GASTROENTEROLOGY

SPECIAL REPORTS

1992:102:699-710

AND REVIEWS

Gastric Chief Cells: Receptors and SignalTransduction Mechanisms JEAN-PIERRE

RAUFMAN

Division of Digestive Diseases, Department of Medicine, State University Health Science Center at Brooklyn, Brooklyn, New York

Elucidation of receptors and mediators regulating gastric pepsinogen secretion has lagged behind understanding of the factors that control acid secretion. During the past decade, as a consequence of the development of in vitro models for studying the control of pepsinogen secretion at the cellular level, much information about chief cell receptors and signal-transduction mechanisms has been obtained, including the identification and characterization of receptors for secretin, vasoactive intestinal polypeptide, cholinergic agonists, gastrin, cholecystokinin, peptide YY, and cholera toxin. Moreover, these cell preparations have permitted secretagogue-induced changes in chief-cell calcium concentration, protein kinase C distribution, and phophoinositide and cyclic nucleotide content to be measured and related to changes in pepsinogen secretion. This article reviews these advances, discusses areas of uncertainty and controversy, and indicates areas for future investigation.

C

ompared with the voluminous literature regarding acid secretion from parietal cells,’ until the last decade little attention was given to the study of pepsinogen secretion. In large measure this was a consequence of the belief that acid secretion is the major determinant of gastroduodenal ulceration and healing and that pepsinogen secretion plays an inconsequential ro1e.‘g3 This misconception regarding the relative importance of acid vs. pepsin, simplified in the expression “no acid, no ulcer”,4 persists despite several reports indicating that pepsin is a necessary cofactor for acid-induced ulceration of the esophagus, stomach, and duodenum.5-7 Technical factors such as the relative difficulty associated with in vivo measurements of pepsinogen secretion and the lag in development of in vitro preparations of functional chief cells have also retarded progress in this area. Aspiration of gastric contents is considered a reliable method for measuring gastric acid secretion,’ but this is not true for pepsinogen

of New York-

secretion. Complicating factors, such as “wash-out” of presecreted pepsinogen from the lumen of gastric glands, may artifactually increase responses to secretagogues. In addition, secretagogue-induced acid secretion may stimulate pepsinogen secretion indirectly by an atropine-sensitive mechanism and modulate the effects of other secretagogues.S’3 Hence, little could be learned regarding cellular control of pepsinogen release before the development of in vitro preparations that obviate factors such as washout and the nonspecific effects of stimulants, like acid, whose secretion may be induced by the same agents that stimulate peptic cells. This article reviews the information regarding cellular control of gastric pepsinogen secretion that has been obtained from improvements in methods for obtaining functional peptic cell preparations. A description of these preparations, with analysis of their relative advantages and disadvantages, is followed by a summary of the information gained from their use. The focus of this summary will be chief cell receptors and signal transduction mechanisms that mediate secretory responses following receptor-ligand interaction. The reader is referred elsewhere’4J5 for reviews of the chemistry, synthesis, processing, storage, and actions of pepsinogen and pepsin. Models for Studying Pepsinogen

Secretion

Gastric Analysis Despite its limitations for the study of pepsinogen secretion (see above), gastric analysis is the only method that can assess the role of physiological neural and endocrine modulation of pepsinogen secretion after stimulation with secretagogues or ingestion of a meal. In vivo studies in cats,” dogs,16J7 and humansI were the first to indicate that gastric mucosal acidification, duodenal acidification, and in0 1992by

the American

Gastroenterological

0016-5065/92/$3.00

Association

700

JEAN-PIERRE RAUFMAN

travenous secretin infusion stimulated gastric pepsinogen secretion. Surgical preparations of gastric fistulae (Thomas cannulae) or denervated pouches (Heidenhain) from dog stomachs improved the collection of gastric secretions but did not obviate problems caused by wash-out and neural and cellular interactions.g~‘3*20 Nevertheless, these preparations were useful for determining that the secretory effect of mucosal acidification was atropine sensitive.g Rabbit Gastric Mucosal Cultures Secretagogue-induced pepsinogen secretion from rabbit gastric mucosal cultures is preserved for more than 24 hours and served as the first in vitro model that could be used to examine the effects of secretagogues in greater detai1.21~22Using this model, it was shown that acetylcholine, secretin, pentagastrin, and cholecystokinin (CCK) stimulated pepsinogen secretion independent of acid secretion.” In contrast to other models,’ in rabbit gastric mucosal cultures acid-induced pepsinogen secretion is not blocked by anticholinergic agents.” Although gastric mucosal cultures were a major innovation in the study of pepsinogen secretion, their use for further elucidation of secretory mechanisms was limited by cellular heterogeneity, the potential for the release of mediators from nonpeptic cells that might modulate the secretory response, and the inability to prepare multiple identical samples to compare responses for various secretagogues and controls at the same time. Gastric Glands Dispersed gastric glands from rabbit stomach, a model developed by Berglindh and Obrinkz3 to study gastric acid secretion, was a major advance in the study of pepsinogen secretion. In this preparaion, (a) pepsinogen secretion is modulated by :hanges in environmental pHz4 but is not dependent In acid secretion;25 (b) basal enzyme secretion is relaively low (4% of total glandular pepsinogen conent is released over 1 hour), whereas a severalfold i ncrease in enzyme secretion can be stimulated by carbamylcholine (carbachol), caerulein, physalaemin, CCK, gastrin, A23187,isoproterenol, adrenalin, noradrenalin, cyclic adenosine monophosphate (CAMP) analogues, and forskolin but not by secretin, prostaglandin (PG) E,, histamine, adenosine, adenosine triphosphate (ATP), AMP, or bombesin;24-34 and (c) basal and CCK-’m d uced pepsinogen secretion are not dependent on extracellular calcium, indicating the importance of intracellular calcium stores to the secretory response.27 Limitations of the rabbit model, such as cost, the failure to observe potentiation of

