Gastrin biosynthesis in canine G cells

Vinzenz Stepan1, Kentaro Sugano2, Tadataka Yamada3, Jung Park2, and Chris J. Dickinson1

Departments of 1 Pediatrics and 2 Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109-0656; 3 GlaxoSmithKline, King of Prussia, Pennsylvania 19406-0939


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Gastrin requires extensive posttranslational processing for full biological activity. It is presumed that progastrin is cleaved at pairs of basic amino acids by a prohormone convertase to form a glycine-extended intermediate (G-Gly) that serves as a substrate for peptidyl-glycine alpha -amidating monooxygenase (PAM), resulting in COOH-terminally amidated gastrin. To confirm the nature of progastrin processing in a primary cell line, we performed [35S]methionine-labeled pulse-chase biosynthetic experiments in canine antral G cells. Radiolabeled progastrin reached a peak earlier than observed for G-Gly or amidated gastrin. G-Gly radioactivity accumulated in G cells and preceded the appearance of radioactivity in amidated gastrin. The conversion of G-Gly to amidated gastrin was enhanced by the PAM cofactor ascorbic acid. To determine whether one member of the prohormone convertase family (PC2) was responsible for progastrin cleavage, G cells were incubated with PC2 antisense oligonucleotide probes. Cells treated with antisense probes had reduced PC2 expression, an accumulation of radiolabeled progastrin, and a delay in the formation of amidated gastrin. Progastrin in antral G cells is cleaved via PC2 to form G-Gly that is converted to amidated gastrin via the actions of PAM.

COOH-terminally amidated gastrin; prohormone convertase; peptidyl-glycine alpha -amidating monooxygenase; glycine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AS IS THE CASE WITH OTHER peptide hormones, the structure of progastrin suggests that the formation of COOH-terminally amidated gastrin (G-NH2) requires extensive posttranslational processing (Fig. 1). Previously we characterized gastrin processing intermediates in tissues (2, 7, 19, 24, 35, 37). Because there are no stable gastrin-producing cell lines, we also characterized progastrin processing mechanisms in vitro (9) and heterologously expressed the progastrin cDNA in endocrine cell lines to define the molecular determinants for progastrin processing in a cellular system (8, 11, 27). On the basis of these studies, we had surmised that progastrin is cleaved at basic amino acids by a prohormone convertase (PC) resulting in the formation of a glycine-extended intermediate (G-Gly). G-Gly then serves as a substrate for peptidyl-glycine alpha -amidating monooxygenase (PAM) to form G-NH2. The posttranslational processing of progastrin is of particular importance because both G-NH2 and G-Gly have biological activities mediated via distinct receptors (20, 30, 38). In complementary studies, other investigators have noted that some PC family members are expressed in the antrum and that PC expression is physiologically regulated (23). However, to date, no investigation has confirmed that a given PC is responsible for progastrin processing in antral G cells. Thus to confirm our in vitro studies and those in endocrine cell lines, we undertook the present gastrin biosynthesis studies using primary cultures of isolated and enriched canine antral G cells.


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Fig. 1.   Proposed model of gastrin processing. Progastrin is initially endoproteolytically cleaved by a prohormone convertase (PC) at Arg57Arg58 and Arg94Arg95. The COOH-terminal Arg94 and Arg95 residues are sequentially removed by carboxypeptidase E, resulting in glycine-extended gastrin of 34 amino acids (G34-Gly) in length. Subsequent cleavage at Lys74Lys75 and COOH-terminal amidation via the action of peptidyl-glycine alpha -amidating monooxygenase (PAM) results in the formation of G17-NH2.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

G cell isolation and culture. Details of the method for isolation and culture of canine antral G cells have been described previously (35). In brief, antral mucosal sheets bluntly separated from freshly excised canine stomach were minced into small pieces sequentially exposed to 0.35 mg/ml collagenase (type I; Sigma, St. Louis, MO) in basal Eagle's medium and to Ca2+- and Mg2+-free Earle's balanced salt solution containing 1 mM EDTA. The cells liberated at the last step of digestion were collected and separated by counterflow elutriation to obtain fractions enriched in G cells. These fractions were plated onto tissue-culture wells previously coated with a supportive collagen layer at a density of 2-3 × 106 cells/well and cultured for 18 h in Ham's F-12/DMEM (1:1, vol/vol; Irvine Scientific, Santa Ana, CA) supplemented with 10% dog serum and gentamicin (100 µg/ml).

