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Endocrinology Vol. 138, No. 3 955-962
Copyright © 1997 by The Endocrine Society

Articles

Short Term Inhibitory Effect of Somatostatin on Gastric Histamine Synthesis1

Frédéric Hollande, Armelle Choquet, Jean Pierre Bali and Richard Magous

Laboratoire de Biochimie des Membranes, Faculté de Pharmacie, Montpellier, France

Address all correspondence and requests for reprints to: Dr. Richard Magous, Laboratoire de Biochimie des Membranes, Faculté de Pharmacie, 15 avenue Charles Flahault, 34060 Montpellier Cedex, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study we investigated the short term effect of somatostatin on histamine synthesis in a cell population isolated from rabbit gastric mucosa and enriched in enterochromaffin-like cells. Somatostatin inhibited basal and gastrin-stimulated histamine synthesis through a dual mechanism involving a decrease in the affinity of histidine decarboxylase (HDC) for its substrate (L-histidine) and a reduction in the number of functional HDC molecules.

H-89 (an inhibitor of cAMP-dependent protein kinase) mimicked somatostatin-induced reduction of HDC affinity, which, on the contrary, was selectively reversed by pertussis toxin (PTX). Furthermore, forskolin was shown to reverse the inhibitory effect of H-89 and to prevent the somatostatin-induced reduction in HDC affinity for L-histidine. Thus, the somatostatin-induced reduction in affinity seems to involve a PTX-sensitive G protein and an inhibition of the cAMP-dependent pathway.

On the other hand, the somatostatin-induced decrease in the number of functional HDC molecules seems to be PTX insensitive and independent from a modulation of the cAMP pathway, and does not seem to involve a significant change in HDC messenger RNA expression or a regulation of protein kinase C. The exact nature of this second mechanism will need further studies to be elucidated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOMATOSTATIN secreted by fundic and antral D cells is an important physiological modulator of various cellular activities in the gastric mucosa. It has been shown to participate in the paracrine control of acid secretion (1, 2), through a direct action on the H+-secreting parietal cell (3, 4) as well as through an interaction with various endocrine cell types, such as the gastrin-producing G cell in the antrum (5) and the histamine-producing enterochromaffin-like (ECL) cell in the fundus (6). Its effect on parietal cells has been directly demonstrated on purified cell populations (3, 4), where somatostatin inhibited both histamine- and gastrin-stimulated acid secretion. On the other hand, the great difficulty in purifying ECL cells has made it hard to show whether somatostatin directly affected histamine synthesis and release. Most studies to date have been performed in vivo (1) or on isolated perfused whole stomachs (7). These studies indicate that somatostatin inhibits gastrin-stimulated, but not spontaneous, histamine release (7). Similar results were recently obtained by our group (8) on isolated rabbit fundic mucosal cells and by Chuang et al. (6) on a population of canine gastric mucosal cells in primary culture containing 3% histamine-storing endocrine (ECL) cells. Finally, recent results obtained by Prinz et al. (9) established the presence of a somatostatin type 2 (SSTR2) receptor on purified ECL cells, which was involved in the somatostatin-induced inhibition of gastrin-stimulated histamine release.

Although it is clear that somatostatin can inhibit the release of histamine stored in secretory granules within the ECL cells, much less is known concerning the effect of this peptide on the activity of L-histidine decarboxylase (HDC), the enzyme responsible for the synthesis of histamine in ECL cells.

Histamine is produced by decarboxylation of L-histidine by means of HDC (EC 4.1.1.22), which is found in all tissues secreting histamine, including brain (10) and gastric mucosa (11). The presence of HDC has been demonstrated in the gastric mucosa of various species, including dog (12), rat (13), and man (14). Although HDC appears to be present in mucosal mast-like cells, the highest levels of activity are generally found in ECL cells (15). Gastric HDC activity is positively regulated by neurohormonal mediators such as gastrin and muscarinic agonists (15, 16), and recent results have shown that the release (17) and synthesis (18) of histamine from the rabbit stomach could be negatively regulated by histamine itself through the activation of an H3 receptor (19).