GASTROENTEROLOGYVol. 102,No. 2

enzyme secretion,26 and the failure of agents such as secretin and PGs to stimulate pepsinogen secretion,26 led to the adaptation of these methods to other species. Dispersed gastric glands from rat35 (Figure 1)and guinea pig3” stomachs have been used to study potentiating interactions between secretagogues, including secretin, and the actions of PGs on pepsinogen secretion. Nevertheless, with any gastric gland preparation, the limitation of cellular heterogeneity remains. Hence, measurement of changes in mediators such as CAMP or calcium will not indicate in which cell-type alterations in the levels of the mediator occur. A potential means of addressing this problem may be digital imaging of individual cells loaded with specific fluorescent probes within a gland.37 Although gastric glands are denervated, the possibility of autocrine or paracrine modulation of responses via cell-cell interactions or the release of modulators into the incubation bath presents an additional limitation. Tissue Culture Monolayers To obviate the cellular heterogeneity of gastric gland preparations and the failure of freshly isolated canine chief cells obtained by elutriation to respond to secretagogues, Ayalon et ale3’plated these cells in monolayer cultures. Advantages of these (96% chief monolayers included near homogeneity cells by histological criteria) and the restoration of presumably important histological features such as cellular polarity (maintenance of the potential difference between the apical and basal surfaces) and

Figure 1. Gastric gland from rat stomach prepared by a modification,35 of the method of Berglindh and ObrinkZS (original magnification X330).

GASTRIC

February 1992

tight junctions between cells (by electron microsc~py).~~ The major limitation of this model appears to be the small secretory response when the monolayers are stimulated with secretagogues and the inability to make multiple concurrent determinations of pepsinogen release from identical samples.38 In terms of the secretory response, at 42 hours after plating, carbachol stimulated an atropine-sensitive 2-s-fold increase in pepsinogen secretion over 60 minutes.3g Similarly, secretin, vasoactive intestinal polypeptide (VIP), and CCK stimulated an increase in enzyme secretion3’ Epinephrine and histamine induced secretion only at high concentrations (100 pmol/L). In this model, pentagastrin did not stimulate secretion, suggesting, as shown later in another modeL4’ that chief cells have distinct receptors for CCK and gastrin. Limited secretory responses prevented the determination of dose-response curves for these agents, and potentiating interactions between secretagogues could not be shown. Few investigators have used this mode1.4144 Peptic Glands and Dispersed Bullfrog Esophagus

Cells From

Simpson et a1.45took advantage of the natural localization of peptic cells to the esophagus and acidsecreting cells to the stomach of the bullfrog Rana catesbeiana to prepare esophageal mucosal strips capable of secreting pepsinogen but not acid.46 This amphibian (R. catesbeiana and Rana tigerina) model of pepsinogen secretion has been used to examine potentiating and other interactions between secretagogues and antagonists as a means of characterizing cellular mediators of secretion47-52 and to study the actions of agents, like bombesin and adrenergic agonists, that are less efficacious in mammalian peptic cells. 47.48.53,54 Although isolated peptic cells prepared from R. catesbeiana esophageal glands by collagenase digestion and calcium chelation can be used to study pepsinogen secretion, treatment with ethylene glycol tetraacetic acid (EGTA) appears to disrupt microfilament networks in the cells, thereby impairing calcium influx after stimulation with a variety of secretagogues. 55Whether this defect occurs in peptic cells isolated from other species by calcium chelation using EGTA has not been determined.