Biosynthesis studies. For pulse-labeling experiments, cultured cells were washed twice with methionine-deficient Ham's F12/DMEM (custom prepared by Irvine Scientific) containing 10% dialyzed dog serum and gentamicin (100 µg/ml). Cells were then incubated with the same media containing [35S]methionine (1,165.5 Ci/mmol; 100-150 µCi/well; New England Nuclear, Boston, MA) for various time intervals at 37°C. To examine the effects of ascorbic acid on the biosynthesis of gastrin, it was added to the media at a concentration of 50 µg/ml and was included in both the pulse and chase media. For pulse-chase studies, 2 h after pulse labeling in the radioactive methionine-deficient media, cells were washed three times with methionine-containing complete media and further incubated in the complete media for up to 6 h.

Analysis of biosynthetic products. After various time intervals of pulse labeling, cells from duplicate wells were harvested by scraping the supportive collagen layer and boiled in 2 ml of water for 5 min. Cell extracts were then applied to affinity columns packed with Affigel-10 beads (Bio-Rad, Richmond, CA) coupled to antisera specific for progastrin-like immunoreactivity, [antibody (Ab) 8207] (37), G-Gly antibody (Ab 8237) (24, 34), and the amidated COOH terminus of gastrin (Ab 1802, kindly provided by Dr. John Walsh, Los Angeles, CA). Ab 8207 was originally generated against GlyArgArg extended gastrins but has cross-reactivity with gastrins with further COOH-terminal extensions, such as GlyArgArgSerAla in radioimmunoassays (37). Ab 8237 and Ab 1802 are specific for G-Gly and amidated gastrin, respectively, and cross-react <1% with progastrin (24). After application of extracts, the affinity columns were washed four times with 2 ml of 50 mM sodium phosphate/0.1 M NaCl (pH 7.4) followed by 2 ml of 50 mM ammonium acetate (pH 5.0) four times. Specifically bound radioactivity was eluted with three column volumes of 2% trifluoroacetic acid (TFA) and lyophilized. The lyophilized samples were dissolved with 100 µl of 0.1 N NaOH and the total volume was adjusted to 1.0 ml with sodium barbital buffer pH 8.6 containing 150 µg of phenol red. After saving a 50-µl aliquot for counting total radioactivity, the remainder of the reconstituted samples were applied to Sephadex G-50 SF columns (1.5 × 75 cm) calibrated with amidated tetratriacontagastrin (G34-NH2), heptadecagastrin (G17-NH2), and dye markers [blue dextran for void volume (Vo), and phenol red for salt peak (Vt)]. Columns were equilibrated and eluted with sodium barbital buffer pH 8.6 containing 0.02% NaN3 and fractions of 1.8 ml were collected.

Aliquots of 1 ml from each fraction were counted in 10 ml of aqueous scintillation cocktail (ACS; Amersham, Arlington Heights, IL) in a liquid scintillation counter (Beckman Instruments, Fullerton, CA). The affinity-purified radiolabeled products were also analyzed by reverse-phase, high-pressure liquid chromatography on a Z-module, µ-Bondapak C8 cartridge (Waters Associates/Millipore, Milford, CA). The column was equilibrated with 0.1% TFA and eluted with a linear gradient of acetonitrile containing 0.1% TFA. Fractions of 4 ml were collected and 2-ml aliquots were counted in 18 ml of ACS.

Inhibition of PC2 expression with antisense oligonucleotides. In previous studies using endocrine cell lines, we noted that one member of the PC family, PC2, was a candidate progastrin processing enzyme and is expressed in antral G cells (11, 23, 28). Thus, in additional experiments, we sought to determine the cDNA sequence of canine PC2 for antisense oligonucleotide synthesis. We constructed two oligonucleotide probes from highly homologous regions of the known rat, human, and mouse sequences (29, 33). Canine antral RNA was obtained with TRIzol (GIBCO-BRL), reverse transcribed from polyA+ RNA using oligo-dT15 (1 µg) as a primer, and AMV reverse transcriptase (13 units/reaction). The reaction mixture, without the reverse transcriptase, was incubated at 41°C for 15 min; the reverse transcriptase was then added, and the reaction was allowed to proceed for 1 h at 41°C. The reverse-transcribed PC2 DNA was amplified by a PCR reaction containing 100 pmol of each amplification primer, 1.5 µg of genomic DNA, 0.01% gelatin, and 2.5 units of Taq polymerase plus (in mM) 1.25 of each dNTP, 10 Tris (pH 8.3), 50 KCl, 1.5 MgCl2, and 2 mM DTT. Reactions were performed in a Perkin-Elmer thermocycler for 30 cycles consisting of dissociation at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 2 min. PCR products were analyzed on a 3% Nusieve, 1% agarose gel, and a 355 base pairs (bp) product sequenced in core facilities at the University of Michigan. A phosphorothioate modified antisense 18 bp oligonucleotide probe (5'-CCUUCCACUGAGAUACAC-3') was synthesized (BioSynthesis, Lewisville, TX). The chosen sequence was near the translation start site of PC2. A control or nonsense oligonucleotide probe containing the same base pairs but in random order was also synthesized (1). A third nonsense, control probe was also synthesized to a different portion of the canine PC2 sequence.

