acid secretagogue-induced stimulation of gastric parietal cell

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 19, Issue of July 5, pp. 11381-11386,1989 Printed in U.S.A.

Acid Secretagogue-induced Stimulation of Gastric Parietal Cell Gene Expression*

(Received for publication, November 10, 1988)

Virginia W. Campbell.$ and Tadataka Yamada From the Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0682

Carbamoylcholine (carbachol), histamine, and gastrin are three principal stimulants of gastric acid secretion. To explore the mechanisms by which these agents exert their actions in parietal cells, we examined their effects on the gene expression of the enzymes responsible for H+ generation. Each secretagogue induced rapid and coordinate increases in steady-state levels of mRNAs encoding carbonic anhydrase I1 and H+,K+-ATPase in isolated canine gastric parietal cells. Furthermore, pronounced increases, with different kinetics, in eftpression of @-actin mRNA were observed. With increasing time after cell isolation, carbonic anhydrase I1 and H+,K+-ATPase, but not &actin, mRNA levels were attenuated, suggesting that parietal cell- specific genes may be dependent upon maintenance of parietal cell contacts within intact mucosal tissue. Pre- treatment of the cells with competitive inhibitors of each secretagogue blocked the increases. Our results indicate that acid secretagogue-specific receptor activation in parietal cells triggers coordinate gene expression of the two enzymes involved in H+ ion generation and that @-actin may be an important regulator of acid secretion.

Gastric acid secretion in mammals is regulated by at least three principal chemical messengers that appear to act at separate receptors on the surface of the highly specialized and differentiated parietal cell (1). Stimulation of this cell by the binding of acetylcholine (or an analogue such as carbachol), histamine, or gastrin to their respective receptors initiates a series of intracellular activating events, morphological trans- formations, and transport processes that result in the secretion of voluminous amounts of HCL (1, 2). The resting parietal cell contains a collapsed secretory canaliculus, lined with stubby microvilli, and cytoplasmic tubulovesicles containing the gastric proton pump, H’,K’-ATPase. Carbonic anhydrase 11, an enzyme thought to be responsible for cata- lyzing the conversion of the OH- generated by the proton pump into HCO; (3), is scattered throughout the cytoplasm of the resting cell. When the cell is stimulated, the tubulovesicles rapidly disappear, and the canaliculi swell with the newly formed acid. The latter structures contain a large

* This work was supported by National Institutes of Health Grant R01-DK34306 and funds from the Michigan Gastrointestinal Peptide Hormone Research Center under National Institutes of Health Grant P30-DK34933. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

j: To whom correspondence should be addressed University of Michigan Gastrointestinal Peptide Hormone Research Ctr., 6510 B Medical Sciences Research Bldg. I, University of Michigan, Ann Arbor, MI 48109-0682.

number of elongated microvilli formed by extensive microfilaments having a central cytoskeletal core of actin filaments stabilized by other proteins (2-4). From morphological and histochemical studies (2,5), it appears that acid secretagogues lead to insertion of the H+,K’-ATPase into the secretory membrane of the elongated microvilli. Associated with the activation of the proton pump, there is a large increase in HCO, production, accompanied by a translocation of carbonic anhydrase I1 to sites near both the canalicular and basolateral membranes (6, 7). Acid then accumulates in the parietal cell canaliculi, from which it is actively extruded into the glan- dular lumen through a channel in the apical membrane of the cell. This process of extrusion is inhibited and the microvillus structure disrupted by substances such as cytochalasin B that block the polymerization of actin into microfilaments (8).

In previous studies we have shown that acid secretagogue stimulation of freshly isolated canine gastric parietal cells is associated with increases in carbonic anhydrase I1 gene transcription (9). We undertook the present studies to examine the pattern of expression of H’,K+-ATPase mRNA, particularly in relation to carbonic anhydrase I1 gene expression, during acid secretion. Our results demonstrate that acid secretagogues such as carbachol, histamine, and gastrin coor- dinately induce the expression of mRNAs encoding both carbonic anhydrase I1 and H+,K’-ATPase. In addition, we observed a remarkable increase in @-actin mRNA content in stimulated cells, suggesting that stimulation leads to induction of genes encoding proteins required not only for H’ generation but also for the cellular morphological transformation involved in the acid secretory process.

