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Endocrinology Vol. 140, No. 1 22-28
Copyright © 1999 by The Endocrine Society

ARTICLES

Glicentin and Oxyntomodulin Modulate Both the Phosphoinositide and Cyclic Adenosine Monophosphate Signaling Pathways in Gastric Myocytes1

Rodier Geneviève, Richard Magous, Tohru Mochizuki, Dung Le Nguyen, Jean Martinez, Jean-Pierre Bali, Dominique Bataille and Claire Jarrousse

INSERM Unité 376 Endocrinologie des Peptides et Régulation Génique (R.G., D.L.N., D.B., C.J.), CHU Arnaud-de-Villeneuve, F-34295 Montpellier cedex 5, France; CNRS EP 612, Signalisation Cellulaire Normale et Tumorale (R.M., J.-P.B.), Faculté de Pharmacie, F-34060 Montpellier, France; Laboratory of Bioorganic Chemistry (T.M.), Shizuoka College of Pharmacy, Shizuoka 422, Japan; CNRS UMR 5810 Laboratoire des Amino Acides Peptides et Protéines (J.M.), Faculté de Pharmacie, F-34060 Montpellier, France

Address all correspondence and requests for reprints to: Dr. Claire Jarrousse, INSERM U376, CHU Arnaud de Villeneuve, 371 rue du doyen Giraud, F-34 295 Montpellier cedex 05, France. E-mail: jarrou{at}u376.montp.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have investigated the transduction pathways mediating the contractile effect of two glucagon-containing peptides, glicentin (GLIC) and oxyntomodulin (OXM), on smooth muscle cells isolated from rabbit antrum. Low concentrations of GLIC induced a biphasic and rapid (first phase at 5–8 sec) Ins(1,4,5)P3 production. By comparison, higher concentrations of OXM or OXM(19–37) were required to obtain biphasic time-courses of Ins(1,4,5)P3 production. In a Ca2+ free medium, the first phase of Ins(1,4,5)P3 production induced by GLIC or OXM was maintained, while the second phase disappeared. In saponin-permeabilized cells, all three peptides induced cell contraction with similar efficacies and potencies. Exogenous Ins(1,4,5)P3 mimicked the contractile effect of the peptides and heparin, which inhibits the Ins(1,4,5)P3 binding to its receptor, prevented contraction stimulated by each effector. We conclude that a Ca2+ mobilization from the intracellular stores is essential in the contractile effects of GLIC and OXM. Using the fluo-3 probe, a [Ca2+]i increase was observed in the presence of GLIC, OXM, or OXM(19–37). The three peptides reduced by 30–40% the cAMP content of cells stimulated by forskolin. This effect was pertussis toxin sensitive as demonstrated with OXM(19–37). Our data constitute important clues for the existence in smooth muscle cells of receptor(s) specific for the GLIC/OXM hormones, coupled via G protein(s) to both Ca2+ and cAMP pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLICENTIN (GLIC) and oxyntomodulin (OXM) are two hormones coreleased by ileum and colon L-cells (1, 2) during digestion (3). Both are glucagon-containing peptides elongated by a C-terminal octapeptide (4, 5), which is essential for their biological activities (6). GLIC differs from OXM by a 32-amino acid N-terminal extension.

A main role for OXM and GLIC in the regulation of gastric acid secretion was evidenced: in the conscious rat, physiological concentrations of OXM (7) or GLIC (8) reduce stimulated gastric acid secretion. The C-terminal common fragment (OXM 19–37) mimicks their effects (7, 9). This role is substantiated by the inverse correlation between their plasma concentration over the nycthemere in man and peak acid output (3). So far, no specific receptor for these peptides has been discovered on the gastric acid-secreting parietal cells (10, 11). An action through the nervous system is likely, as demonstrated on jejunal secretions (12).