After isolation, G cells (1-2 million/well) were plated in matrigel-coated 12-well plates and cultured in Ham's F-12/DMEM supplemented with 10% dog serum and gentamicin (100 µg/ml) for 24 h. Cells were then washed twice with Opti-M (GIBCO) and transfected with either control or antisense oligonucleotide probes (0.1 µM/well) using Superfect transfection reagent (4 µl/well) (GIBCO-BRL). After 10 h, cells were washed with Opti-M media and transfected again, as described above. After an overnight incubation, cells were washed twice with methionine-free DMEM and incubated in methionine-free media for 30 min. Cells were then pulse-labeled with methionine-free DMEM supplemented with [35S]methionine (150 µCi/well). After a 2-h pulse incubation, cells were washed twice with media containing 5× methionine and then incubated in the same media for up to 6 additional hours (chase). Media were collected, cells washed once with ice-cold PBS (GIBCO), and cells were lysed in 500 µl of lysis buffer of (in M) 0.05 HEPES (pH 7.4), 0.15 NaCl, 10% glycerol, 1% Triton X-100, 0.0015 MgCl, 0.001 EDTA, 0.001 NaVO4, 0.01 Na-pyrophosphate, and 0.01 NaF, plus 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 mg/ml AEBSF. Cell lysates were used for immunoprecipitation using Sepharose A beads and either Ab 5135 for G-NH2 (kind gift of J. Walsh, Los Angeles, CA) or Ab 8207 for progastrin. Beads were then dissolved in 0.1 M NaOH, added to 10 ml of aqueous scintillation cocktail, and counted.

To quantify the uptake of DNA into our G cells, we performed green fluorescent protein (GFP) studies. G cells were plated in 12-well plates with coverslips, coated with matrigel, and allowed to settle overnight. Cells were washed twice with serum-free media and then transfected overnight with 2 µg of the pCMS-Egfp Vector (Clontech, Palo Alto, CA) containing GFP, with the use of Superfect transfection reagent (Quiagen). Media were changed again to serum-free media and 5 h later the cells were washed twice with PBS, fixed for 30 min with 4% paraformaldehyde, washed with PBS, and stored at 4°C. Immunohistochemistry was performed with the gastrin-specific Ab 5135 (1:2,000). Cy5 donkey anti-rabbit IgG (Jackson Laboratories) was used as a secondary antibody. GFP was expressed in >98% of cells. When we specifically stained transfected cells for gastrin, we noted that dual staining of cells was in excess of 95%, suggesting that almost all G cells were efficiently transfected (data not shown).

To quantify PC2 expression by Western blot, cells were lysed as described above and incubated on a rotary shaker overnight at 4°C with a PC2 antibody (kind gift of Nabil Seidah, Montreal, PQ, Canada). Aliquots of Sepharose A (Pharmacia) were then added and the solutions were mixed for one additional hour. After centrifugation, pellets were washed twice with lysis buffer. Samples were then resuspended in 20 µl of electrophoresis buffer [consisting of (in ml) 1 glycerol, 0.5 2-mercaptoethanol, 3 10% SDS, 2 0.1% bromophenol bleu, plus 1.25 ml of 1 M Tris buffer and 0.6 g urea], boiled for 5 min, applied to a 8% SDS-polyacrilamide gel for 10 ml, and transferred to a nitrocellulose membrane. Membranes were incubated in Tris-buffered saline (0.15 mol/l NaCl) containing 0.3% Tween and 5% nonfat dry milk for 1 h at room temperature to block nonspecific binding sites. Blots were then incubated with the primary antibody (PC2) at a final dilution of 1:1,000 in Tris-buffered saline for 2 h, washed twice and incubated with a peroxidase-linked secondary antibody (Zymed rabbit anti-mouse-horseradish peroxidase, 1:1,250) for 60 min. Immunoreactive bands were visualized using the standardized immunoblotting detection system (Amersham).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The time-dependent incorporation of [35S]methionine into immunoadsorbable material is depicted in Fig. 2. Radioactivity incorporated into the progastrin-specific fraction was quite low, but a gradual rise was detected over the initial hour of incubation. In contrast, the radioactivity incorporated into the G-Gly fraction increased almost linearly during the 2-h pulse incubation. Notable G-NH2-specific radioactivity did not rise above baseline until 2 h of incubation. No radiolabeled progastrin, G-Gly, or G-NH2 was detectable in the media during a 2-h labeling period.