The intracellular pathways activated by somatostatin are obviously dependent on the type of receptor involved. The type 2 receptor (SSTR2), which has been identified on ECL cells, seems coupled to an inhibition of gastrin-stimulated histamine secretion through a blockade of the gastrin-induced calcium signal (9). In other tissues, such as brain, activation of this receptor was shown to be blocked by pertussis toxin (20), and coimmunoprecipitation experiments have shown a selective association of the SSTR2 receptor with Go and Gi proteins (20). These G proteins can be coupled to the regulation of calcium channels, but can also inhibit the production of cAMP by adenylate cyclase. Interestingly, somatostatin inhibits gastric parietal cell activity through a mechanism involving inhibition of cAMP production and at least partly dependent on a pertussis toxin-sensitive inhibitory guanine nucleotide-binding protein (4). A modulation of this intracellular pathway could also be involved in the somatostatin-induced inhibition of HDC. Studies on enriched protein fractions have shown that HDC activity could be regulated by the cAMP-dependent protein kinase (PKA) (21), which is known to be regulated by somatostatin in numerous tissues (4, 22). Furthermore, the HDC sequence possesses two sites for phosphorylation by PKA (13). The regulation of histidine decarboxylase could be tissue specific, as brain and lung enzymes were shown to be inhibited after phosphorylation by cAMP-dependent protein kinase, whereas gastric HDC displayed, on the contrary, a slight increase in activity under the same phosphorylating conditions (21, 23).

ECL cell histamine is rapidly released from secretory granules after stimulation by various neurohormonal factors, such as gastrin (8, 9) and acetylcholine (8). Several studies have shown that a longer exposure to these agents induced an increase in the activity of HDC, leading to a reconstitution of the histamine reserve. However, little is known about the short term regulation of HDC activity, which represents an important physiological issue because it determines the amount of histamine rapidly available for further mobilization by hormones or neurotransmitters. We recently reported that both gastrin and muscarinic agonists were able to induce a rapid stimulation of histamine synthesis (24). In the present study, we examined whether somatostatin could exert a rapid inhibition of HDC activity in a population of cells isolated from rabbit fundic mucosa and enriched in ECL cells. Furthermore, we studied the effect of somatostatin on the kinetic parameters of HDC in these cells, and we tried to assess which intracellular mechanism is involved in this inhibition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Unsulfated gastrin analog [NLe15]-HG-17, was a gift from Prof. L. Moroder (Max Planck Institut für Biochemie, Martinsried, Germany). Somatostatin-(1–14), pertussis toxin, staurosporin, forskolin, carbamoylcholine (carbachol), bicinchoninic acid protein assay kit, RNA mol wt markers, phenylmethylsulfonylfluoride, leupeptin, aprotinin, and agarose were purchased from Sigma Chemical Co. (St. Louis, MO). H-89, dihydrochloride [N-(2-(p-bromocinnamyl-amino)ethyl)-5-iso-quinolinesulfonamide, HCl] was provided by Calbiochem (La Jolla, CA). The Quickprep messenger RNA (mRNA) purification kit and protein A-Sepharose CL-4B were obtained from Pharmacia LKB (France). Guanidium isothiocyanate, phenol, and chloroform for isolation of total RNAs were purchased from Bioprobe (Montreuil-sous-bois, France). [2,5-3H]Histamine dihydrochloride, L-[2,5-3H]histidine, Hybond N nitrocellulose membranes, and Megaprime DNA labeling system were purchased from Amersham (Les Ulis, France). Histamine dihydrochloride, L-histidine monohydrochloride monohydrate, pyridoxal 5'-phosphate monohydrate, formaldehyde, formamide, and BSA were obtained from Fluka (Mulhouse, France). Prestained high mol wt protein markers were purchased from Bio-Rad (Hercules, CA). Standard medium in HDC activity experiments consisted of Earle’s Balanced Salt Solution without bicarbonate, containing 0.2% BSA and 10 mM HEPES (pH 7.4). Earle’s Balanced Salt Solution was obtained from Biomerieux (France). Antihistamine antibody was purchased from Milab (Malmo, Sweden). Antibodies against human gastric M1 mucin epitopes were kindly provided by Dr. J. Bara, U-55 INSERM (Paris, France) (25).