CHIEF CELLS

701

nearly homogeneous (>90%) population of functional chief cells was developed by Raufman et al. in 1984.5” This method has been used by several laboratories to study the control of pepsinogen secretion. Advantages of this preparation include (a) relative ease of preparation using readily available reagents and instruments; (b) maintenance of cell polarity, with the nucleus and mitochondria at one pole and zymogen granules at the opposite, secretory pole (Figure 2); (c) functional integrity of the cells in terms of a severalfold increase in pepsinogen secretion upon stimulation; (d) the ability to study multiple homogeneous cell samples under identical conditions; and (e) the ability to measure cellular changes in second messengers, such as cAMP,~~ calcium,58*5g and products of phosphoinositol hydrolysis.60 Disadvantages include (a) loss of physiological neuronal and cellcell interactions; (b) potential damage caused by treatment with collagenase or calcium chelation55 [although the calcium chelation step is not essential for the preparation of dispersed cells (Raufman and Raffanielo, April 1990, unpublished observation)]; and (c) the limited life span (hours) of the cells in terms of maintaining an adequate secretory response. Signal-Transduction

Pathways

The focus for this review is the cartoon shown in Figure 3, indicating recognized receptors and signal-transduction mechanisms mediating pepsinogen secretion from dispersed chief cells from the guinea pig stomach. Species differences regarding the pres-

Dispersed Chief Cells From Mammalian Stomach Despite the difficulties with chief cells obtained from dog stomachs, a method for fractionating dispersed gastric mucosal cells from guinea pig stomachs on a Percoll density gradient and obtaining a

ENdOPLASMlC

RETICULUM

MITOCH‘O’NDRIA

Figure 2. Electron micrograph of a dispersed chief cell from guinea pig stomach prepared by the method of Raufman et al.56 (original magnification X21,000).

702 JEAN-PIERRE RAUFMAN

GASTROENTEROLOGY Vol. 102, No. 2

CHIEF CELL

CCK -

PHOSPHOLIPID

GASTRIN e-e CARBACHOL

SECRETIN

CHOLERA

*

c,

TOXIN@ -10

-11

PYYINPY

-9

-0

-7

-6

-5

-4

+==+ CONCENTRATION

(log

M)

Figure 4. Relative potencies and efficacies for pepsinogen secretion stimulated by agents whose actions on dispersed chief cells Figure 3. Illustration showing interaction of various secretaappear to be mediated by activation of the adenylyl cyclase sysgogues with receptors and signal-transduction mechanisms in dispersed chief cells from guinea pig stomach.10~50~5’~s8.80.05,8’1~ tem. Data are derived from the equation R = R, [c/(c + K)], where R is the calculated response, R,, is the maximal re72~‘s*71*‘S61~8z~~8,80 Features of this model are discussed in the text. sponse to the agent, K is the concentration of the agent causing a Gs, stimulatory guanine nucleotide-binding protein; Gi, inhibihalf-maximal response, and c is the concentration of the agent. tory guanine nucleotide-binding protein. R, and K were obtained from references 56, 57, and 65 and normalized to show relative potencies and efficacies with 10 nmol/L secretin = 100%.

ence of specific receptors and mechanisms for pepsinogen secretion are indicated and discussed where appropriate. Agents That Activate Adenylyl

Cyclase

Interaction of secretin, VIP, cholera toxin, and PGs with receptors linked to the adenylyl cyclase system results in pepsinogen secretion from dispersed chief cells from the guinea pig stomach. Doseresponse curves for the stimulation of pepsinogen secretion by these agents are shown in Figures 4 and 5. Adrenalin and noradrenalin, agents known to act via activation of adenylyl cyclase in other tissues; CAMP analogues such as 8-bromo-CAMP and dibutyryl-CAMP; and forskolin, a diterpene that binds to the catalytic unit of adenylyl cyclase, have been reported to stimulate pepsinogen secretion from rabbit gastric glands.26*33,“’Secretin, a peptide that stimulates CAMP-mediated enzyme release from other tissues,62 does not stimulate pepsinogen secretion from rabbit gastric glandsz6 but is a potent secretagogue in dispersed glands from rat and guinea pig stomachs.35p3” Because of the cellular heterogeneity of gastric gland preparations, early studies evaluating cellular mechanisms of secretin-induced pepsinogen secretion relied on showing potentiating interactions as indirect evidence for CAMP-mediated mechaof enzyme secretion occurs nisms.35 Potentiation with the combination of two secretagogues that have different cellular mechanisms of action but not with two secretagogues that have the same mechanism of

action. Thus, in rat gastric glands, the observation that a combination of secretin, VIP, or a CAMP analogue plus carbachol or a calcium ionophore resulted in potentiation of pepsinogen secretion provided indirect evidence that secretin- and VIP-induced secretion was mediated by cAMP.~~

ioogo-0

ao-

.-E 70:: E

??Cellular CAMP

r -II

-10

-9

CHOLERA

-8

-7

? -6

TOXIN (log Ml

Figure 5. Relation of cholera toxin-induced increases in CAMP, pepsinogen secretion, and inhibition of binding of radiolabeled cholera toxin. Reprinted with permission.”