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Fig. 2.   Incorporation of [35S]methionine ([35S]Met) into immunoadsorbable progastrin, G-Gly, and G-NH2. Cells were labeled by incubation with [35S]Met for 30 min, 1 h, and 2 h. After labeling, cells were harvested and extracted by boiling. Radiolabeled products were purified by chromatography on affinity columns specific for progastrin, G-Gly, and G-NH2, respectively. cpm, Counts/min.

In previous studies (9, 14), we characterized the conversion of G-Gly to G-NH2 via the action of PAM in vitro. As was the case with peptide amidation found in other tissues, gastrin amidation was dependent on the presence of ascorbic acid. Thus additional experiments were performed to determine the effect of this cofactor on gastrin biosynthesis. As shown in Table 1, ascorbic acid remarkably enhanced [35S]methionine incorporation into amidated gastrin and, at the same time, reduced incorporation of radioactivity into G-Gly. Radioactivity in the progastrin fraction was essentially unchanged by the presence or absence of ascorbic acid.

                              
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Table 1.   Effect of ascorbic acid on incorporation of [35S]methionine into immunadsorabable progastrin, G-Gly, and gastrin

To explore the precursor-product relationship among the radioactively labeled components of gastrin biosynthesis, we pulse-labeled the cells with [35S]methionine and then chased the radioactivity with nonradioactive methionine. As depicted in Fig. 3, incorporation of [35S]methionine into G-Gly continued to increase after pulse labeling and remained high throughout the chase period of 6 h in the absence of ascorbic acid. Radioactive labeling of the G-NH2-specific fraction remained almost negligible until 3 h of chase incubation and then emerged suddenly at 6 h. In contrast, when the media were supplemented with 50 mM ascorbate, G-Gly radioactivity remained low at 3 h and decreased by the end of the chase period, whereas G-NH2-specific radioactivity appeared much faster than in the absence of ascorbate and continued to increase throughout the chase period.


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Fig. 3.   Time course of [35S]methionine radioactivity incorporation into immunoadsorbable G-Gly and G-NH2 after pulse labeling. Boiled cell extracts were prepared immediately after 2 h of incubation in medium containing [35S]methionine (pulse) and after transferring to nonradioactive medium containing unlabeled methionine (chase). Cell extracts were applied to immunoaffinity columns specific for progastrin, G-Gly, and G-NH2. The affinity-purified materials were reconstituted in 1 ml of barbital buffer as described in Analysis of biosynthetic products and 50-µl aliquots were assayed in a liquid scintillation counter. After background counts of 40 counts/min were subtracted, specific radioactivity was expressed as total counts in 1 ml of reconstituted affinity eluate. Open and closed circles represent ascorbate-supplemented (Vit C+, 50 µg/ml) and ascorbate-deficient groups (Vit C-), respectively.

On gel filtration, radioactivity associated with the progastrin fraction formed a predominant peak in the column void with several ill-defined peaks around the elution position of G34 data (not shown). These peaks were present irrespective of the addition of ascorbic acid and gradually disappeared during the chase period.

As shown in Fig. 4A, in the absence of ascorbate, radioactivity associated with the G-Gly fraction at the initiation of the chase period was separated into several peaks, including those eluting just after the column void and just before G34 and G17. By 3 h of chase incubation, a dominant peak of radioactivity was observed at an elution position between G34 and G17, which corresponded to the elution position of G17-Gly (34), and a shoulder of radioactivity corresponding to G34-Gly was also present. However, after an additional 3 h of incubation, this shoulder was diminished, but the peak corresponding to G17-Gly remained. Radioactivity associated with the G-NH2 fraction (Fig. 4B) did not elute in any discernible peaks until 6 h of chase incubation when a peak was observed at the elution position of G17-NH2 with a small shoulder corresponding to G34-NH2 being present.