EXPERIMENTAL PROCEDURES

Materials-Atropine, carbamoylcholine chloride (carbachol), cimetidine, collagenase (type I), dithiothreitol, EDTA, Ficoll, formaldehyde, histamine, HEPES,’ Sarkosyl, salmon sperm DNA, sodium dodecyl sulfate, /3-mercaptoethanol, ethidium bromide, and bovine serum albumin (BSA) were purchased from Sigma. Famotidine (Pep- cid@ MSD) was supplied by Merck Sharp and Dohme and proglumide and CR1409 by Rotta Laboratories (Milan, Italy). Cesium chloride, phenol (ultrapure), agarose (ultrapure), and proteinase K were from Bethesda Research Laboratories. Polyvinylpyrrolidone was obtained from Eastman Kodak Company and dextran sulfate and Percoll from Pharmacia LKB Biotechnology Inc. Gastrin hepatodecapeptide (G17) was obtained from Peninsula Laboratories (Belmont, CA). Earle’s balanced salt solution (EBSS) and Hanks’ balanced salt solution were supplied by Irvine Scientific (Santa Ana, CA). Guanidinium isothiocyanate and formamide were purchased from Fluka (Hauppauge, NY and radiolabeled dCTP (3000 Ci/mmol) from Amersham Corp.

Cell Preparation-Canine gastric parietal cells were prepared from freshly obtained canine fundic mucosa according to methods described previously (9, IO). The mucosal layer of the fundus was bluntly separated from the submucosa and minced in cold Hanks’ balanced

’ The abbreviations used are: HEPES, 4-(2-hydroxyethyl)-l-piper- azineethanesulfonic acid; BSA, bovine serum albumin; EBSS, Earle’s balanced salt solution; UBCP, ubiquitin carboxyl-terminal precursor.

11381

11382 Stimulation of Parietal Cell Gene Expression

salt solution containing 0.1% BSA. The cells were then dispersed by sequential exposure to collagenase (0.35 mg/ml) and EDTA (1 mM), and parietal cells were enriched by centrifugal elutriation using a Reckman JE-GB elutriation system. Our best preparations contained 70% parietal cells, as determined by hematoxylin and eosin and by periodic acid-Schiff's reagent staining. In some experiments we uti- lized preparations of parietal cells that were further purified by centrifugation through density gradients generated with 50% Percoll a t 30,000 X g for 20 min. The cell fraction banding a t p = 1.05 consisted of 95-100% parietal cells. Since the responses of gene- specific mRNA content in these cells were identical to those of the enriched elutriated fractions, we used the latter for the bulk of our studies.

Preparation of RNA from Cells-Isolated cells (2 X 106/ml) were suspended in 10 ml of Earle's balanced salt solution containing 10 mM HEPES buffer (pH 7.4) and 0.1% BSA. After addition of acid secretagogue, or of secretagogue and inhibitor, the cells were incubated for various times a t 37 "C in 95% air, 5% CO, and then pelleted by centrifugation a t 500 X g for 10 min and homogenized in a dounce homogenizer after lysis in 1 ml of 5 M guanidinium isothiocyanate, 1% Sarkosyl, 20 mM EDTA, 1% @-mercaptoethanol, 50 mM Tris, pH 7.5. The homogenates were layered over a 1.2-ml cushion of 5.7 M CsCI, and RNA was pelleted by centrifugation for 16 h a t 36,000 rpm in an SW 60 rotor (Beckman Instruments, Palo Alto, CA). The pellets were resuspended in 200 pl of 50 mM sodium citrate, pH 7.5, 100 mM NaC1, 1 mM EDTA. Proteinase K was added to a concentration of 200 pg/ml and sodium dodecyl sulfate to 0.2% and the solution incubated at 37 "C for 30 min. RNA was then extracted with phenol/ chloroform/isoamylalcohol (25:24:1), followed with chloroform/isoamylalcohol (24:1), and precipitated with ethanol. Each RNA sample was redissolved in 50-100 pl of 50 mM sodium citrate, 1 mM EDTA, pH 7.5, and RNA concentrations were estimated by measurement of absorbance a t 260 nm.