GASTRIC

February 1992

Direct confirmation that CAMP mediates secretininduced pepsinogen secretion was obtained using dispersed chief cells from the guinea pig stomach.57 There is a close relationship between concentrations of secretin and VIP that stimulate pepsinogen secretion and those that increase chief-cell CAMP. However, whereas the secretin dose-response curves are monophasic, those for VIP are biphasic, indicating that these agents interact with more than one class of receptor (Figure 4). To elucidate the nature of these interactions, changes in pepsinogen secretion and chief-cell CAMP levels were related to binding of radiolabeled secretin and VIP by computer analysis (Ligand; Biosoft, Cambridge, England).57 Using this approach, four classes of chief-cell receptors for secretin and VIP have been identified (Table 1). These classes include (a) a high-affinity VIP, low-affinity secretin receptor that mediates a small increase in CAMP and pepsinogen secretion; (b) a low-affinity VIP, low-affinity secretin receptor that is not associated with an increase in CAMP or pepsinogen secretion; (c) a high-affinity secretin, low-affinity VIP receptor that mediates the major increase in CAMP and pepsinogen secretion; and (d) a low-affinity secretin receptor that does not bind VIP and does not stimulate an increase in CAMP or pepsinogen secretion (Table 1).57 In contrast to in vivo studies63@’ and in vitro studies using rabbit gastric glands,‘” prostanoids increase cellular CAMP and pepsinogen secretion from dispersed glands and chief cells from the guinea pig stomach.36s65 Natural and synthetic PGE has also been reported to stimulate pepsinogen secretion from canine gastric chief-cell monolayers. In the guinea pig, prostaglandins potentiate carbachol- and calcium ionophore-induced pepsinogen secretion.36 The relative order of potency for the actions of pros-

Table 1. Characteristics of Receptors for Vasoactive Intestinal Polypeptide and Secretin on Dispersed Chief Cells From Guinea Pin Stomach Increase in chief cell

Receptor class

KdO

Sites/ chief cell

CAMP

Pepsinogen secretion

High-affinity VIP Low-affinity secretin

0.8 nmol/L 0.1 pmol/L

5 x 103

z-fold

o.5-fold

Low-affinity VIP Low-affinity secretin

1 .O pmol/L 1.0 pmol/L

5 x 106

None

None

High-affinity secretin Low-affinity VIP

1.0 nmol/L 0.5 pmol/L

6 x 103

lo-fold

a-fold

Low-affinity secretin

1.0 pmol/L

6X lo5

None

None

‘Concentration of peptide required Data from Sutliff et a1.57

to occupy 50% of binding sites.

CHIEF CELLS

703

tanoids on CAMP and enzyme secretion from guinea pig chief cells is PGE > PGA > PGB (Figure 4).36 Nevertheless, comparison of dose-response curves for PG-induced increases in pepsinogen secretion and CAMP indicates the possibility that CAMP is not the only mediator of PG action.65 That is, detectable increases in pepsinogen secretion occur with PG concentrations about lo-fold less than those necessary to increase CAMP. Although this may represent spareness of mediator in the sense that stimulation of a small number of receptors with a modest increase in CAMP is sufficient to maximally stimulate enzyme secretion,66 an alternative explanation is that an additional mediator plays a role. To date, however, CAMP is the only mediator in chief cells that has been reported to increase upon stimulation with prostanoids. Further analysis of this problem has been limited by the inability of investigators to show binding of radiolabeled PCs to dispersed chief cells.“5 Examination of the actions of cholera toxin, an agent that ribosylates stimulatory guanine nucleotide-binding (G) proteins, also indicates close correlation between concentrations of the toxin that increase chief-cell CAMP levels and those that stimulate pepsinogen secretion (Figure 5). Analysis of radioligand-binding data indicates that the toxin interacts with only one receptor (Kd = 3 nmol/L; 8.7 X lo5 sites/cell).67 Although it has been suggested that some actions of cholera toxin are mediated by PGs,“’ measurement of chief-cell PGE, production and the use of inhibitors of PG synthesis exclude a role for these agents in mediating cholera toxin-induced pepsinogen secretion.6g Phosphodiesterase inhibitors stimulate pepsinogen secretion from dispersed chief cells by increasing cellular CAMP concentrations7’ Of three inhibitors examined, the relative order of potency for increasing CAMP and pepsinogen secretion is Ro 201724 > 3-isobutyl-l-methylxanthine > theophylline.70 Differential actions of these phosphodiesterase inhibitors on the dose-response curves for secretagogues can be used to enhance the response with agents and to evaluate cellular compartmentation of cAMP.~’ Agents That Inhibit the Activation Adenylyl Cyclase

of

Little is known regarding the actions of peptides that decrease secretagogue-induced pepsinogen secretion by inhibiting the activation of adenylyl cyclase. Somatostatin and peptides in the peptide YY (PYY)/neuropeptide Y (NPY) family have been reported to decrease stimulated but not basal secretion by mechanisms involving inhibitory G proteins, So-

