine parietal cells and its inhibition by prostaglandins. Agents and Actions 8, MALINOWSKA, D. H., SACHS, G. & CUPPOLETTI, J. (1988). Gastric H' secretion: histamine (c-ampmediated) activation of protein phosphorylation. Biochimica et biophysica acta 972, MANGEAT, P., GESPACH, C., MARCHIS-MOUREN, G. & ROSSELIN, G. (1982). Differential effects of histamine, vasoactive intestinal polypeptide, prostaglandin E, and somatostatin on cyclic AMPdependent protein kinase activation in gastric glands isolated from the guinea-pig fundus and antrum. Regulatory Peptides 3, MANGEAT, P., MARCHIS-MOUREN, G., CHERET, A. M. & LEWIN, M. J. M. (1980). Specific activation of cyclic AMP-dependent protein kinase(s) by H2-histamine agonists in isolated gastric mucosal cells from guinea-pig. Biochimica et biophysica acta 629, MARDH, S., SONG, Y. H., CARLSSON, C. & BJORKMAN, J. (1987). Mechanisms of stimulation of acid production in parietal cells isolated from the pig gastric mucosa. Acta physiologica scandinavica 131, MICHELL, R. H. (1987). How do receptors at the cell surface send signals to the cell interior? British Medical Journal 295, MIEDERER, S. E., SCHEPP, W., DEIN, H. J. & RUOFF, H. J. (1986). Effect of glucagon on adenylate cyclase activity and acid production of isolated human parietal cells. Klinische Wochenschrifte 64, MISIEWICZ, J. J. (1988). Future trends in the management of peptic ulcer disease. Scandinavian Journal of Gastroenterology 23, suppl. 146, MODLIN, I. M., SCHAFER, D. E., TYSHKOV, M., BALLANTYNE, G. H., FRATESI, G. R., ROBERTS, J. R. & ZDON, M. J. (1986). Forskolin (cyclic adenosine monophosphate)-dependent protein phosphorylation in isolated gastric glands. Archives of Surgery 121, MORI, S., MORISHITA, Y., SAKAI, K., KURIMOTO, S., OKAMOTO, M., KAWAMOTO, T. & KUROKI, T. (1987). Electron microscopic evidence for epidermal growth factor receptor (EGF-R)-like immunoreactivity associated with the basolateral surface of gastric pariental cells. Acta pathologica japonica 37, MUALLEM, S., BLISSARD, D., CRAGOE, E. J. & SACHS, G. (1988). Activation of the Na+/H+ and Cl-/HCO3- exchange by stimulation of acid secretion in the parietal cell. Journal of Biological Chemistry 263, MUALLEM, S., FIMMEL, C. J., PANDOL, S. J. & SACHS, G. (1986). Regulation of free cytosolic Ca2+ in the peptic and parietal cells of the rabbit gastric gland. Journal of Biological Chemistry 261, MUALLEM, S. & SACHS, G. (1984). Changes in cytosolic free Ca2+ in isolated parietal cells. Differential effects of secretagogues. Biochimica et biophysica acta 805, MUALLEM, S. & SACHS, G. (1985). Ca2+ metabolism during cholinergic stimulation of acid secretion. American Journal of Physiology 248, G NEGULESCU, P. A. & MACHEN, T. E. (1988a). Intracellular Ca regulation during secretagogue stimulation of the parietal cell. American Journal of Physiology 254, C NEGULESCU, P. A. & MACHEN, T. E. (1988b). Release and reloading of intracellular Ca stores after cholinergic stimulation of the parietal cell. American Journal of Physiology 254, C NISHIZUKA, Y. (1986). Studies and perspectives of protein kinase C. Science 233, NISHIZUKA, Y. (1988). The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334, NYLANDER, O., BERGQVIST, E. & OBRINK, K. J. (1985). Dual inhibitory actions of somatostatin on isolated gastric glands. Acta physiologia scandinavica 125,