Antisomatostatin antibody was kindly provided by Dr. D. Dussossoy (Sanofi Recherche, Montpellier, France). Polyclonal antiserum against rat HDC was a generous gift from Dr. Hiroshi Wada, Osaka University Faculty of Medicine (Osaka, Japan). Donkey antirabbit IgG (Fc) was purchased from Jackson ImmunoResearch (West Grove, PA). Plasmid pCMV5 containing a rat HDC complementary DNA (cDNA) insert of 2.3 kilobases in the EcoRI site (13) was provided by Dr. D. R. Joseph, Laboratories for Reproductive Biology, University of North Carolina (Chapell Hill, NC).

Preparation of isolated rabbit gastric nonparietal cells
Cells were isolated from the fundus of 3-month-old male New Zealand rabbits killed by cervical dislocation. Cell isolation was carried out following the collagenase/EDTA procedure as previously described (26). Cell separation was performed by counterflow centrifugation with a Beckman Elutriator rotor JE6-B (Palo Alto, CA). Three fractions were collected at a rotor speed of 2100 rpm by increasing the flow rate from 17 to 22, 44, and 68 ml/min. The first fraction, named F1 cells, mainly contained mucus and endocrine cells and was devoid of parietal and chief cells. Cell viability (trypan blue exclusion) was always greater than 90%.

Immunocytochemistry
Slides were prepared by cytocentrifugation from F1 cells. For immunocytochemical studies, cells were fixed in methanol for 3 min at -20 C and incubated for 45 min at room temperature with antihistamine or antisomatostatin polyclonal antibodies, or with antisera raised against peptide M1 of human gastric mucin (25) (1:100 diluted). Immunostained cells were localized with isotypic anti-IgG Igs coupled to fluorescein isothiocyanate. For mast cells visualization, cells were fixed in isopropanol for 5 min at room temperature and stained with toluidine blue; mast cells were quantified as a percentage of the total number of cells.

HDC activity assay
Enriched cells (0.4 ml; 1.5 x 106 cells/ml in Earle’s medium) were incubated for 30 min at 37 C with or without the agents to be tested (final incubation volume, 0.5 ml). The incubation was stopped, and cells were lysed by several steps of freeze-thawing. Lysates were then homogenized and tested in triplicate for HDC activity. HDC activity was determined as previously described (16, 24).

Determination of Km and Vmax of HDC
The kinetic properties of the enzyme were determined under the same conditions, but using increasing concentrations of unlabeled histidine with a constant level of [3H]histidine. Lineweaver-Burk plots have been drawn from the data and used for determinations of both the Michaelis constant (Km) and the maximum velocity (Vmax) of the enzyme.

Incorporation of {alpha}-[ring-4-3H]fluoromethylhistidine into the monomers of HDC
For this determination, we followed the protocol of Taguchi et al. (27), with minor modifications. Briefly, cells (107 cells/sample, 1.5 x 106/ml) were preincubated alone or with pertussis toxin (200 ng/ml) or H-89 (20 µM) in medium A without BSA during 4 h at 37 C under 95% O2-5% CO2 and constant shaking. They were thereafter incubated, under the same conditions, for 30 min in the presence or absence of somatostatin (1 µM) and/or forskolin (10 µM). After a brief centrifugation (500 x g, 2 min), cell pellets were resuspended in 50 µl of a mixture of lysis buffer and a buffer containing 0.1 M potassium phosphate, 0.1 mM NaEDTA, and 0.02 mM dithiothreitol (pH 6.5; 1:1). Cells were then lysed by three consecutive steps of freeze-thawing. Homogenates were spun for 5 min at 300,000 x g in a Beckman Airfuge, and 5 µl of each supernatant were diluted and assayed for protein content. An equivalent amount of proteins from each supernatant (typically 50 µg) was then incubated with 0.4 mM {alpha}-[ring-4-3H]fluoromethylhistidine (0.532 mCi/mg) with or without unlabeled {alpha}-fluoromethylhistidine (10 mM) for 45 min at 20 C under constant shaking. Samples were subjected to sodium borohydride reduction (3 mg/ml NaBH4), denatured, and run on 10% SDS-PAGE along with mol wt markers. After fixation in 10% methanol-10% acetic acid (vol/vol), the gels were cut into 2-mm slices, and 1 ml Soluene 350 was added to each slice. The mixtures were heated to 60 C for 30 min, and then 0.5 ml glacial acetic acid was added. Five milliliters of ACS-II were added to this suspension, and the radioactivity of samples was counted in a liquid scintillation counter.