Physiological concentrations of OXM delay gastric emptying of a liquid meal in human (13), and supraphysiological concentrations of GLIC decrease the amplitude of the antral contractions induced by a solid meal in dog (14). These effects may be either neurally mediated or due to a direct action on smooth muscles. On a smooth muscle cell (SMC) preparation isolated from rabbit antrum and devoid of neural elements, a direct contractile effect of these hormones was observed (15). GLIC was 15-fold more potent than OXM or its (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) fragment, suggesting the existence of a glicentin-preferring binding site. Such a binding site appears to be a G protein-coupled receptor because the contraction of permeabilized SMC induced by these peptides was prevented by GDPßS, an inhibitor of the G-protein cycle (16).

The role of calcium in the excitation-contraction coupling of smooth muscles is well established. The phospholipase C activation by muscarinic, {alpha}1-adrenergic, peptidergic, adenosine, or some opioid transmitters (17) is believed to initiate contraction by generating inositol phosphates and diacylglycerol followed by Ca2+ release from intracellular stores. In parallel, the cAMP pathway is under the negative control of the muscarinic, adenosine, or opioid transmitters (17). The aim of this study was to investigate the modifications induced by GLIC, OXM, and their C-terminal moiety OXM (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) on the calcium/phosphoinositide and the cAMP pathways in rabbit antral smooth muscle cells and their possible implication in the contractile effect of these hormones.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Collagenase A from Clostridium histolyticum, creatine phosphate, creatine phosphokinase and pronase were purchased from Boehringer Mannheim (Meylan, France). Soybean trypsin inhibitor (STI), ATP, 3-isobutyl-1-methylxanthine (IBMX), carbachol, saponin, BSA, glutaraldehyde, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and the adhesive protein extracted from the blue mussel were purchased from Sigma Chemical Co. (St. Louis, MO). Pertussis toxin (PTX) was provided by France Biochem (Meudon, France), Ins(1, 4, 5)P3 RRA kit was provided by N.E.N. (Les Ulis, France). Fluo 3/AM was provided by Molecular Probes, Inc. (Eugene, OR).

Peptides
Human glicentin was obtained by solid phase synthesis as previously described (18). [Nle27]-oxyntomodulin was synthesized according to a procedure previously published (19). Replacing methionine in position 27 by norleucine did modify neither the immunological nor the biological properties of the molecule (19). OXM (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) was synthesized in our laboratory (15). Somatostatin-14 was provided by Neosystem (Strasbourg, France).

Isolation of smooth muscle cells
Smooth muscle cells (SMC) were isolated from the muscle layer of the rabbit antrum following the method of Moummi (20). Fed New Zealand male rabbits weighed 2–3 kg. The muscle layer was cut into small pieces. The pieces were incubated for a 30–40 min period at 30 C in 10 ml of medium A (in mM, NaCl 132, KCl 5.4, MgSO4 1.2, CaCl2 1, Na2HPO4 5, NaH2PO4 1, HEPES 25, glucose 11, BSA 0.2%, phenol red 0.02%, pH 7.4) containing 0.25% collagenase (0.76 U/mg), 0.04% pronase (7 U/mg) and 0.03% soybean trypsin inhibitor and gased with 100% O2. The tissue diluted in medium A was filtered through a nylon mesh. The remaining tissue was reincubated in fresh medium A and dispersed cells were obtained by gentle stirring for 30–40 min. Cells dissociated in enzyme-free medium were centrifuged at 150 g for 5 min. The cell pellet was resuspended in medium B (in mM, NaCl 116, KCl 5.4, MgSO4 0.81, CaCl2 1.79, NaH2PO4 1, HEPES 10, glucose 5.5, pH 7.4). Medium B was without BSA for Ins(1, 4, 5)P3 measurements or supplemented with 0.5% BSA for cAMP determinations. Viability (estimated by Trypan blue exclusion) was always greater than 90%. In rabbit, the circular vs. longitudinal muscle layers represented respectively 2/3 and 1/3.