26 632 P. J. HANSON AND J. F. HATT ODDSDOTTIR, M., GOLDENRING, J. R., ADRIAN, T. E., ZDON, M. J., ZUCKER, K. A. & MODLIN, I. M. (1988). Identification and characterization of a cytosolic 30 kda histamine stimulated phosphoprotein in parietal cell cytosol. Biochemical and Biophysical Research Communications 154, ODDSDOTTIR, M., MODLIN, I. M., ZUCKER, K. A., ZDON, M. J. & GOLDENRING, J. R. (1987). A calmodulin dependent protein kinase in parietal cells. Biochemical and Biophysical Research Communications 148, PARK, J., CHIBA, T. & YAMADA, T. (1987). Mechanisms for direct inhibition of canine gastric parietal cells by somatostatin. Journal of Biological Chemistry 262, PEARCE, J. B., BAIRD, A. W., WILLIAMSON, E. M. & EVANS, F. J. (1981). Inhibition of histamineinduced acid secretion in rat isolated gastric mucosa by esters of phorbol and 12-deoxyphorbol. Journal of Pharmacy and Pharmacology 33, PFEIFFER, A., ROCHLITZ, H., HERZ, A. & PAUMGARTNER, G. (1988). Stimulation of acid secretion and phosphoinositol production by rat parietal cell muscarinic M, receptors. American Journal of Physiology 254, G PFEIFFER, A., SAUTER, G. & RoCHLITZ, H. (1987). Functional and biochemical interaction of dibutyryl cyclic AMP with the phosphoinositide system in isolated rat parietal cells. Biochemical and Biophysical Research Communications 147, PUURUNEN, J., LOHSE, M. J. & SCHWABE, U. (1987). Interactions between intracellular cyclic AMP and agonist-induced inositol phospholipid breakdown in isolated gastric mucosal cells of the rat. Naunyn-Schmiedeberg's Archives of Pharmacology 336, PUURUNEN, J., RUOFF, H. J. & SCHWABE, U. (1987). Lack of direct effect of adenosine on the parietal cell function in the rat. Pharmacology and Toxicology 60, PUURUNEN, J. & SCHWABE, U. (1987). Effect of gastric secretagogues on the formation of inositol phosphates in isolated gastric cells of the rat. British Journal of Pharmacology 90, REYL, F. J. & LEWIN, M. J. M. (1982). Intracellular receptor for somatostatin in gastric mucosal cells: decomposition and reconstitution of somatostatin-stimulated phosphoprotein phosphatases. Proceedings of the National Academy of Sciences of the USA 79, REYL, F., SILVE, C. & LEWIN, M. J. M. (1979). Somatostatin receptors on isolated gastric cells. In Hormone Receptors in Digestion and Nutrition, ed. ROSSELIN, G., FROMAGEOT, 0. & BONFILS, S., pp Amsterdam: Elsevier/North-Holland Biomedical Press. ROSENFELD, G. C. (1983). Pirenzepine (LS 519): a weak inhibitor of acid secretion by isolated rat parietal cells. European Journal of Pharmacology 86, ROSENFELD, G. C. (1984). Isolated parietal cells: adrenergic response and pharmacology. Journal of Pharmacology and Experimental Therapeutics 229, ROSENFELD, G. C. (1986). Prostaglandin E2 inhibition of secretagogue-stimulated ["1C]aminopyrine accumulation in rat parietal cells: a model for its mechanism of action. Journal of Pharmacology and Experimental Therapeutics 237, RUOFF, H.-J., WAGNER, M., GUNTHER, C. & MASLINSKI, S. (1982). Adrenergic stimulation of isolated rat gastric mucosal cells. Effect on adenylate cyclase and ['4C]aminopyrine uptake. Naunyn- Schmiedeberg's Archives of Pharmacology 320, SCARPIGNATO, C., TRAMACERE, R., ZAPPIA, L. & DEL SOLDATO, P. (1987). Inhibition of gastric acid secretion by adenosine receptor stimulation in the rat. Pharmacology 34, SCHEPP, W., HEIM, H.-K. & RUOFF, H.-J. (1983 a). Comparison of the effect of PGE, and somatostatin on histamine stimulated 14C-aminopyrine uptake and cyclic AMP formation in isolated rat gastric mucosal cells. Agents and Actions 13, SCHEPP, W. & RUOFF, H.-J. (1984). Adenylate cyclase and H+ production of isolated rat parietal cells in response to glucagon and histamine. European Journal of Pharmacology 98, SCHEPP, W., RUOFF, H. J. & MASLINSKI, S. (1983b). Aminopyrine accumulation of isolated parietal cells from the rat stomach. Effect of histamine and interaction with endogenous inhibitors. Archives internationales de pharmacodynamine et de thirapie 265, SCHEPP, W., SCHNEIDER, J., SCHUSDZIARRA, V. & CLASSEN, M. (1986). Naturally occurring opioid peptides modulate H+ production by isolated rat parietal cells. Peptides 7, SCHEPP, W., TATGE, C., SCHUSDZIARRA, V. & CLASSEN, M. (1988a). Substance P exerts a direct inhibitory effect on isolated rat parietal cells. Gastroenterology 94, A405. SCHEPP, W., TATGE, C., SCHUSDZIARRA, V. & CLASSEN, M. (1988b). The neuropeptide TRH inhibits acid formation by isolated rat parietal cells. Gastroenterology 94, A405.