Identification of HDC mRNA
Cells (107 cells/sample; 1.5 x 106/ml) were incubated for 30 min with or without 1 µM somatostatin. Total RNAs were then prepared by the guanidium isothiocyanate-phenol-chloroform method as described by Chomczynski and Sacchi (28). Total (50 µg) RNA was denatured, separated on 1.2% agarose-formaldehyde gels, hybridized overnight with a [{alpha}-32P]deoxy-CTP-labeled HDC cDNA probe, and exposed to Kodak X-Omat films (Eastman Kodak, Rochester, NY) as described previously (29).

Quantification and analysis of data
Autoradiograms from mRNA localization were scanned with a Hoefer GS-300 scanning densitometer (Hoefer Instruments, San Francisco, CA). The density of the single band identified as HDC mRNA was measured on each lane and expressed as a percentage of a control sample (100%) run on the same gels. The molecular masses of the bands were determined using RNA molecular mass standards run on the same gels.

Statistics
Values are the mean ± SEM. Data were analyzed for statistical significance with unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunofluorescence studies were performed on F1 cells using antibodies raised against human M1 gastric mucin, somatostatin, or histamine; 68.5 ± 3.8% of cells proved immunopositive with antibodies raised against M1 peptides of human gastric mucin, and less than 2% were immunostained with antisomatostatin antibody. Moreover, about 20% of the total cell number were stained with antihistamine antibody, whereas less than 0.5% of cells were positive with toluidine blue, a marker of mast-like cells. Nonspecific staining was very low in all experiments.

In our cell preparation enriched in mucous and endocrine cells, HDC was found to decarboxylate histidine with an apparent Km of 92.42 ± 4.32 µM. Under the conditions described in Materials and Methods, basal HDC activity measured in unstimulated cells was 0.240 ± 0.017 pmol histamine formed/mg protein·h (n = 12) (16). The formation of [3H]histamine was dose dependently inhibited by {alpha}-fluoromethylhistidine, a specific inhibitor of HDC (30), and maximal inhibition (<10% residual activity) was reached with 1 mM {alpha}-fluoromethylhistidine.

Effect of somatostatin on basal HDC activity
Basal HDC activity was not modified after a 30-min preincubation period in the presence of antisomatostatin antibody (1:100), indicating that endogenous somatostatin did not influence HDC activity. However, a preliminary study performed with a single dose of exogenous somatostatin (1 µM) indicated that this peptide was able to inhibit significantly basal HDC activity, and this inhibition was reversed in the presence of antisomatostatin antibody (1:100; not shown). Furthermore, somatostatin inhibited HDC activity in a dose-dependent manner (EC50, 7.03 ± 1.82 nM). The inhibition was already significant with 0.1 nM somatostatin, reached a maximum for 1 µM somatostatin (26.8 ± 3.4% inhibition; n = 8; P < 0.01 compared to basal HDC activity), and decreased for higher somatostatin concentrations (Fig. 1AGo).



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  		Figure 1. Inhibition of HDC activity by somatostatin. Cells (1.5 x 106/ml) were incubated for 30 min with the indicated concentration of somatostatin alone (A) or in the presence of 5 nM gastrin (B), and HDC activity was then determined as described in Materials and Methods. Values are expressed as a percentage of inhibition compared to the basal level (A) or as a percentage of the activity measured in the presence of 5 nM gastrin (B). Data are the mean ± SEM from eight (A) or five (B) separate experiments.

 
Effect of somatostatin on gastrin-stimulated HDC activity
Gastrin-stimulated HDC activity was inhibited dose dependently by somatostatin (Fig. 1BGo), with an IC50 (2.3 ± 0.3 nM; n = 5) comparable to that found for the inhibition of nonstimulated activity (Fig. 1AGo). Furthermore, the 34% increase in HDC activity induced by 5 nM gastrin (16) was almost completely abolished by 1 µM somatostatin.