In some experiments, muscle cells were permeabilized with the nonionic detergent saponin (21). Fifty micrograms per ml saponin was added for 7–8 min in a cytosol-like medium containing 180 nM Ca2+ (0.5 mM CaCl2, 1 mM ethylene glycol-bis [(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)], 20 mM NaCl, 100 mM KCl, 5 mM MgSO4, 1 mM NaH2PO4, 10 mM HEPES, 2% BSA, pH 7.4. The cells were washed by centrifugation at 150 g and resuspended in the presence of ATP (1.5 mM), and of an ATP-regenerating system consisting of creatine phosphate (5 mM) and creatine kinase (10 U/ml).

Measurement of contraction
The contraction of isolated muscle cells was estimated by measurement of 100 cells in successive microscopic fields. Briefly 450 µl of cell suspension (250 000 cells/ml) were incubated for 30 sec at 30 C with or without (control) agents to be tested. The reaction was stopped by addition of glutaraldehyde (final concentration 2.5%). Contraction was expressed as the percent decrease in mean cell length and compared with control cells.

Ins (1, 4, 5) P3 measurement
The concentration of Ins(1, 4, 5)P3 was determined by RRA. Four hundred fifty microliters of cell suspension (106 cells/ml) were incubated at 30 C with peptides for various periods of time (0–30 sec). In this time frame, addition of lithium in the medium was useless (22). The reaction was stopped by addition of 100 µl ice-cold 60% HClO4. After centrifugation at 4 C (10,000 x g, 1 min), the supernatant was adjusted to pH 7.0 with 3 M KOH/0.15 M HEPES. After a 10-min incubation at 4 C and a 4-min centrifugation at 3000 x g, 100 µl of supernatants were incubated for 1 h at 4 C with 3H-Ins(1, 4, 5)P3 (7 nCi) and Ins(1, 4, 5)P3 binding proteins. After centrifugation (5 min at 10,000 x g, 4 C), the pellets were treated for 10 min with 50 µl 0.15 N NaOH at 20 C. After neutralization (10 µl of 1 N HCl), radioactivity of the pellet was counted.

The absence of BSA in the incubation medium may induce a peptide loss due to sticking. This loss was quantified by RIA for each peptide and an appropriate correction of the peptidic concentration was introduced.

Monitoring of intracellular calcium
Changes in intracellular calcium were monitored by use of the calcium sensitive probe fluo-3. Briefly, cells were attached on glass coverslips precoated with a bioglue and placed in a dish bottom. Cells were loaded at room temperature for 25 min with 6 µM fluo-3/AM, then washed with the medium used for the study: 116 mM NaCl, 3.7 mM KCl, 1.6 mM NaH2PO4, 4.2 mM NaHCO3, 0.36 mM MgCl2, 1 mM CaCl2, 20 mM HEPES, 5.5 mM glucose, pH 7.4. A 1.75 mM CaCl2 caused the contraction of numerous cells. The coverslip was set on the stage of an inverted epifluorescence microscope (Nikon, Tokyo, Japan). The chamber was perfused with a medium maintained at 35 C and a 2 ml/min flow rate. The field was illuminated at 485 nm with a Xenon lamp. Images were captured at 530 nm using a DM 510 dichroïc mirror, a CCD camera connected to an image intensifier and an image processor (Argus Hamamatsu) (Hamamatsu, Hamamatsu City, Japan). Images were acquired every second and divided by image number 1 which corresponds to the basal state. The calculated ratio represented the intracellular calcium variations relative to the basal level.

cAMP intracellular level
Intracellular cAMP content of isolated muscle cells was measured by RIA as follows: 450 µl of cell suspension (500 000 cells/ml) in medium B containing 0.5% BSA were preincubated for 10 min at 30 C in the presence of 0.1 mM isobutylmethylxanthine (IBMX). The agents were then incubated for 30 or 60 sec. The incubation was stopped by addition of ice-cold 60% HClO4. After centrifugation (10,000 x g, 4 C, 1 min), neutralization of the resulting supernatant (with 9 N KOH) and centrifugation (5 min at 10,000 x g), the supernatant was succinylated; cAMP was measured by RIA according to a method previously described (23). In some experiments, cells were treated with 300 ng/ml PTX for 1 h at 31 C under 95% O2/5% CO2.