704 JEAN-PIERRE RAUFMAN

GASTROENTEROLOGY

matostatin inhibits bombesin- and bethanechol-induced pepsinogen release from frog peptic cells.50,52 However, somatostatin receptors and signal-transduction mechanisms have not been characterized further in chief cells. Nanomolar concentrations of PYY, a peptide released from endocrine cells in the small and large intestine, and NPY, a structural analogue of PYY found in neurons, cause 50% inhibition of pepsinogen secretion stimulated by agents such as secretin, VIP, PGE,, and forskolin whose actions are mediated by increases in cellular cAMP.‘l These inhibitors have no effect on secretion caused by agents such as carbachol, CCK, and calcium ionophores whose actions are mediated by phospholipid turnover.‘* Peptide YY and NPY cause a 50% reduction in secretinand VIP-induced increases in CAMP but do not affect binding of VIP to chief-cell receptors.‘l These results indicate that PYY and NPY modulate the coupling between secretin and VIP receptors and the adenylyl cyclase system. Receptors for PYY and NPY have been identified on dispersed chief cells from the guinea pig stomach.” Specific binding of PYY and NPY to Y-2 subtype receptors on dispersed chief cells from guinea pig stomach has been reported.” Using this model, high-affinity (1.8X lo3sites/cell; K, = 1.7nmol/L) and low-affinity (5.1X lo* sites/cell; K, = 83.3 nmol/L) receptors have been identified. Moreover, internalization of 70% of bound radioligand can be shown following a so-minute incubation at 37°C. Comparison of the dose-response curves for PYY-induced inhibition of lz51-PYY binding and inhibition of secretin-induced increases in CAMP suggests that these inhibitory actions are mediated by interaction with the high-affinity sites.” Although a physiological role for PYY as a postprandial inhibitor of pepsinogen secretion has not been proven, several studies indicate that circulating concentrations of PYY after a meal reach the nanomolar range.73 The mechanism whereby PYY and NPY inhibit adenylyl cyclase activity is not well understood. Preliminary data’lindicate that this action can be blocked by preincubating chief cells with pertussis toxin, an agent that causes ribosylation of inhibitory G proteins, thereby uncoupling them from receptors.‘* Further work is needed to establish that PYY and NPY activate inhibitory G proteins and to determine whether these inhibitory peptides play a physiological role in modulating pepsinogen secretion in the postdigestive period. Agents That Stimulate Phospholipid

Turnover

Interaction of CCK, CCK analogues gastrin-I and -11, and cholingergic agents such as carbachol

Vol. 102, No. 2

with chief-cell receptors results in the activation of phospholipases that cause phopholipid turnover, thereby generating inositol trisphosphate (IP,) and diacylglycerol (DAG)60*75 and stimulating pepsinogen secretion (Figure 6).Inositol triphosphate stimulates release of calcium from intracellular stores, thereby increasing cellular calcium concentration.58*767g Diacylglycerol binds and activates protein kinase C.80As discussed below, the relative contributions of increases in cellular calcium concentration and activation of protein kinase C to the secretory process has yet to be determined. Although early studies using dispersed chief cells from the guinea pig5” suggested that supramaximal concentrations of CCK and carbachol cause less-than-maximal pepsinogen secretion, a finding observed in other exocrine tissues,62 subsequent studies from our laboratory (October 1990, unpublished observation) and others40,81*82 have indicated that this phenomonon is not as pronounced as originally thought (Figure 6). Muscarinic cholinergic receptors on dispersed chief cells from guinea pig pancreas have been characterized by examining the binding of [N-methyl3H]scopo1amine.8* Computer analysis (Ligand) of competitive binding data using unlabeled acetylcholine, carbachol, and muscarine indicates the presence of high-affinity (4.3X lo3sites/cell; K, = 12,53, and 44 pmol/L, respectively) and low-affinity (1.6X lo3sites/cell; K, = 2.2,4.6,and 2.7mmol/L, respectively) receptors. ” Interactions of cholinergic ago-

A! -12

-10

-9

-6

CONCENTRATION

-7

-6

(log

-5

-4

-3

M)

Figure 6. Relative potencies and efficacies for pepsinogen secretion stimulated by agents whose actions on dispersed chief cells appear to be mediated by activation of phospholipid turnover. Data are derived from the equation R = R,, [c/(c + K)], where R is the calculated response, R,, is the maximal response to the agent, K is the concentration of the agent causing a half-maximal response, and c is the concentration of the agent. R,, and K were obtained from references 49,56,81,82,93, and 94 and normalized to show relative potencies and efficacies with 5 nmol/L CCK-8 = 100%.