Modification by somatostatin of HDC kinetic parameters
Somatostatin significantly affected the enzymatic behavior of HDC by changing both the Michaelis constant (Km) and the Vmax, as determined using double reciprocal Lineweaver-Burk analysis. After a 30-min incubation of cells with 1 µM somatostatin, the affinity of HDC for its substrate, L-histidine, was reduced by 40% (the Km was increased from 92.4 ± 4.3 µM in the absence of somatostatin to 129.1 ± 4.2 µM in its presence; P < 0.05), and the Vmax decreased by about 20% (from 0.240 ± 0.017 to 0.193 ± 0.008 pmol histamine formed/mg protein·h; P < 0.05; Fig. 2Go).



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  		Figure 2. Lineweaver-Burk plot of basal and somatostatin-inhibited HDC activity. Cells (1.5 x 106/ml) were incubated with or without 1 µM somatostatin, and HDC activity was determined using increasing concentrations of L-histidine, as described in Materials and Methods. Values are expressed as 1/V = f(1/S), where V is the enzymatic activity, and S is the L-histidine concentration. Data are from 1 of 5 (somatostatin) or 12 (basal) similar experiments.

 
Effect of pertussis toxin on the somatostatin-induced inhibition of HDC activity
Somatostatin has been shown in several other models to induce a pertussis toxin-sensitive inhibition of cAMP production (4, 22). Therefore, we first analyzed the effect of pertussis toxin on the somatostatin-induced inhibition of HDC activity. Preincubation of cells for 4 h with pertussis toxin (200 ng/ml) did not significantly change basal HDC activity (2.33 ± 1.07% inhibition), but resulted in a 50% reduction of the somatostatin effect (14.5 ± 0.4% maximal inhibition, instead of 26.8 ± 3.4% with somatostatin alone; P < 0.05; Fig. 3Go), and decreased its potency (EC50, 28.80 ± 0.95 nM with pertussis toxin vs. 7.03 ± 1.82 nM for somatostatin alone; P < 0.05). Preincubating cells with pertussis toxin for longer periods of time (up to 8 h) did not increase its effect on somatostatin action.



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  		Figure 3. Effect of pertussis toxin (PTX) on somatostatin-inhibited HDC activity. Cells (1.5 x 106/ml) were preincubated for 4 h at 37 C with or without pertussis toxin (200 ng/ml) and then incubated for 30 min with the indicated concentration of somatostatin. HDC activity was determined as described in Materials and Methods. Values are expressed as a percentage of inhibition compared to the basal level (0.240 ± 0.017 pmol histamine formed/mg protein·h-1). Data are the mean ± SEM from five to eight separate experiments.

 
Involvement of cAMP- or Ca2+/phospholipid-dependent protein kinases in the somatostatin-induced inhibition of HDC activity
To further explore the potential mechanism of somatostatin inhibition, the effects of inhibitors of cAMP-dependent protein kinase and of Ca2+/phospholipid-dependent protein kinase (PKC) were studied, alone or in combination with somatostatin. Interestingly, staurosporin (0.1 µM), a potent PKC inhibitor, was without effect on basal HDC activity (Table 1Go), whereas H-89, a selective inhibitor of cAMP-dependent protein kinase, inhibited basal HDC activity in a dose-dependent manner down to 15.1 ± 3.0% of its basal value for a concentration of 20 µM (Fig. 4Go); higher concentrations (200 µM) of H-89 did not significantly increase this inhibition. Moreover, it was interesting to note that the inhibitory effect of H-89 (20 µM) was not additive to that of somatostatin; when both compounds were used together, the inhibition reached a value similar to that caused by somatostatin alone (Table 1Go). Under the same experimental conditions, staurosporin was unable to modify somatostatin-induced inhibition of HDC activity (Table 1Go).


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  		Table 1. Involvement of cAMP- or Ca2+/phospholipid-dependent protein kinases in the somatostatin-induced inhibition of histidine decarboxylase activity

 


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  		Figure 4. Effect of H-89 on basal HDC activity. Cells (1.5 x 106/ml) were preincubated for 4 h with the indicated concentrations of H-89 before measurement of HDC activity. Values are expressed as a percentage of inhibition compared to the basal level (0.240 ± 0.017 pmol histamine formed/mg protein·h-1). Data are the mean ± SEM from four separate experiments.