Quuntitation of Specific mRNAs-For Northern blot hybridization analysis (ll), total RNA from cells was separated on a 1.25% formaldehyde-agarose gel and blotted to nitrocellulose. The samples were analyzed in replicate sets. T o confirm that each lane of the gel contained equal amounts of RNA, ethidium bromide was added to each RNA sample before loading onto the gel, and the stained ribosomal RNA bands in the gel were photographed before transfer to ensure that equivalent amounts of RNA were loaded onto each lane. RNA immobilized on replicate nitrocellulose filters was hybridized as described (9) to various labeled cDNAs. Relative amounts of gene-specific mRNA visualized from these Northern analyses as autoradiographic images on Kodak X-Omat film were quantified by digital densitometry on a Loats Image Analysis System (Westminster, MD). Exposure times of the film were chosen to be within the linear range for densitometric scanning.

T o confirm the reliability of our quantification based on densitometric scanning of autoradiograms produced by Northern blot analysis of total RNA, we compared the data from parallel autoradiograms produced from Northern blot analysis to those resulting from dot blot analysis of identical samples of total RNA after hybridization to the same "P-labeled cDNA probe. We also analyzed some dot blots by cutting out spots and quantifying by scintillation counting the radio- active signals derived from the hybridized 32P-labeled cDNA probe and compared this data to densitometric scanning of autoradiograms of the same dot blots. Dot blots for comparison to Northern blots and for some additional experiments were prepared by denaturing total parietal cell RNA a t 55 "C for 15 min in 7.4% formaldehyde, 6 X SSC (0.9 M NaCI, 90 mM sodium citrate, pH 7.0). Duplicate, successive, 2-fold serial dilutions of denatured RNA (2.8, 1.4, and 0.7 pg) were then spotted onto nitrocellulose and hybridized (9) to the ""P-labeled cDNA probe.

The probes used for hybridization analysis were carbonic anhydrase I1 (pMCAII) cDNA from mouse anemic spleen (12, 13) (a gift from Dr. Peter Curtis of the Wistar Institute of Anatomy and Biology and Dr. P. J. Venta of the University of Michigan), rat gastric H+,K+- ATPase cDNA (14) (a gift from Dr. Gary Shull of the University of Cincinnati), @-actin cDNA derived from chicken brain (15) (a gift from Dr. Beverly Mitchell of the University of Michigan), a human cDNA (pKT218) encoding ubiquitin fused to an ubiquitin extension protein named ubiquitin carboxyl-terminal precursor (UBCP) (16) (a gift from Dr. P. Kay Lund of the University of North Carolina), and the cDNA fragment of the ubiquitin fusion protein, lacking ubiquitin sequences and encoding only the UBCP sequences, which we used as a control in our experiments. These probes were labeled with ["PI

dCTP to a specific activity of 1 X lo9 cpm/pg by the random priming procedure (17).

RESULTS

Hybridization Assays for Specific mRNA-We verified the quantitative accuracy of our dot blot and Northern blot hybridization assays by densitometric scanning of the autoradiographic images. As shown in Fig. L4, when less than 3 pg/ spot was probed with gene-specific "P-labeled cDNA, the density of the film image produced by the probe, which hybridized to the isolated gastric parietal cell RNA, was directly proportional to the amount of RNA spotted onto nitrocellulose. The comparative densitometric scanning 'of autoradiographic images derived from hybridization of the same "P-labeled cDNA probe @-actin) to RNA samples analyzed in parallel by each method is shown in Fig. 1B.

Secretagogue Induction of Gene-specific mRNAs-The addition of 0.1 mM carbachol (a concentration shown to be the maximal stimulatory dose (10)) to isolated canine gastric parietal cells caused rapid increases in steady-state levels of mRNAs encoding both carbonic anhydrase I1 and H',K'- ATPase. As shown by the Northern blot analyses depicted in Fig. 2, A and B, carbachol increased steady-state levels of H',K'-ATPase mRNA to a peak after 20 min of incubation, with similar kinetics as the increase observed in specific

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FIG. 1. Verification of hybridization assays for specific mRNA. A, serial dilutions of the same RNA sample from isolated canine parietal cells were spotted onto nitrocellulose and hybridized with each "P-labeled cDNA probe. The radioactivity visualized from the representative autoradiogram obtained with the @-actin cDNA probe shown a t right was analyzed by densitometry. B, a representative comparison of dot blot and Northern blot assays of the same RNA samples after hybridization to the same "P-labeled cDNA probe and analysis by densitometry. RNA from cells stimulated for various periods of time with 0.1 mM carbachol was spotted (squure symbols) or transferred after electrophoresis on a formaldehyde gel (circle symbols) onto nitrocellulose before hybridization with a 32P-labeled cDNA probe for @-actin. A linear transformation of the densitometric data is also depicted.