Statistics
The statistical significance was established by the use of a Mann and Whitney nonparametric test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course of Ins(1, 4, 5)P3 production
GLIC, OXM, or their common fragment OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) induced maximal contraction of antral SMC 30 sec after peptide application (15). Accordingly, the time courses of Ins(1, 4, 5)P3 formation were assessed from 5 to 30 sec.

1) Effect of glicentin
The intracellular Ins(1, 4, 5)P3 content of unstimulated cells (control) was 3.0 ± 0.3 pmol/106 cells (n = 22). It has been shown that a maximal contraction of cells was obtained at a 10-10 M GLIC concentration (15). At GLIC concentrations (3.3 10-11 and 3.3 10-10 M), giving submaximal and maximal contractions, the time courses of Ins(1, 4, 5)P3 content were biphasic (Fig. 1Go). A return to baseline was observed at 30 sec. A rapid increase in Ins(1, 4, 5)P3 content occurred within 5 or 8 sec, depending on the peptide concentration. A significant (P < 0.05) decrease between 5 and 8 sec or 8 and 12 sec, depending on the peptide concentration, preceded the second phase which occurred within 20 sec.



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  		Figure 1. Time course of Ins (1 4 5 )P3 production in antral muscle cells in response to glicentin (GLIC), oxyntomodulin (OXM), OXM (19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 ) or carbachol. The means ± SEM were expressed as pmol/106 cells above control values (basal = 3 ± 0.3 pmol/106 cells). Upper panel, 0.03 nM GLIC ({square}) n = 4 and 0.3 nM GLIC ({blacksquare}) n = 4; 0.3 nM OXM ({circ}) n = 4 and 3 nM OXM (•) n = 4. Lower panel, 30 nM OXM (19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 ) ({blacktriangleup}) n = 6 and 0.1 µM carbachol ({diamondsuit}) n = 5. a, P < 0.05; b, P < 0.02. c, P < 0.01.

 
2) Effect of oxyntomodulin
It was previously observed that a maximal contraction of antral SMC was obtained at 3 10-9 M OXM, this hormone being 15-fold less potent than GLIC (15). For a 3 10-10 M OXM concentration, which produced a half maximal contraction of cells, the Ins(1, 4, 5)P3 content progressively increased (Fig. 1Go), a maximal level being obtained at 20 sec. For a concentration giving a maximal contraction, 3 10-9 M OXM, the Ins(1, 4, 5)P3 production was rapid. A maximal production was reached at 5 sec and sustained at a constant level beyond 30 sec.

3) Effect of OXM (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)
OXM (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) was as potent as OXM in inducing SMC contraction (15). OXM (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37), (3 10-8 M, concentration giving a maximal contraction), induced a rapid production of Ins(1, 4, 5)P3 within 8 sec stimulation, followed by a steady state (Fig. 1Go).

4) Effect of carbachol
Carbachol is known to induce contraction of the gastric SMC (20) through PLC activation and Ins(1, 4, 5)P3 formation (24). At 10-7 M, a dose that triggers a maximal contraction of these cells (20), carbachol induced a rapid production of Ins(1, 4, 5)P3 within 5–12 sec (Fig. 1Go). A maximal Ins(1, 4, 5)P3 increment above baseline was +5.5 ± 0.15 pmol Ins(1, 4, 5)P3/106 cells.