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Fig. 4.   Gel filtration of newly synthesized and affinity-purified G-Gly (A) and G-NH2 (B). Biosynthetic products at 2 h of pulse and at 3 and 6 h of chase incubation were purified by G-Gly and G-NH2-specific immunoaffinity columns. Affinity eluates were reconstituted in 1 ml of barbital buffer and 0.95 ml of the reconstituted samples were analyzed by chromatography on a Sephadex G-50 superfine column (1.5 × 75 cm) as described in Analysis of biosynthetic products. Aliquots (1 ml) of the eluted fractions were counted and the radioactivity in each aliquot, after subtracting background counts of 40 counts/min, is depicted. Vo indicates the elution position of blue dextran. The elution fractions of human G34-NH2, G17-NH2, and [35S]Met are also indicated.

In the presence of ascorbate, markedly different gel-filtration profiles for radioactivity associated with G-Gly and G-NH2 were noted (Fig. 4B). For the duration of chase incubation, radioactivity associated with the G-Gly fractions eluted in small, poorly differentiated peaks near the column void and the elution volumes of G17-Gly and G34-Gly. In contrast, radioactivity associated with G-NH2 eluted in distinct peaks. At the initiation of chase incubation, the major peak coeluted with G34-NH2, but over the next 6 h, the peak corresponding to G17-NH2 became predominant with only a shoulder indicating the presence of radioactivity associated with G34-NH2. To confirm the identity of the gel filtration peaks, radioactive products extracted from cells incubated in chase medium for 6 h, with or without ascorbate supplementation, were purified by affinity chromatography and analyzed by HPLC. As depicted in Fig. 5, the majority of the radioactivity coeluted with G17-NH2 and G34-NH2. The elution profile of the radioactivity specific for G-Gly was almost identical with that of G-NH2 radioactivity, in accordance with our previous observations that G17-Gly and G34-Gly elute at virtually the same positions as the corresponding molecular forms of G-NH2 on the HPLC protocol used for these studies.


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Fig. 5.   Elution profile of newly synthesized and affinity purified G-Gly and G-NH2 on high-pressure liquid chromatography. Affinity-purified radioactive products specific for G-Gly and G-NH2 were reconstituted in 4 ml of 0.1% trifluoroacetic acid (TFA) and applied to a µ-Bondapak C8 reverse-phase column. The column was washed with 0.1% TFA and eluted with a linear gradient of acetonitrile. Fractions of 4 ml were collected, and 2-ml aliquots were counted. The elution fractions of G17-NH2 and G34-NH2 are indicated.

To explore the cleavage of progastrin by PC2, we first amplified 355 bp of canine PC2 reverse transcribed mRNA by PCR. The identified PC2 sequence was 91% homologous with human PC2 and had much less homology than other PC family members such as PC1/PC3 (42%) (29, 32) and PC5 (63%) (22). Searches of GenBank revealed no other sequences with higher degrees of homology. Utilizing this sequence information, we synthesized an antisense canine PC2 oligonucleotide probe located near the translation start site of PC2 and incubated G cells with control or antisense PC2 probes as described above in a separate set of experiments. As depicted in Fig. 6, PC2 antisense oligonucleotide probes significantly inhibited PC2 expression as quantified by Western blot. In additional pulse-chase experiments, the inhibition of PC2 expression was associated with an overall decrease and delay in the appearance of labeled G-NH2 in cell extracts (Fig. 7). As expected, the decrease in PC2 expression was associated with an increase in labeled progastrin and a delay in its disappearance from cell extracts compared with control (Fig. 8). To ensure that the observed effects of PC2 inhibition on gastrin biosynthesis did not alter the secretion of G-NH2 or progastrin from G cells, we quantified these radiolabeled peptides in the chase media. As was the case for the cell extracts, PC2 antisense expression was associated with a decrease in G-NH2 labeling and an increase in progastrin labeling in chase media compared with controls (Fig. 9). The use of a control nonsense probe from a different portion of the canine PC2 sequence did not alter PC2 expression by Western blot or progastrin processing.