Stimulation of Parietal Cell Gene Expression 11383

FIG. 2. mRNA levels in isolated canine gastric parietal cells treated with carbachol. Aliquots of total RNA (4.5 pg) extracted a t indicated times following addition of 0.1 mM carbachol to isolated parietal cells from a single dog were subjected to Northern blot analysis using "P-labeled cDNA probes for carbonic anhydrase I1 (CAII ) (A) , H',K'- ATPase ( R ) , &actin ( C ) , a complete ubiquitin fusion protein (D), or a fragment of the fusion protein ( E ) , an ubiquitin extension protein (UBCP) . The blots were analyzed by densitometry of the autoradiograms. Similar data were obtained with cells from three other dog preparations.

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FIG. 4. mRNA levels in isolated canine gastric parietal cells treated with gastrin. RNA was extracted from cells from a single dog which had been 3 n o incubated for the indicated times in 10 tm nM gastrin, and 4.5 pg samples were analyzed by Northern blotting. Hybridiza- tion to replicate nitrocellulose blots was with '*P-labeled cDNA probes for carbonic anhydrase I1 (CAII ) (A) , H+,K'- ATPase ( B ) , &actin ( C ) , or an ubiquitin extension protein (D) (UBCP). The :! :z blots were analyzed by densitometry of e * the autoradiograms. Similar data were qm

obtained with cells from three other dog preparations.

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carbonic anhydrase I1 mRNA (9). Carbonic anhydrase I1 mRNA levels returned to basal over the course of 1 h, and the H+,K+-ATPase mRNA declined toward basal from its peak in parallel fashion. The addition of 0.1 mM carbachol to parietal cells induced a dramatic and sustained increase in 8- actin mRNA levels over the course of 1 h, suggesting that time of exposure to secretagogue did not alter the responsiveness of the cells in expressing 8-actin (Fig. 2C). Hybridization of a cDNA encoding an ubiquitin fusion protein to a Northern blot of RNA from parietal cells stimulated with carbachol is

shown in Fig. 20. Of the three mRNA sequences which hybridized to the probe, one of about 600 bases in length remained constant for the duration of the study and served as an adequate control. Two larger mRNA species which hybridized with the ubiquitin fusion protein probe, in contrast to the 600 base mRNA species, increased in a time-dependent manner upon stimulation of the parietal cells with carbachol. We confirmed that the 600-base mRNA species serving as our control was specific for the UBCP sequence (16, 17) by hybridization of a replicate Northern blot to the UBCP-

11384 Stimulation of Parietal

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FIG. 5. Inhibition of carbachol, histamine, or gastrin effects on induction of gene expression by specific receptor antago- nists. mRNA levels of carbonic anhydrase I1 (CAZI), H+,K+-ATPase, or 8-actin in parietal cell extracts were analyzed by densitometry of dot blots. A, RNA was derived from cells that were unstimulated (basal) and from cells that were incubated in EBSS only for 40 min ( N S ) , in 0.1 mM carbachol for 40 min, in 0.1 mM carbachol for 40 min after 20-min preincubation in 0.1 mM atropine, and in atropine for 60 min. B, RNA was from unstimulated cells (basal), and from cells that were incubated in EBSS only for 40 min ( N S ) , in 0.1 mM histamine for 40 min, in histamine for 40 min after 20-min preincubation in 0.1 mM cimetidine, in cimetidine for 60 min, in histamine for 40 min after 20-min preincubation in 0.1 mM famotidine, and in famotidine for 60 min. C, RNA was isolated from unstimulated cells (basal) and from cells that were incubated in EBSS only for 40 min ( N S ) , in 10 nM gastrin (G17) for 60 min, in gastrin for 60 min after 10-min preincubation in 1 mM proglumide, in proglumide for 60 min, in gastrin for 60 min after 10-min preincubation in 0.1 mM CR1409, and in CR1409 for 60 min. Results were obtained with a single dog preparation.