February 1992

nists with the high-affinity muscarinic receptors appepsinogen secretions1 Relative affinities of the muscarinic antagonists atropine, DAMP, pirenzepine, and AF-DX-116 indicate that the muscarinic receptors on dispersed chief cells are of the M,, subtype.” In some tissues, such as guinea pig pancreatic acini 62 CCK- and gastrin-related peptides interact with the same receptors. In contrast, guinea pig chief cells possess distinct receptors for CCK and gastrin4’ One class of receptors, designated the C receptor, interacts with caerulein and sulfated and unsulfated CCK-7 and CCK-8.40 The other class, designated the G receptor, interacts with CCK-4, -5, and -6 and with sulfated and unsulfated gastrin.40 Maximal stimulation of the G receptor results in about 60% as much pepsinogen secretion as that of the C receptor (Figure 6). Moreover, G-receptor agonists do not inhibit the actions of C-receptor agonists as would be expected from a partial agonist acting at the same receptor, providing further evidence for distinct receptors. Receptor antagonists such as proglumide-10 and L364,718 are more potent in blocking the C receptor than the G receptor.40 Binding of radiolabeled CCK and gastrin to approximately 12,000 sites/chief cell [K,(CCK) = 640 pmol/L; K,(gastrin-I) = 2.8 nmol/L] can be shown in the guinea pig model. *’However, further analysis of binding parameters using CCK- and gastrin-receptor antagonists indicates that interaction of CCK and gastrin with these binding sites does not mediate pepsinogen secretion. a’The reason for the failure to characterize the receptors that mediate the secretory actions of CCK and gastrin on chief cells or the function of the binding sites that have been detected for these ligands is not presently known. Bombesin and its mammalian analogue gastrin-releasing peptide have been reported to stimulate pepsinogen secretion from dispersed chief cells from guinea pig stomach. 83The pattern of interaction with other secretagogues indicates that the actions of bombesin and gastrin-releasing peptide are mediated by changes in intracellular calcium concentration. Nevertheless, the efficacy of these agents for stimulating enzyme secretion from guinea pig chief cells is much less than that observed using frog peptic models,50v54 receptors for these agents have not been characterized on chief cells, and changes in intracellular calcium concentration have not been measured in the mammalian model. Role of calcium as a second messenger. Several factors, such as the secretagogue activity of calcium ionophores (A23187, ionomycin) and potentiating interactions between secretagogues, indicate that calcium is an important mediator of pepsinogen

pear to stimulate

GASTRICCHIEF CELLS 705

secretion. With the availability of fluorescent probes for measuring changes in intracellular calcium concentration directly, several investigators explored the role of this divalent cation in signal transduction.58*76-7gMethodological differences, such as the choice of fluorescent probe (quin2, fura-2, or aequorin), the amount of probe loaded into the cells, and differences in cell preparations probably account for the range in basal calcium concentrations reported in various studies. Values for basal calcium concentration range from 105 nmol/Ls8 to 282 nmo1/Le4 in guinea pig chief cells and 100 nmo1/L76 to 140 nmo1/L77 in rabbit chief cells. There is a close correlation between the ability of carbachol, CCK, gastrin, and calcium ionophores to increase chiefcell calcium concentration and their ability to increase pepsinogen secretion.58*767g Maximal concentrations of these agents have been reported to cause a a-s-fold increase in chief-cell calcium concentraRemoving calcium from the extracellular tion. 58*76-7g medium or adding a calcium channel blocker such as La3+ has little effect on the initial increase (O-5 minutes) in cellular calcium seen with these agents, indicating that as in other tissues,85 this initial increase is caused by release of calcium from intracellular stores.75,7g Prior stimulation with one agent (e.g., carbachol) decreases the increase in cellular calcium concentration after stimulation with another agent (e.g., CCK), indicating that these agents release calcium from the same intracellular po01.~~,~’Sustained pepsinogen secretion (>5 minutes) requires extracellular calcium and is blocked by La3+, indicating that as in other tissues,85 this sustained elevation in cell calcium is caused by influx of calcium from the medium.75v7g A twofold increase in chief-cell IP, has been observed following stimulation with CCK or carbacho1,77 and treatment of permeabilized chief cells with IP, induces release of calcium from intracellular stores.75 These observations suggest that as in other tissues, interaction of CCK and carbachol with chief-cell receptors results in the activation of phospholipase C and hydrolysis of phosphoinositol.75s77 A product of this hydrolysis, IP, binds to intracellular receptors, thereby causing calcium release and increasing intracellular free calcium c0ncentration.85 A recent reporta indicates that alcohol (0.3 to 0.9 mol/L) causes a 2.5-fold increase in pepsinogen secretion by stimulating an increase in cellular calcium concentration. Chief-cell IP, levels are not altered by ethanol, but the increase in cellular calcium is blocked by removing calcium or adding La3+ to the incubation solution. 84Although these results suggest that ethanol induces pepsinogen secretion by stimulating an influx of extracellular calcium through