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Fig. 6.   Western blot of PC2 expression. Top: representative blot using antisense or control oligonucleotide probes. Bottom: blots were analyzed by densitometry, and PC2 expression was significantly reduced (48 ± 3% of control, P < 0.01, n = 6) in cells incubated with the antisense probe.



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Fig. 7.   Biosynthesis of G-NH2. After a 0.5-h incubation in methionine-free media, G cells were pulsed with [35S]Met in methionine-free media. After 2 h, cells were washed and incubated for an additional 6 h in methionine-containing media (chase). Boiling water cell extracts were then immunoprecipitated with antibody (Ab) 5135, which is specific for fully processed, amidated gastrin. A: representation of several other individual experiments with the closed and open circles depicting the use of control and PC2 antisense probes. B: means ± SE of the antisense experiments expressed as %control (number of experiments in parentheses). All time points from -1.5 to 6 were significantly different from control, P < 0.01.



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Fig. 8.   Biosynthesis of progastrin. After a 0.5 h incubation in methionine-free media, G cells were pulsed with [35S]Met in methionine-free media. After 2 h, cells were washed and incubated for an additional 6 h in methionine-containing media (chase). Boiling water cell extracts were then immunoprecipitated with Ab 8207 that is specific for progastrin. The panel at the top is representative of several other individual experiments with the closed and open circles depicting the use of control and PC2 antisense probes. B: means ± SE of the antisense experiments expressed as %control (number of experiments in parentheses). All time points from 0 to 6 were significantly different from control, P < 0.05.



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Fig. 9.   Gastrin products in media. Immunoprecipitated radiolabeled G-NH2 (Ab 5135) and progastrin (Ab 8207) products secreted into the media are expressed as %control at the chase times indicated (number of experiments in parentheses). Results are reported as %control oligo (means ± SE). All time points from 1 to 4 were significantly different from control (P < 0.01).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanism of gastrin biosynthesis has long been a focus of interest because of the physiological significance of gastrin in the regulation of gastrointestinal function. Moreover, gastrin requires processing of the inactive precursor, progastrin, for full biological activity. From the earliest biochemical studies, multiple molecular forms of gastrin were characterized both in circulating blood and in tissues (12, 26), leading to the notion that gastrins of 17 amino acids in length were derived from larger molecular forms of gastrin by proteolytic processing (3, 12). The posttranslational processing of progastrin is of particular importance because both G-NH2 and G-Gly have biological activities mediated via distinct receptors (20, 30, 38). However, the lack of a stable gastrin-producing cell line has slowed the investigation of gastrin processing mechanisms. Thus we previously characterized two important gastrin processing reactions, endoproteolysis and COOH-terminal amidation, in vitro and in cell lines heterologously expressing progastrin (8, 11, 27). In the present study, we utilized isolated enriched G cells in primary culture that permits a high degree of peptide-specific radiolabeling under reproducible and controlled conditions (36). For the first time, these studies demonstrated in G cells that G-Gly is converted to G-NH2 via an ascorbate-dependent process and firmly linked one member of the PC family, PC2, to progastrin cleavage.

During the initial phases of these studies, we encountered problems in demonstrating the incorporation of radioactivity into the G-NH2 fraction although we had no difficulty in labeling G-Gly. From previous work (9, 14, 18), we noted the dependence of the gastric PAM activity and gastrin amidation on ascorbic acid. Thus we reasoned that the absence of ascorbate in our culture system may have been the source of our difficulties. As observed in our studies, G-Gly was synthesized and accumulated before any evidence of de novo amidated gastrin synthesis in the ascorbate-deficient medium. Similar observations of diminished amidated peptide synthesis in the presence of ascorbate-deficient media have been reported in other cell culture systems (13) but not in G cells in primary culture. After completion of these studies, two sodium-dependent vitamin C transport proteins (SVCT1 and SVCT2) were characterized (25, 41). SVCT1 is expressed primarily in gut epithelial cells and is felt to be responsible for absorption of dietary ascorbate. SVCT2 is expressed primarily in neuroendocrine cells. In the present study we added 50 µg/ml (280 µM) of L-ascorbate to the media. The Km's for the ascorbate transporters, SVCT1 and SVCT2, are ~250 and 28 µM, respectively. Thus we were well within the range of either Km. Previous studies (17) have examined the dose-dependence of ascorbate on the amidation of melanocyte-stimulating hormone (MSH) in primary cultures of intermediate lobe pituitaries and noted a transporter-mediated, Na+-dependent uptake of ascorbate with a Km of 35 µM consistent with expression of SVCT2 in neuroendocrine cells. Unfortunately, the identity of the vitamin C transporter in G cells and other gut endocrine cells remains to be elucidated. Thus when the medium was supplemented with ascorbate in our studies, G-NH2 synthesis could be demonstrated clearly. The absence of changes in the pool of radioactively labeled G-Gly under the latter conditions may reflect a rapid turnover into the amidated forms of gastrin. These results suggest a precursor-product relationship between G-Gly and G-NH2 and, in addition, provide evidence for the importance of an ascorbate-dependent amidating enzyme in vivo in the posttranslational processing of gastrin.