encoding cDNA fragment (16) (Fig. 2 E ) . The addition of 0.1 mM histamine to isolated gastric parietal

cells induced a modest but reproducible increase over steady- state basal levels in carbonic anhydrase I1 and H',K'-ATPase mRNAs. A representative experiment of mRNA obtained from cells from a single dog is shown in Fig. 3. Levels of mRNA for each enzyme peaked around 20 min after secreta-

Cell Gene Expression

0 HOUR 1 HOUR 4 HOURS

.. .J L c C A II

0 10 20 40 60 0 10 20 40 60 TIME (MIN)

0 10 20 40 60 TIME (MIN) TIME (MIN)

0 2 0 4 0 6 0 TIME (MIN)

FIG. 6. Time-dependent reduction in magnitude of response of carbonic anhydrase I1 (CAZZ) mRNA levels to carbachol stimulation. Levels of carbonic anhydrase I1 mRNA in RNA extracts from cells stimulated with 0.1 mM carbachol were analyzed by densitometry of autoradiograms derived from Northern blotting experiments. RNA was from cells stimulated immediately (closed squares), 1 h (closed circles), or 4 h (closed triangles) after elutriation. Results were obtained with cells from a single dog preparation.

gogue addition, with a decline toward basal after 1 h. In contrast, levels of @-actin mRNA increased steadily and substantially over the course of 1 h. The level of the mRNA which hybridized to the "P-labeled UBCP cDNA remained near basal for the duration of the study.

As shown in Fig. 4, when isolated parietal cells were stimulated with 10 nM gastrin (G17), steady-state levels of carbonic anhydrase I1 and H+,K+-ATPase mRNAs exhibited sustained increases that reached levels of about 2-fold over basal within 1 h. &actin mRNA levels steadily increased by more than 3-fold during the same period. During the period when the cells were exposed to gastrin, the levels of mRNA hybridizing to the UBCP cDNA probe remained unchanged and served as an adequate control.

To investigate whether the increases in expression of carbonic anhydrase 11, H',K+-ATPase, and p-actin mRNAs were triggered by the binding of acid secretagogues to specific receptors on the gastric parietal cell surface, we incubated cells in each stimulant after pretreatment for 20 min with a specific receptor antagonist or with a vehicular control. Iso- lated cells, incubated in 0.1 mM carbachol for 40 min after pretreatment with 0.1 mM atropine, showed a substantial reduction in steady-state levels of carbonic anhydrase 11, H+,K+-ATPase, and j3-actin mRNAs as compared to the levels observed in stimulated cells not pretreated with atropine (Fig. 5). Similarly, cells incubated in 0.1 mM histamine for 40 min after preincubation in either, 0.1 mM cimetidine or 0.1 mM famotidine exhibited a substantially reduced level of each specific RNA as compared with the levels measured in cells not pretreated with Hz receptor inhibitor. Decreased levels of specific mRNA were also measured in isolated cells incubated in 10 nM gastrin (G17) for 60 min after preincubation in either 1 mM proglumide or 0.1 mM CR1409 (19, 20), as compared to specific mRNA levels in cells treated with G17 without inhibitor.

Influence of Time after Cell Isolation on Differentiated Func-

Stimulation of Parietal Cell Gene Expression 11385

A

0 2 4 8 1 6 2 4 TIME (HOURS)

120 1

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TIME (HOURS)

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FIG. 7 . Time-dependent changes in basal levels of specific mRNAs in canine gastric parietal cells. mRNA levels of carbonic anhydrase I1 (CAZZ) ( A ) , H+,K+-ATPase (CAZZ), or p-actin in parietal cell extracts (C) were analyzed from densitometric scans of dot blots of total cellular RNA. RNA was derived from cells suspended in EBSS containing 10 mM HEPES buffer (pH 7.4), 0.1% BSA and incubated at 37 "C in 95% air, 5% CO, for the indicated times after elutriation. Results were obtained with cells from a single dog preparation.

tion Gene Expression-As we have reported previously (9), carbonic anhydrase I1 gene expression appeared to decrease with time after cell isolation. To further investigate these findings, we examined the changes in steady-state levels of carbonic anhydrase I1 mRNA in cells stimulated with 0.1 mM carbachol at various intervals following cell elutriation. We observed, as shown in Fig. 6, that carbonic anhydrase I1 mRNA exhibited the same time-dependent increases within each subset of cells after stimulation with carbachol, followed by a return to basal within 1 h. However, there was a marked and successive decline in the magnitude of the response of cells studied after several hours as compared to freshly isolated cells.