 
We then studied the effect of an increase in the intracellular cAMP level on basal or somatostatin-inhibited HDC activity. (Bu)2cAMP (at concentrations up to 1 mM) and forskolin, an activator of adenylyl cyclase, did not affect basal HDC activity. However, forskolin partially reduced maximal somatostatin inhibition of HDC activity. The maximal reversion was obtained with 10 µM forskolin, which approximately reduced the somatostatin inhibitory effect by 50% (Table 1Go). On the other hand, the inhibitory effect of 20 µM H-89 was completely reversed by 10 µM forskolin (Table 1Go).

Involvement of a pertussis toxin-sensitive G protein and a cAMP-dependent intracellular pathway in the somatostatin-induced modifications of HDC kinetic parameters
Our first results demonstrated 1) that somatostatin induced a inhibition of HDC activity through an action on both the number of functional enzyme molecules and on the affinity of these molecules for their ligand; and 2) that an intracellular mechanism involving a pertussis toxin-sensitive G protein and a down-regulation of the cAMP-dependent pathway seemed involved in this inhibition. We then investigated whether this latter intracellular mechanism was selectively responsible for the modification of one of the HDC kinetic parameters affected by somatostatin. For this purpose, we studied the effects of H-89 (20 µM) and forskolin (10 µM), alone or in combination with 1 µM somatostatin, and of pertussis toxin (200 ng/ml) on the kinetic parameters of HDC (Table 2Go). It was interesting to note that H-89 alone increased the Km of HDC to a value similar to that found with somatostatin. When both compounds were used together, the Km and Vmax were similar to those found with somatostatin alone. Furthermore, forskolin alone did not change the kinetic parameters of the enzyme, but prevented the somatostatin-induced decrease in the affinity of HDC for its substrate, without affecting its Vmax (Table 2Go). Finally, preincubation of cells with pertussis toxin had no effect on basal HDC activity, but reversed the somatostatin-induced increase in the Km of HDC for L-histidine, without significantly changing the effect of this peptide on the Vmax (Table 2Go).


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  		Table 2. Effects of somatostatin, pertussis toxin, and H 89, alone or in combination, on the kinetic parameters of histidine decarboxylase

 
Effect of somatostatin on the incorporation of {alpha}-[ring-4-3H]fluoromethylhistidine into HDC monomers
When cytoplasmic proteins (prepared from the ECL cell-enriched cell population) were incubated with {alpha}-[ring-4-3H]fluoromethylhistidine and subjected to SDS-PAGE, a single peak of radioactivity was localized in a slice corresponding to a Mr of 50–56 kDa (Table 3Go), similar to that of the HDC monomers. This peak was abolished when samples were incubated with an excess of unlabeled {alpha}-fluoromethylhistidine. Moreover, the intensity of the peak varied depending on the treatment. Indeed, the peak was reduced by more than 30% when cells were incubated with 1 µM somatostatin (Table 3Go). This reduction was left unaffected by treatment of cells with pertussis toxin or forskolin, indicating that neither a Gi-like protein nor the intracellular cAMP level was involved in this somatostatin-induced effect. Finally, H-89 (PKA inhibitor) was unable to affect the intensity of the peak compared to that in untreated cells (Table 3Go).


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  		Table 3. Effects of somatostatin, pertussis toxin, forskolin, and H 89 on the incorporation of [ring-4-3H]{alpha}-fluoromethylhistidine into HDC monomers