Our studies in vitro, and now in isolated G cells, would seem to confirm the importance of ascorbate to gastrin amidation. The role of dietary ascorbic acid in peptide amidation has been examined in guinea pigs that, like humans, cannot synthesize vitamin C. As might be expected, guinea pigs with ascorbic acid deficiency manifest significantly higher serum and antral G-Gly-to-G-NH2 ratios than control animals (18). Consistent with the notion that prohormones processing mechanisms are shared with other neuroendocrine cell-types was the finding that MSH amidation was also impaired in the same ascorbate-deficient guinea pigs (15). However, it was unknown whether the failure to amidate is a direct effect of ascorbate deficiency because these animals showed other signs of scurvy, such as growth failure (18). Our studies would suggest that amidation is directly dependent on ascorbate.

It is tempting to speculate on the effects of vitamin C deficiency on progastrin processing because G-Gly has trophic actions mediated by receptors distinct from the gastrin/CCKB receptor that mediates the biological actions of G-NH2 (30). There is some support for the notion that altered levels of gastrin amidation may effect gut function. Because PAM requires copper as well as ascorbic acid, we inhibited PAM activity and gastrin amidation by copper chelation (10). As expected, copper chelation of rats for 3 days decreased levels of antral G-NH2 and increased levels of G-Gly. Serum levels of G-NH2 were unchanged, but G-Gly levels in serum were increased. Additionally, copper chelation also increased basal and G-NH2-stimulated gastric acid output. Significantly increased acid secretion, despite normal circulating concentrations of G-NH2,suggested that gastric acid secretory mechanisms were upregulated by altered peptide amidation and chronic increases in plasma G-Gly. It is currently unknown whether relative deficiencies in vitamin C in the human diet might alter the ratio of G-Gly to G-NH2 and gut function.

Consistent with the findings of others (3, 39, 40), we also noted in our pulse-chase experiments that gastrin precursors were susceptible to processing endopeptidases. In the absence of ascorbate, the larger molecular forms of G-Gly appeared to be converted to G17-Gly over time, indicating sequential cleavage at amino-terminal dibasic residues. Under these circumstances, most of the G-NH2 that was synthesized was G17-NH2 and probably resulted from amidation of G17-Gly. On the other hand, in the presence of ascorbic acid, when a more rapid conversion of G-Gly to G-NH2 was made, rapid conversion of G-Gly to G-NH2 was made possible, and synthesis of larger molecular forms of G-NH2 was demonstrated. Subsequent conversion of these forms to G17-NH2 confirms the conventionally hypothesized mechanisms of gastrin biosynthesis involving the proteolytic cleavage of larger molecular forms of gastrin to smaller ones.

To examine the proteolytic cleavage of progastrin, we (11) and others (23, 28) demonstrated that several members of the PC family were expressed in gastric antral tissues. Macro et al. (23) noted that PC expression in the stomach was physiologically regulated, suggesting a role for PCs in progastrin processing. In complementary studies (6, 8, 11, 27), we noted that PC2 and PC1/PC3 processed progastrin at the COOH-terminal Arg94Arg95 residues to produce G-NH2 in endocrine cell lines. However, PC2, but not PC1/PC3, is expressed in canine G cells (11). To determine whether PC2 or some other unknown protease was responsible for progastrin processing in gastric G cells, we sought to inhibit PC2 expression and observe the resultant effects on processing. Our previous work (11) suggested that PC2, but not PC1/PC3, was capable of cleaving progastrin at Lys74Lys75 to produce G17-NH2. Because G17-NH2 is the major molecular form of G-NH2 found in the canine antrum, we hypothesized that PC2 was a gastrin processing enzyme (4).