The Ins (1, 4, 5)P3 production in a Ca2+-free medium
A Ca2+ influx is by itself able to activate phospholipase C (25, 26). Accordingly, we determined the Ins(1, 4, 5)P3 content in cells incubated in a Ca2+-free medium containing 0.2 mM EGTA. In such a medium, the basal Ins(1, 4, 5)P3 cellular content decreased from 3.0 ± 0.3 to 1.7 ± 0.3 pmol/106 cells (n = 6). The 3 10-10 M GLIC-induced biphasic Ins(1, 4, 5)P3 production observed when cells were incubated with a Ca2+ containing medium, became monophasic in the absence of extracellular Ca2+ (Fig. 2Go). The first phase exhibited a 240% increase over baseline, in the presence or the absence of extracellular calcium. The second phase disappeared when extracellular Ca2+ was omitted (Fig. 2Go).



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  		Figure 2. Time course of Ins (1 4 5 )P3 production in antral muscle cells in a Ca2+-free medium + EGTA 0.2 mM. Left panel, 0.3 nM GLIC n = 4. Right panel, 3 nM OXM, n = 4. The means ± SEM were expressed as pmol/106 cells above control values (basal = 1.7 ± 0.3 pmol/106 cells). b, P < 0.02.

 
Similarly, in the absence of extracellular Ca2+, OXM 3 10-9 M induced a transient increase in Ins(1, 4, 5)P3 level within 8 sec. The first phase exhibited a 240% and 205% increase, in the presence or in the absence of extracellular calcium, respectively.

Thus, OXM and GLIC shared a similar profile of Ins(1, 4, 5)P3 production when extracellular calcium was omitted.

Contraction of permeabilized cells
To investigate whether the production of inositol phosphates induced by GLIC and OXM was a critical step to initiate contraction, we used the permeabilized cell model.

Ins(1, 4, 5)P3 elicited a concentration-dependent contraction consistent with its intracellular effect (27), as estimated at 30 sec (Fig. 3Go). Ins(1, 4, 5)P3-induced cell shortening was already reached at 10 sec (9.8 ± 2.1% of cell length, n = 5), and was maintained for longer times (10.9 ± 1.4% and 8.9 ± 0.8% of cell length for 20 and 30 sec, respectively). The EC50 value for Ins(1, 4, 5)P3 was about 1 nM.



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  		Figure 3. Contractile response of muscle cells permeabilized with saponin. Dose-response curve with GLIC ({square}) n = 5–7, OXM ({circ}) n = 6, OXM (19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 ) ({blacktriangleup}) n = 7 and Ins (1 4 5 )P3 (*) n = 5. Results represented the means ± SEM estimated at 30 sec.

 
GLIC, OXM, and OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) also induced a concentration-dependent contraction of permeabilized cells (Fig. 3Go). Maximal contraction was similar: 11.2 ± 1.5% decrease in cell length for GLIC, 10.9 ± 1.5% for OXM and 10.9 ± 0.8% for OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). GLIC, OXM, and OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) EC50 were about 70 pM. Heparin (10 µg/ml), which inhibits the binding of Ins(1, 4, 5)P3 to its receptor (28, 29), did not significantly modify the basal cell length (149 ± 3 µm vs. 148 ± 2 µm in control, n = 5). The maximal contraction induced by Ins(1, 4, 5)P3 was inhibited by heparin (Table 1Go). Heparin prevented by more than 80% the maximal contraction induced by the three peptides (Table 1Go).


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  		Table 1. Contractile activity of GLIC, OXM, or OXM(19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 ) in the presence of heparin or permeabilized cells

 
The intracellular calcium
A direct proof of an intracellular calcium increase in response to the peptides was provided by the use of a fluorescent probe. Figure 4Go represents the variations of intracellular calcium induced by 10-8 M GLIC, 5 10-8 M OXM, or 5 10-8 M OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). All peptides triggered a rapid increase of intracellular free calcium, similar to that induced by 10-6 M carbachol taken as a reference. In the case of OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37), the return to baseline required a longer period of time (about 5 min).



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  		Figure 4. Intracellular calcium changes in muscle cells preincubated with 6 µM fluo3/AM. The effect of GLIC 10 nM, OXM 50 nM and OXM (19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 ) 50 nM was compared with that of carbachol 1 µM. Results were reported to the fluorescence measured during the prestimulation period.