706

IEAN-PIERRE RAUFMAN

La3+-sensitive channels, nonspecific ethanol-induced damage to chief-cell membranes and other discrepancies have been observed previously by other investigators.86 Role of protein kinase C in signal transduction. The role of protein kinase C as a mediator of secretagogue-induced pepsinogen secretion is less clear than that of calcium. Several studies have shown that phorbol esters such as phorbol12-myristate, 13-acetate (PMA) that bind and activate protein kinase C in vitro” stimulate pepsinogen secretion.48,76,7gMoreover, PMA-induced secretion is potentiated by the addition of a calcium ionophore,48.‘6.‘9 Consequently, it has been suggested that sustained pepsinogen secretion with agents such as carbachol and CCK is mediated by DAG by-products of phosphoinositol hydrolysis that activate protein kinase C.48,76,7g Increased chief-cell DAG levels have been observed after stimulation with CCK.GO Nevertheless, direct evidence that activation of protein kinase C plays a role in mediating carbacholand CCK-induced pepsinogen secretion is lacking. Although PMA stimulates pepsinogen secretion, this is not necessarily attributable to activation of protein kinase C alone. For example, PMA-induced increases in chief-cell calcium concentration have been reported.” Moreover, even if PMA stimulates pepsinogen secretion by activating protein kinase C, this is not evidence that this enzyme plays a role in mediating the actions of carbachol or CCK. Efforts to clarify this issue are limited by methodological problems, including the lack of markers such as protein kinase C phosphorylation substrates that have been observed in other tissues” or specific inhibitors of the kinase that can be used in viva.” Recent experiments in our laboratory indicate that although redistribution of chief-cell protein kinase C activity from the cytosol to the membrane is observed with PMA or cell-permeant DAGs, redistribution of enzyme activity is not observed when cells are stimulated with carbachol or CCK.*’ These data suggest that activation of protein kinase C may not be an important mediator of carbachol- or CCK-induced pepsinogen secretion. Interactions Pathways

Between Signal-Transduction

Several observations indicate that interactions or “cross-talk” between signal-transduction pathways occurs in dispersed chief cells. In cells that are first incubated with cholera toxin and then stimulated with carbachol, CCK, or calcium ionophores, a twofold augmentation in cellular levels of CAMP is observed (Figure 7), accompanied

GASTROENTEROLOGY Vol.

6+%%+

90 93 96

102,

No.

2

99 102 10

TIMEhin)

Figure 7. Effect of preincubation with cholera toxin on subsequent actions of carbachol and CCK on CAMP levels in guinea pig chief cells. Reprinted with permission.”

by potentiation of pepsinogen secretion.g0 This response is abolished by removing extracellular calcium or adding inhibitors of calmodulin.gO As with other potentiating responses, this calcium- and calmodulin-dependent interaction between second messenger systems may serve as a means of fine-tuning the secretory response. The precise mechanism whereby a calcium-calmodulin complex modulates the adenylyl cyclase system is as yet undetermined, but it is possible that phosphorylation of a component of the adenylyl cyclase system by a calciumand calmodulin-dependent protein kinase plays a role. Furthermore, it will be important to determine whether this form of interaction is limited to cholera toxin or can occur with “physiological” agents. Phorbol esters like PMA that activate protein kinase CBo inhibit prostanoid-induced increases in chief-cell CAMP by 4O%.‘l This effect is specific to prostanoids (that is, it does not occur with other stimulants of chief-cell adenylyl cyclase such as secretin, VIP, cholera toxin, or forskolin) and is blocked by adding inhibitors of protein kinase C.” Technical difficulty in identifying chief-cell prostanoid receptors has prevented the elucidation of the cellular site of protein kinase C action in this regard. Heterologous desensitization of pepsinogen secretion is another form of cellular control that requires interaction between signal-transduction mechanisms.g2~g3When dispersed chief cells are incubated with carbachol or CCK and washed and incubated for a second time with these agents or others such as