To examine whether time after cell isolation affected expression of all parietal cell genes or only those related to differentiated function, we measured steady-state levels of a variety of mRNAs from unstimulated gastric parietal cells at intervals after cell elutriation (Fig. 7 ) . Levels of accumulated carbonic anhydrase I1 and H+,K+-ATPase mRNAs in unstimulated cells were rapidly attenuated over time, declining to below 50% of basal within 8 h. In contrast, the level of P-

actin mRNA stabilized near basal for several hours and remained close to 70% of basal 24 h after the cells were isolated.

DISCUSSION

Isolated canine gastric parietal cells have been used widely as a model system to explore the regulation of gastric acid secretion by chemical transmitters (1, 10). The major recog- nized secretagogues, acetylcholine, histamine, and gastrin, mediate their interdependent effects via separate neurocrine, paracrine, and endocrine routes, respectively. When removed from its endogenous environment, the isolated parietal cell may be used to study the effect of a single stimulant and the specificity of its receptor in regulating acid secretion. We chose to use this reductionist approach to examine the effect of each acid secretagogue in regulating the expression of genes required for acid secretion. Activation of the parietal cell to secrete acid is accompanied not only by changes in metabo- lism, but also in morphology, particularly as related to the intracellular sites of various proteins, including carbonic anhydrase I1 and H+,K+-ATPase (4, 6, 21). In addition, there are changes in ion transport properties in both the basolateral membrane and apical (secretory) membrane surfaces (1, 2). These changes predictably would involve changes in expression of many genes expressing differentiated function in the parietal cell.

Gastric H',K'-ATPase is a membrane-bound enzyme lo- cated at the secretory surface and has been identified as the enzyme responsible for gastric proton transport (3). Its action has been shown to alkalinize the cell interior (5,22). Carbonic anhydrase, an enzyme abundant in the cytoplasm of the parietal cell, is also present at the apical plasma membrane, an ideal position to catalyze the generation of HCO; from the resultant OH- ions. In previous work (9), we have shown that carbonic anhydrase gene expression in parietal cells is regulated by each of the principal acid secretagogues, supporting the importance of the enzyme in the secretory process. In the current work therefore, we examined the effects of secretagogues on expression of the gene encoding the proton pump itself, the H',K+-ATPase. Our experiments demonstrated that H+,K'-ATPase mRNA levels followed similar kinetics as carbonic anhydrase I1 mRNA levels upon secretagogue stimulation of the cells. These findings suggest that the gene expression of these two enzymes may be regulated in coordinate fashion by secretatogues during the process of acid secretion.

A second goal of our studies was to explore the role of actin gene expression in stimulated parietal cells. Actin filaments frame a cortical network between the tubular system and the luminal secretory membrane. With the onset of acid secretion, the tubular system becomes incorporated into the secretory canalicular membrane and villous processes containing microfilaments fill the secretory surface. It has been suggested that this morphological reorganization of membranes and cytoskeletal matrix could regulate acid secretion by the translocation of membranes containing the proton pump from the endocellular compartment to the secretory surface (4). Our results showing a rapid and sustained induction of actin gene expression by each of the three secretagogues suggest the importance of actin in the secretory process and support the hypothesis that P-actin, its synthesis, polymerization, and movement occurring in response to parietal cell activation, may be an important regulator of acid secretion.