 
Intracellular cAMP level
Because the cAMP system is of importance in the control of SMC contraction, we tested the effect of GLIC and OXM on the adenylate cyclase activity.

1) Effect of GLIC, OXM, and OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) on the basal cAMP content
In the presence of 0.1 mM IBMX, the basal cAMP content of antral SMC was 47 ± 6 pmol per 106 cells (n = 18). After a 30-sec stimulation, GLIC, OXM or their C-terminal fragment had no significant effect on the intracellular cAMP content (Table 2Go).


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  		Table 2. Variations of the cAMP content in the presence of GLIC, OXM, or OXM(19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 )

 
In the absence of IBMX, a 30-sec stimulation by 10-7 M OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) did not induce any significant change in the cAMP content: 85 ± 7% of the control values (n = 5). These results were confirmed after a 2-min stimulation: the cAMP content was identical to that observed at 30 sec either in the control (107 ± 8%, n = 6) or in the presence of OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) (121 ± 10%, n = 6).

2) Effect of GLIC, OXM and OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) on forskolin stimulated cAMP production
Forskolin is known to induce a direct stimulation of the catalytic unit of adenylate cyclase (30). The cellular cAMP content was stimulated by forskolin with maximal effect at 5 10-7 M (+29 ± 5 pmol cAMP/106 cells over baseline, n = 5) and up to 10-5 M.

GLIC, OXM, and their OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) fragment counteracted significantly the stimulatory effect of 10-6 M forskolin on adenylate cyclase (Fig. 5Go). No significant inhibition was observed at 10-10 M of GLIC or OXM and a maximal inhibition was achieved at 10-9 M for both hormones, averaging 34% and 31% for GLIC and OXM. OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37), tested at 10-7 M, also induced a significant decrease in cAMP content (Fig. 5).



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  		Figure 5. Effect of GLIC (n = 11), OXM (n = 5–8), and OXM (19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 ) (n = 4) on the cAMP production induced by 1 µM forskolin for 60 sec. The medium contains 0.1 mM IBMX. The means ± SEM were expressed in % of forskolin effect. a, P < 0.05; b, P < 0.02.

 
Effect of PTX treatment
A possible mediation by G proteins coupled to the GLIC/OXM binding site(s) involved in the negative control of the peptides was investigated using PTX, which inhibits receptor activation by the G{alpha}i and G{alpha}o proteins through ADP-ribosylation. Somatostatin was shown to inhibit the intestinal SMC adenylate cyclase via a receptor coupled to G{alpha}i and G{alpha}o proteins (31).

2.10-7 M somatostatin or OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) reduced 10-6 M forskolin-induced cAMP production from 100 ± 18% to 33 ± 7% (P < 0.01, n = 5) and 47 ± 6% (P < 0.01, n = 5), respectively (Fig. 6Go). PTX-pretreated cells did not modify the forskolin activity. It abolished the inhibitory effect of somatostatin as well as that of OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) (Fig. 6Go). Thus, it is likely that a Gi/Go link between the activated binding site and adenylate cyclase mediates the action of OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37).



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  		Figure 6. Effect of PTX (300 ng/ml, 1 h) in the presence or the absence of 0.2 µM OXM (19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 ) or 0.2 µM somatostatin on forskolin (1 µM) induced cAMP accumulation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a preceding report, we showed that, besides their physiological role on gastric acid secretion, GLIC and OXM were able to induce a contraction of isolated smooth muscle cells from rabbit stomach with a higher potency for GLIC (15). The contribution of G protein for mediating the signal (16) suggested the existence of a G protein-coupled receptor. In this report, we investigated further the intracellular pathways which link this hypothetic receptor to contraction.