Although previous investigators have noted that PC2 and gastrin were coexpressed in the antrum, no study, to date, had directly linked PC2 activity to progastrin processing in antral G cells. Thus we sought to inhibit PC2 expression and observe the resultant effects on progastrin processing in canine antral G cells. These studies presented several substantial technical challenges. First, there are no currently available gastrin-producing cell lines. Second, G cells in primary culture tend to dedifferentiate after several days in culture and cease to efficiently process progastrin (21). Therefore, strategies designed to inhibit PC expression requiring several days (e.g., transfection with antisense expression vectors and subsequent selection with agents such as neomycin) would not be feasible. Third, there are no known PC2-specific inhibitors. Fourth, and perhaps most importantly, freshly isolated G cells contain substantial amounts of PC2. To overcome this challenge, it might be helpful to know the half-life of PC2 in G cells. Previous studies (31) examining the biosynthesis of PC2 have demonstrated that PC2 is secreted from cells along with the peptide products as its primary mode of degradation. Thus one would expect cells with high rates of basal secretion to have short PC2 half-lives. Canine antral G cells generally do not secrete >2-5% of their total intracellular peptide product with bombesin stimulation (16, 36). However, we and others (36), noted an increase in the basal secretion with isolation that returns to lower values with short-term culture (2-4 days).

In summary, it would be quite difficult for us to estimate the half-life of PC2 in freshly isolated G cells. However, given the fact that very little (~1%/h) of the total gastrin content is secreted, we would expect substantial residual PC2 to be found in our cells at the start of the gastrin biosynthetic experiments. Thus we feel that much of the PC2 found on Western blotting after incubation with antisense oligos is PC2 synthesized before incubation. This is consistent with our finding of much lower levels of PC2 mRNA (<15% of control, data not shown) versus PC2 protein detected by Western blotting (<50% of control). A further confounding variable is that although one might expect that older peptide products would be secreted before newly synthesized products, in some gut endocrine cell systems (canine antral D cells) the newly synthesized peptide is preferentially secreted (5). Thus prolonged treatment of cells might be necessary to completely eliminate PC2. Thus inhibition of PC2 synthesis must be highly efficient to result in substantive decreases in PC2 protein at the time of the biosynthetic studies. Finally, any method of PC2 inhibition must not interfere with the cellular machinery necessary for progastrin processing.

Given these constraints, we utilized PC2-specific antisense phosphorothioate oligonucleotide probes. We chose this method because these probes are a well-characterized and generally accepted method for gene functionalization and target validation (1). To complete these studies, we sequenced a portion of the canine reverse-transcribed PC2 mRNA sequence. Not surprisingly, canine PC2 is highly homologous to other mammalian PC2 sequences, such as human (91%) (32). More importantly, the chosen PC2 sequence for antisense oligonucleotide probes had little homology to other PC family members or other known proteins. This suggests that any observed changes in progastrin processing were due to changes in PC2 expression and not due to unintentional inhibition of other PC family members. To eliminate the possibility that the addition of probes nonspecifically inhibited PC2 expression, we utilized a second (control) probe containing the same base pairs found in the antisense probe but in random order. We also utilized another control probe containing base pairs found in a different region of the PC2 sequence but in random order. As depicted in Fig. 6, a specific PC2 antisense probe significantly inhibited PC2 expression in canine antral G cells in primary culture. Although we would have preferred that our probes completely eliminate PC2 expression, this is generally not achievable in primary cell lines (1), especially given other constraints listed above.

PC2 antisense probes also inhibited formation of G-NH2 and lead to an accumulation of progastrin in cell extracts and media. These data for the first time confirm that PC2 cleaves the COOH-terminal Arg94Arg95 residues in progastrin within antral G cells. However, because we did not achieve complete inhibition of PC2 expression or progastrin processing, we cannot rule out the possibility that other PC family members also cleave progastrin in G cells. Nevertheless, we suspect that the low levels of observed progastrin processing are due to residual PC2 activity. On the basis of the present studies, we propose that PAM and PC2 process progastrin in G-cells. Moreover, the conversion of G-Gly to G-NH2 via the action of PAM is ascorbate dependent.


    FOOTNOTES

Address for reprint requests and other correspondence: C. J. Dickinson, Pediatric Gastroenterology, 1150 W. Medical Center Drive, A520 MSRB I, Ann Arbor, MI 48109-0656 (E-mail: chrisjd{at}umich.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 16, 2002;10.1152/ajpgi.00167.2001

Received 30 April 2001; accepted in final form 4 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 282(5):G766-G775
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