 
Effect of somatostatin on HDC mRNA expression
As we showed that somatostatin, on a short time scale, was able to decrease the number of active HDC molecules (as demonstrated by the decrease in the Vmax of HDC and in the stoichiometrical binding of {alpha}-[ring-4-3H]fluoromethylhistidine), we examined whether this effect resulted from a down-regulation of the expression of HDC mRNA. However, a densitometric analysis of the single 2.7-kilobase band representing HDC mRNA did not detect any significant difference in HDC mRNA expression in somatostatin-treated cells (1 µM; 30 min) and untreated cells (not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Somatostatin is a well known inhibitor of numerous endocrine and exocrine secretions. It has been identified as a paracrine regulator of gastric acid secretion, through a direct action on parietal cells, a regulation of gastrin secretion from G cells, as well as an inhibition of histamine secretion from ECL cells. However, little is known about the role of this peptide on HDC, the enzyme responsible for histamine synthesis and thus for reconstitution of the histamine pool of ECL cells. Gastric HDC appears highly regulated, mainly by gastrin, cholinergic agonists (15, 16), or histamine itself (18). However, little is known about the role of somatostatin in the regulation of gastric histamine synthesis. Although somatostatin is generally considered a modulator of stimulated activity in various cellular models (3, 4, 22, 31), the present study demonstrates a direct inhibitory effect of this peptide on the enzyme responsible for histamine synthesis in unstimulated rabbit ECL cells. This effect is unlikely to be due to an effect of somatostatin on another cell type, secreting a paracrine factor capable of modulating ECL cell HDC activity; indeed, besides ECL cells, mucous cells represent the only cell type occurring in high number in our cell population, and their exocrine secretions do not influence ECL cell activity. Furthermore, the absence of any effect on basal HDC activity of an antibody directed against somatostatin argues against any involvement of endogenous somatostatin in these experiments. The only mediators that might be secreted by some of the contaminating cells and could act on HDC activity would be PGs. Yet, the maximal concentration of PGs reached in the incubation medium (2 pM) was clearly below the dissociation constants described for PG receptors in gastric mucosa (32), which precludes their participation in somatostatin-induced inhibition of histamine synthesis in ECL cells. Finally, this result is in accordance with the recent identification of somatostatin receptors on rat ECL cells (9).

We concentrated in this study on the rapid effect of somatostatin on HDC activity. Such a short term regulation of the enzyme is essential to quickly control the amount of histamine available for mobilization by neurohormonal regulators and thus to quickly switch on or off a major stimulatory pathway for acid secretion. We have recently shown that HDC activity was rapidly stimulated by gastrin through the activation of a gastrin/cholecystokinin-B receptor (16, 24). We now report the presence of a rapid inhibitory regulation of the same enzymatic activity by somatostatin. It interesting to note that both the potency and efficiency of somatostatin inhibition were unaffected in the presence of gastrin, which may indicate that these two peptides affect two completely different intracellular pathways.

Interestingly, somatostatin was found to decrease both the affinity of HDC for its substrate (as shown by an increase in the Km) and the number of functional enzyme molecules. A short term regulation of the affinity of HDC for L-histidine has previously been postulated and may involve the cAMP-dependent intracellular pathway (21). Furthermore, several reports have already shown that the number of HDC molecules could be enhanced by gastrin, muscarinic agonists (16, 33), glucocorticoids, and activators of protein kinase C (34), whereas a long term incubation of mastocytoma P-815 cells with activators of cAMP-dependent protein kinase also enhanced de novo synthesis of the enzyme (35). Thus, the effect of somatostatin on both parameters in our model could reflect the coupling of somatostatin receptors to several transducing pathways in gastric ECL cells.

The intracellular couplings of somatostatin receptors are multiple and appear to be tissue specific; they are coupled to different guanine nucleotide-binding proteins (22, 36, 38), and some of the intracellular effects of somatostatin are prevented by treatment with pertussis toxin (4, 22, 37), whereas others are not (31). Our observation that somatostatin inhibition of basal HDC activity is only partly reversed by pertussis toxin suggests that somatostatin receptors on ECL cells could be coupled to two different guanine nucleotide-binding proteins (G proteins), as previously shown for the somatostatin receptor SSTR2 in Chinese hamster ovary cells (38). Moreover, the fact that pertussis toxin reverses the effect of somatostatin on the Km of HDC while its effect on the Vmax is unchanged is incompatible with the involvement of a single type of guanine nucleotide-binding protein in the somatostatin-induced inhibition of HDC activity. Interestingly, a dual mechanism has been postulated to explain the inhibitory effect of somatostatin on acid secretion from gastric parietal cells (4).

In various models, somatostatin has been shown to induce a dose-related inhibition of cAMP production (4, 31), generally through the activation of pertussis toxin-sensitive guanine nucleotide-binding proteins (37). In our cell population, the partial reversal by forskolin of the somatostatin-induced inhibition of HDC activity as well as the lack of additivity between the inhibitory effects of somatostatin and H-89 indicate that an inhibition of the cAMP-dependent pathway is partly responsible for somatostatin action. The somatostatin-induced inhibition of the cAMP-dependent pathway appears to be sensitive to pertussis toxin and to mediate specifically a decrease in the affinity of HDC for L-histidine. Furthermore, as this somatostatin-induced decrease in affinity is mimicked by an inhibitor of cAMP-dependent protein kinase (H-89), it seems reasonable to postulate that this effect of somatostatin could be due to a decrease in the degree of phosphorylation of HDC on the sites sensitive to this kinase, previously identified by Joseph et al. (13).