We observed that GLIC and OXM increased Ins(1, 4, 5)P3 production in the cells at concentrations inducing contraction. The biphasic and fast production (within 5–8 sec) of Ins(1, 4, 5)P3 by low concentrations of GLIC is consistent with a direct coupling of the receptor with PLC (26). A similar profile required a higher OXM concentration, an observation compatible with the presence of a preferential GLIC receptor. Because OXM is known to cross-react with the glucagon and tGLP1 receptor, whereas OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) does not (32), it was of major interest to compare the two peptides. Like OXM, OXM (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) displayed an Ins(1, 4, 5)P3 production on antral SMC. Because OXM and OXM (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) were equally efficient and potent on contraction whereas glucagon or tGLP-1 had no effect on both contraction and cAMP content (15), we can exclude any implication of glucagon or tGLP-1 receptors in the Ins(1, 4, 5)P3 produced by OXM. Inositol phosphate production is due to direct PLC activation, as demonstrated by the rapid rise of Ins(1, 4, 5)P3 content upon GLIC or OXM application on cells incubated in a Ca2+-free medium. This rise was thus not related to an intracellular Ca2+ increase due to a calcium influx which secondarily can activate PLC (25, 26). The Ins(1, 4, 5)P3 production in function of time is well documented for acetylcholine and cholecystokinin on SMC (24), and for substance P, bradykinin, bombesin, angiotensin II, and purinergic agonists in different cell types (33). The permeabilized cells do not disrupt the agonist-receptor-messenger chain (34), and respond to concentrations of Ins(1, 4, 5)P3 in antral SMC identical to those previously reported in guinea pig intestinal SMC (35). We demonstrate that the contractile effects of GLIC, OXM, and OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) are largely related to an increase in inositol phosphate production since the peptide effects are mimicked by exogenous Ins(1, 4, 5)P3 and suppressed by blocking the Ins(1, 4, 5)P3 receptors. Comparison of the results obtained with the permeabilized vs. intact cells shows the disappearance in the permeabilized cells of the higher potency of GLIC vs. the shorter peptides. This higher potency might result of an additional mechanism triggered specifically by GLIC and depending on the transmembrane potential.

Another signaling pathway, related to cAMP, plays a crucial role in regulating SMC contraction through inhibition or activation of adenylate cyclase. Our data show that GLIC, OXM and OXM (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) also modulated this system by inhibiting the forskolin stimulated cAMP content. This inhibition of cAMP production appears to implicate a Gi or Go PTX-sensitive protein, as demonstrated with OXM(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). Somatostatin, which inhibits adenylate cyclase activity in guinea pig intestinal SMC through activation of a Gi/Go protein-coupled sstr3 receptor (31) exhibited the same activity in the rabbit antral SMC.

Thus, the contractile effect of GLIC and OXM on antral SMC could be mediated through both stimulation of the phosphoinositide hydrolysis and inhibition of the cAMP production. A double transduction pathway has already been described to explain SMC contraction. The muscarinic M3 receptor mediates PLC activation (36), whereas the M2 receptor mediates inhibition of cAMP production (37). In addition, bradykinin B2 receptor (38), or the {delta} opioid receptor on circular muscle activates Ins(1, 4, 5)P3 production and inhibition of adenylate cyclase activity via the same receptor (39). In the same way, both transduction pathways are used by the adenosine A1 receptor (40), or the somatostatin sstr3 receptor (31) on the same SMC type.

Taken together, our present and previous findings (16) constitute important clues for the existence of a GLIC/OXM receptor coupled to G proteins, which recognizes their common C-terminal moiety. In the gastrointestinal physiology and pathophysiology (6, 41), activation of the two described pathways by both hormones on their different target cells have now to be considered.


   Acknowledgments
 
We thank Alain Kervran for his precious help and François Beauclair for the peptide synthesis. We are indebted to Michel Pucéat for his technical and intellectual contribution. We thank Catherine Legraverend for helpful discussions.


   Footnotes
 
1 This work was supported by La Fondation pour la Recherche Médicale. Back

Received May 21, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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