February

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gastrin, calcium ionophores, or phorbol esters that act by or mimic the results of phospholipid turnover, pepsinogen secretion and the increase in intracellular calcium are reduced in the second incubation.“‘95 Enzyme secretion stimulated by agents whose actions are mediated by activation of adenylyl cyclase is not affected by prior incubation with carbachol or CCK.94*Q5 Desensitization appears to be a form of negative modulatory control of the secretory response.” Other Receptors and Mediators Atria1 naturetic factor binds to dispersed chief cells and increases cyclic guanosine monophosphate (cGMP) levels by a protein kinase C-sensitive mechanism.‘” However, cGMP analogues and agents such as atria1 naturetic factor and nitroprusside that increase chief cell cGMP content do not alter pepsinogen secretion. The function, if any, of the increase in these cyclic nucleotides is currently unknownQ6 Recent reports evaluating the effects of cGMP in pancreatic acini suggest that this agent may play a role in calcium gating across cell membranes.g7 Whether this finding applies to chief cells remains to be determined. Summary and Conclusions This review indicates the recent progress in elucidatingreceptorsandsignal-transductionmechanisms in gastric chief cells. Whereas the current edition of the most commonly used gastroenterology text states that “no peptide receptors have been demonstrated on chief cells,“” the present review shows that this is no longer the case. Nevertheless, much remains to be learned regarding the cellular control of pepsinogen secretion. Many areas for future investigation are apparent from the cartoon of a guinea pig chief cell shown in Figure 3. These include further elucidation of the physiological actions of inhibitory factors such as somatostatin and PYY/NPY and the role of G proteins in mediating these actions; elucidation of the role of cGMP in cellular signaling; identification and characterization of chief-cell receptors for PGs; characterization of receptors and signal-transduction mechanisms in chief cells from other mammals, particularly humans; isolation, characterization, and cloning of chief-cell CCK, gastrin, secretin, VIP, PYY, and muscarinic receptors; and more detailed analysis of secretagogue-induced increases in products of phospholipid turnover, including measurement of calcium changes in single chief cells. Nonetheless, a major focus of inquiry could be the confluence of arrows shown in the center of the chief cell (Figure 3). That is, how do increases in putative

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second messengers such as CAMP, calcium, and DAG stimulate pepsinogen secretion? Moreover, how do these signaling pathways interact to produce potentiation or desensitization of enzyme secretion? Answering these questions will require the study of messenger-induced activation of chief-cell protein kinases and the subsequent phosphorylation events that are currently thought to mediate secretion in other tissues.gQ,‘OOTo date, in gastric chief cells and even in other tissues, relatively little is known regarding the activation or targets of calcium-calmodulin kinases, protein kinase C, or even CAMP-dependent protein kinases. References Sol1 AH, Berglindh T. Physiology of isolated gastric glands and parietal cells: receptors and effecters regulating function. In: Johnson LR, ed. Physiology of the gastrointestinal tract. Volume 1.2nd ed. New York: Raven, 1987:883-909. 2. Boyd EJS, Wormsley KG. Etiology and pathogenesis of peptic ulcer. In: Bockus Gastroenterology. Volume 2.4th ed. Philadelphia: Saunders, 1985:1013-1059. 3. Sol1 AH. Duodenal ulcer and drug therapy. In: Sleisenger MH, Fordtran JS, eds. Gastrointestinal disease. Volume 1.4th ed. Philadelphia: Saunders, 1989:814-879. 4. Schwartz K. Ueber penetrierende Magen und Jejunalgeschwure. Beitr Klin Chir 1910;67:96-128. 5. Taylor WH. Proteinases of the stomach in health and disease. Physiol Rev 1962;42:519-553. 6. Alphin RS, Vokac VA, Gregory RL, Bolton PM, Tawes JW. Role of intragastric pressure, pH, and pepsin in gastric ulceration in the rat. Gastroenterology 1977;73:495-500. 7. Joffe SN, Roberts NB, Taylor WH, Bacon JH. Exogenous and endogenous acid and pepsins in the pathogenesis of duodenal ulcers in the rat. Dig Dis Sci 1980;25:837-841. 8. Baron JH. Clinical tests of gastric secretion. New York: Oxford University, 1979. 9. Johnson LR. Regulation of pepsin secretion by topical acid in the stomach. Am J Physiol 1972;223:847-850. 10. Bynum TE, Johnson LR. Stimulation of human pepsin output by topical hydrochloric acid. Dig Dis 1975;20:607-612. 11. Liebman WM, Samloff IM. Effect of topical acid on duodenal pepsinogen secretion in the rat. Dig Dis 1978;23:989-992. 12. Nakajima S, Nakamura M, Magee DF. Effect of secretin on gastric acid and pepsin secretion in response to various stimuli. Am J Physiol 1969;216:87-91. 13. Johnson LR. Effect of gastric mucosal acidification on the action of pepsigogues. Am J Physiol 1973;225:1411-1415. 14. Hersey SJ. Pepsinogen secretion. In: Johnson LR, ed. Physiology of the gastrointestinal tract. 2nd ed. New York: Raven, 1987:947-957. 15. Samloff IM. Pepsinogens, pepsins, and pepsin inhibitors. Gastroenterology 1971;60:586-604. 16. Stening GF, Johnson LR, Grossman MI. The effect of secretin on acid and pepsin secretion in cat and dog. Gastroenterol1.

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Received January 29, 1991. Accepted May 3, 1991. Address requests for reprints to: Jean-Pierre Raufman, M.D., SUNY-Health Science Center, Box 1196, 450 Clarkson Avenue, Brooklyn, New York 11203-2098. Supported by National Institutes of Health grant DK-34189.