On the other hand, further experiments corroborated the hypothesis that the cAMP-dependent pathway was not involved in the somatostatin-induced decrease in the maximal velocity of HDC. In these experiments, we estimated the amount of functional HDC molecules in ECL cells using tritiated {alpha}-fluoromethylhistidine, which is known to bind covalently and stoichiometrically to the active site of each HDC subunit (39). Our results showed that somatostatin induced, after a short incubation time, a reduction in the number of functional HDC molecules, whereas the effectors at the cAMP-dependent pathway were unable to affect this number. These results emphasize the existence of a dual intracellular pathway mediating the effects of somatostatin on the affinity and number of HDC molecules in ECL cells.

Interestingly, all of the {alpha}-fluoromethylhistidine-associated radioactivity was found in a single peak of 50–56 kDa. The absence of any higher Mr peak seems to indicate that the potential precursor of HDC (73 kDa) (13) is either unstable or unable to bind {alpha}-fluoromethylhistidine, and therefore probably L-histidine, in ECL cells.

It is difficult to conclude what is the intracellular target for the somatostatin-induced regulation of the number of functional HDC molecules. Although no major changes in HDC mRNA expression proved detectable, it still seems possible that the stability of these mRNA could be regulated by somatostatin. Alternatively, somatostatin could affect the translation of these mRNA into new HDC molecules, although the rapidity of the effect seems to preclude this hypothesis. Finally, somatostatin could also regulate the posttranslational processing of HDC from the precursor stage to the 53- to 55-kDa molecule, which seems important for activation of the enzyme.

The present discovery of a rapid effect of somatostatin on the unstimulated HDC activity in gastric endocrine cells emphasizes the major role of this paracrine factor as the main inhibitor counteracting the stimulatory actions of gastrin and vagal neurotransmitters in the overall regulation of stomach functions. Somatostatin is known to inhibit gastrin-stimulated histamine release. The originality of its effect in this model is that it also rapidly modulates the activity of the enzyme synthesizing histamine on unstimulated or stimulated ECL cells. This effect of somatostatin is also interesting, because the peptide controls two distinct parameters of the same enzyme (i.e. the maximal velocity and the Michaelis constant) through two different intracellular pathways, resulting in an overall decrease in the enzymatic activity. From our results, it seems that the effect of somatostatin on the affinity of HDC is about twice as important as that induced on the maximal velocity. In this respect, the short term regulation is different from long term effects, which very often involve mainly a modification in the number of molecules (i.e. affecting the maximal velocity), for example through changes in mRNA expression.

The decrease in the affinity of HDC for L-histidine seems to result from a Gi-like-mediated down-regulation of the cAMP-dependent pathway, which could induce a decrease in the phosphorylation level of HDC on sites sensitive to the cAMP protein kinase. On the other hand, the somatostatin-induced decrease in the number of HDC molecules seems independent from a regulation of cAMP- and Ca2+/phospholipid-dependent protein kinases and is not due, on a short time scale, to a significant down-regulation of HDC mRNA expression. Further biochemical studies will be necessary to better understand the relative importance of the two intracellular mechanisms involved in the effect of somatostatin on HDC.


   Acknowledgments
 
The authors thank Dr. M. Garbarg and Prof. J. C. Schwartz (INSERM U-109, Paris, France) for their helpful advice in the development of the histidine decarboxylase assay. They are very thankful to Dr. D. R. Joseph (Laboratories for Reproductive Biology, University of North Carolina, Chapell Hill, NC) for the generous gift of rat HDC cDNA. They are also very grateful to Dr. H. Wada (Osaka University Faculty of Medicine, Osaka, Japan) for the gift of polyclonal antirat HDC antibody, and to Dr. J. Bara (INSERM U-239, Faculté Xavier Bichat, Paris, France) for the gift of antimucin antibodies.


   Footnotes
 
1 This work was supported by grants from Centre National de la Recherche Scientifique, INSERM, and Université de Montpellier I. Back

Received July 22, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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