When assessing the increases in carbonic anhydrase 11, H+,K+-ATPase, and P-actin mRNA levels, we considered the possibility that the stability of these mRNAs may be affected by the action of an acid secretagogue during the time that the

11386 Stimulation of Parietal Cell Gene Expression

parietal cell is under its stimulation. We reasoned that gastrin, which has been thought to exert its major effect on the highly differentiated parietal cell as an acid secretagogue, is a trophic hormone as well (23) and may selectively stabilize specific mRNAs in the manner that epidermal growth factor has been shown to stabilize &actin mRNA in differentiated cells (24, 25). This hypothesis seems unlikely, however, in view of the fact that specific increases in levels of the same mRNAs also occur in response to both carbachol and histamine, stimulants of acid secretion which are not cellular growth factors. More- over, in previous work (9) we demonstrated the ability of gastric secretagogues to induce parietal cell carbonic anhydrase I1 mRNA expression at the level of gene transcription.

We initially used the cDNA probe for a ubiquitin fusion protein as a control probe for our experiments with the expectation that ubiquitin gene expression would remain unchanged in parietal cells during stimulation with acid secretagogues. Unexpectedly, of the three mRNA species which hybridized with the cDNA probe, only one that encoded ubiquitin fused to an extension protein sequence (UBCP) remained constant for the duration of our experiments. The function of the small, basic ubiquitin extension protein in the cell is unknown, although it is present as a stable product in many cell types and appears to be cosynthesized as a fusion product with ubiquitin. Its structure and sequence suggest that it plays a role in a fundamental cellular process (18). We were surprised to discover that two larger mRNA species hybridizing with the ubiquitin cDNA probe were induced in parietal cells in response to secretagogue stimulation. It is likely that these mRNAs encode ubiquitin precursors which contain several repeats of the ubiquitin amino acid sequence (26-28). The precise physiological functions of ubiquitin in the gastric parietal cell are not known, and the finding that its gene expression was induced by secretagogue stimulation seems to suggest that ubiquitin serves an important function for the cell during acid secretion. Conjugation of ubiquitin to intracellular proteins is known to mediate their selective degradation in eukaryotes, and an increase in ubiquitin may be required to aid in clearing the parietal cell of unnecessary proteins during rapid morphological changes.

Our data indicate that the observed changes in gene expression resulted from activation of specific receptors by acid secretagogues. We confirmed that the induction by carbachol of carbonic anhydrase 11, H+,K+-ATPase, and p-actin gene expression was mediated via a cholinergic receptor by showing that atropine inhibited the effect of carbachol on the expression of these genes (29). Similarly, our findings suggest that histamine action on parietal cells is mediated by a pharma- cologically typical Hz receptor (29); both cimetidine and famotidine inhibited the observed effects of histamine induction of gene expression. Moreover, the effect of gastrin on gene expression was inhibited by the compounds proglumide and CR1409, both of which have gastrin receptor antagonistic activity (19, 20).

In examining the responsiveness of the cells to stimulation with carbachol, it was clear that the resultant magnitude of increase in carbonic anhydrase I1 mRNA was attenuated rapidly after cell isolation. In addition, basal levels of mRNA for both carbonic anhydrase I1 and H+,K+-ATPase declined rapidly. A likely possibility is that carbonic anhydrase I1 and H+,K+-ATPase are transcripts of differentiated function

genes that require interactions between parietal cells and the surrounding extracellular matrix for their expression (30-32). Normal cell-cell communication is disrupted during the isolation of cells, and some of the regulators of gene expression may actually depend upon intracellular signals originating from adjacent cells. As suggested by others working with isolated hepatocytes (31, 32), the complete and accurate activation of differentiated function genes may actually require the establishment of a basal level of mRNA synthesis and accumulation as well as in increase in RNA polymerase I1 activity that is somehow dependent on the mature tissue structure. In contrast to declining levels of carbonic anhydrase I1 and H+,K+-ATPase mRNAs, basal levels of p-actin mRNA remained fairly stable for more than 8 h and a subsequent decline occurred only after this relatively prolonged plateau period. P-actin is a cytoskeletal protein, and its gene expression appears to be subject to separate regulatory signals (33) from differentiated function genes, a phenomenon that has been observed in other in vitro systems (32, 33). From our studies we conclude that isolated cells provide a convenient and useful system for analyzing the regulation of expression of genes required for acid secretion, even though they lack some crucial regulatory factors provided in vivo and, thus, are limited in their ability to model accurately and completely the activation of parietal cell gene expression by acid secretagogues.

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