27 REGULATION OF GASTRIC ACID SECRETION 633 SHALTZ, L. J., BOOLS, C. & REIMANN, E. M. (1981). Phosphorylation of membranes from the rat gastric mucosa. Biochimica et biophysica acta 673, SHAW, G. P. & HANSON, P. J. (1986). Inhibitory effect of 12-o-tetradecanoylphorbol 13-acetate on acid secretion by rat stomach in vivo. FEBS letters 201, SHAW, G. P., HATT, J. F., ANDERSON, N. G. & HANSON, P. J. (1987). Action of epidermal growth factor on acid secretion by rat isolated parietal cells. Biochemical Journal 244, SOLL, A. H. (1978). The actions of secretagogues on oxygen uptake by isolated mammalian parietal cells. Journal of Clinical Investigation 61, SOLL, A. H. (1980a). Secretagogue stimulation of [14C]aminopyrine accumulation by isolated canine parietal cells. American Journal of Physiology 238, G SOLL, A. H. (1980b). Specific inhibition by prostaglandins E2 and 12 of histamine-stimulated ['4C]aminopyrine accumulation and cyclic adenosine monophosphate generation by isolated canine parietal cells. Journal of Clinical Investigation 65, SOLL, A. H. (1981). Extracellular calcium and cholinergic stimulation of isolated canine parietal cells. Journal of Clinical Investigation, 68, SOLL, A. H. (1982). Potentiating interactions of gastric stimulants on ['4C]aminopyrine accumulation by isolated canine parietal cells. Gastroenterology 83, SOLL, A. H., AMIRIAN, D. A., THOMAS, L. P., REEDY, T. J. & ELASHOFF, J. D. (1984). Gastrin receptors on isolated canine parietal cells. Journal of Clinical Investigation 73, SOLL, A. H. & BERGLINDH, T. (1987). Physiology of isolated gastric glands and parietal cells: receptors and effectors regulating function. In Physiology of the Gastrointestinal Tract, 2nd edn, ed. JOHNSON, L. R., pp New York: Raven Press. SOLL, A. H. & WHITTLE, B. J. R. (1981). Prostacyclin analogues inhibit canine parietal cell activity and cyclic AMP formation. Prostaglandins 21, SOLL, A. H. & WOLLIN, A. (1979). Histamine and cyclic AMP in isolated canine parietal cells. American Journal of Physiology 237, E SONG, Y.-H., MARDH, S., NYREN, 0. & LOOF, L. (1988). Adrenaline stimulates acid production in isolated pig and human parietal cells. Scandinavian Journal of Gastroenterolog.y 23, SONNENBERG, A., BERGLINDH, T., LEWIN, M. J. M., FISCHER, J. A., SACHS, G. & BLUM, A. (1979). Stimulation of acid secretion in isolated gastric cells. In Hormone Receptors in Digestion and Nutrition, ed. ROSSELIN, G., FROMAGEOT, P. & BONFILS, S., pp Amsterdam: Elsevier/North Holland Biomedical Press. SOUMARMON, A., ABASTADO, M., BONFILS, S. & LEWIN, M. J. M. (1980). Cl transport in gastric microsomes. An ATP-dependent influx sensitive to membrane potential and to protein kinase inhibitor. Journal of Biological Chemistry 255, THOMPSON, W. J., CHANG, L. K. & ROSENFELD, G. C. (1981). Histamine regulation of adenylyl cyclase of enriched rat gastric parietal cells. American Journal of Physiology 240, G TSAI, B. S., KESSLER, L. K., SCHOENHARD, G., COLLINS, P. W. & BAUER, R. F. (1987). Demonstration of specific E-type prostaglandin receptors using enriched preparations of canine parietal cells and [3H]misoprostol free acid. American Journal of Medicine 83, TSUNODA, Y. (1987). Ca2+ currents and acid secretion in the isolated parietal cell involved in response to gastrin, compound 48/80 and ethylenediamine tetraacetic acid. Biochemistry and Cell Biology 65, TSUNODA, Y. & MATSUMIYA, H. (1987). Calcium-activated membrane depolarization via modulation of chloride efflux from parietal cells during gastrin stimulation. FEBS Letters 222, TSUNODA, Y., TAKEDA, H., ASAKA, M., NAKAGAKI, I. & SASAKI, S. (1988). Initial and sustained calcium mobilizations in the parietal cell during stimulations with gastrin, inositol trisphosphate, phorbol ester and exogenous diacylglycerol. FEBS Letters 232, TSUNODA, Y. & WIDER, M. D. (1987). Porcine ileal polypeptide causes an increase in cytoplasmic Ca2+ in both parietal and chief cells resulting in acid and pepsinogen secretion. Biochimica et biophysica acta 905, URUSHIDANI, T., HANZEL, D. K. & FORTE, J. G. (1987). Protein phosphorylation associated with stimulation of rabbit gastric glands. Biochimica et biophysica acta 930, WALZ, D. A., WIDER, M. D., SNOW, J. W., DASS, C. & DESIDERIO, D. M. (1988). The complete amino acid sequence of porcine gastrotropin, an ileal protein which stimulates gastric acid and pepsinogen secretion. Journal of Biological Chemistry 263,

28 634 P. J. HANSON AND J. F. HATT WOLLIN, A. (1984). Carbonic anhydrase activity and aminopyrine uptake in isolated gastric mucosal cells. American Journal of Physiology 247, G WOLLIN, A., SOLL, A. H. & SAMLOFF, I. M. (1979). Actions of histamine, secretin and PGE2 on cyclic AMP production by isolated canine fundic mucosal cells. American Journal of Physiology 237, E WOOTEN, M. W. & WREN, R. W. (1984). Phorbol ester induces intracellular translocation of phospholipid/ca2+-dependent protein kinases and stimulates amylase secretion in isolated pancreatic acini. FEBS Letters 171,