Alain Botella

Comparative effects of galanin on isolated smooth muscle cells from ileum in five mammalian species

Effect of galanin and CCK8 were studied on isolated smooth muscle cells obtained from pig, guinea-pig, rat, rabbit and dog ileum circular muscle layer. Galanin as well as CCK8 induced a concentration-dependent contraction of pig, rat, rabbit and guinea-pig ileum smooth muscle cells. Maximal contraction ranged between 23.7 +/- 1.9% and 26.1 +/- 3.1% decrease in cell length from control in the presence of both peptides. This maximal contraction was obtained at 1 nM galanin in pig, rat, rabbit, 1 nM CCK8 in rat, rabbit, guinea-pig, at 10 nM galanin in guinea-pig and 10 nM CCK8 in pig. Concentrations of galanin inducing a half maximal contraction (EC50) ranged between 8 pM and 80 pM in these species. In dog, CCK8 induced a concentration-dependent contraction of ileum smooth muscle cells, with a maximal contraction (24.5 +/- 2.3%) at 1nM and an EC50 of 50 pM while galanin inhibited cell contraction induced by CCK8. The CCK-induced contraction was abolished at 10 nM galanin and 10 nM VIP. Concentrations of galanin and VIP inducing a half-maximal relaxation of contracted cells were 2 pM and 3 pM respectively. It is concluded that galanin may induce cell contraction of pig, guinea-pig, rat and rabbit ileum circular muscle layer and cell relaxation of dog ileum by a direct myogenic effect.

Stimulatory (EP1 and EP3) and inhibitory (EP2) prostaglandin E2 receptors in isolated ileal smooth muscle cells

Isolated smooth muscle cells from the circular layer of pig and guinea-pig ileum were used to study the effect of prostaglandin E2 (PGE2) and three PGE2 receptor (EP) agonists; iloprost (EP1), butaprost (EP2) and enprostil (EP3). In pig cells, PGE2 and enprostil induced cell contraction (22.1 and 21.5% shortening of cell length, obtained at 10 nM for PGE2 and 1 nM for enprostil, respectively). Iloprost and butaprost had no contractile effect. However, the cholecystokinin octapeptide (CCK-8; 10 nM)-induced contraction was inhibited when cells were preincubated with iloprost or butaprost. In guinea-pig cells, PGE2, butaprost and iloprost induced cell contraction, whereas enprostil had no effect (23.1% for 10 nM PGE2, 22.8% for 1 nM butaprost and 22.6% for 10 nM iloprost). Preincubation with SC19220 (EP1 antagonist) inhibited the PGE2-, butaprost- and iloprost-induced contractions. When the contractile effect of PGE2, butaprost and iloprost was inhibited by addition of SC19220, these agents inhibited the cell contraction induced by CCK-8 (1 nM). Smooth muscle cells from guinea-pig and pig ileum express two PGE2 receptor subtypes that induce opposite effect. EP1 and EP3 receptors mediate cell contraction in guinea-pig and pig, respectively, whereas EP2 receptors mediate cell relaxation in both species.

Galanin contracts and relaxes guinea pig and canine intestinal smooth muscle cells through distinct receptors

BACKGROUND/AIMS Galanin induces a contraction or a relaxation of digestive smooth muscle. Receptors mediating these effects have not been pharmacologically characterized. The aim of the study was to evaluate properties of two specific galanin antagonists M15 and M35 on galanin effects on muscle cells. METHODS Isolated muscle cells were obtained separately from circular and longitudinal layers of guinea pig and dog ileums. Contraction was expressed as percentage decrease in cell length from control. RESULTS Galanin induced a contraction of cells from guinea pig circular layer (50% effective concentration [EC50], 80 pmol/L) and dog longitudinal layer (EC50, 100 pmol/L). The antagonists inhibited galanin-induced contraction. The most potent was M15 (50% inhibitory concentration [IC50], 80 pmol/L in guinea pig; 90 pmol/L in dog) which was > M35 (IC50, 4 nmol/L in guinea pig; 1 nmol/L in dog). In dog circular layer, galanin inhibited cholecystokinin-induced contraction by relaxing the cells (EC50, 3 pmol/L). The antagonists inhibited this relaxation. The most potent was M35 (IC50, 60 pmol/L) which was > M15 (IC50, 900 pmol/L). CONCLUSIONS Galanin antagonists M15 and M35 inhibit the contraction and the relaxation induced by galanin with different potency, suggesting the presence of distinct galanin receptors in gastrointestinal tract that each mediates a specific effect.

Intracolonic glycerol induces abdominal contractions in rats: role of 5-HT3 receptors.

Summary— Serotonin and 5‐HT3 receptors may be involved in the activation of nociceptive afferent pathways by rectal distension. In rats, intracolonic infusion of glycerol is able to trigger nociceptive inputs as evidenced by the occurrence of abdominal constrictions. This work was designed to evaluate the influence of 5‐HT3 receptor antagonists on this reflex and to approach the site of action by comparing their relative efficacies according to the route of administration. Male Wistar rats (250–350 g) were surgically prepared for abdominal electromyography and a catheter was placed in the colonic lumen. Five days after surgery, electrical activity of abdominal muscles was recorded before and during (20 min) intracolonic infusion of glycerol (60% glycerol + 40% saline, rate 0.75 mL/h). Cilansetron was administered intraperitoneally, 15 min before glycerol infusion, at doses of 5 to 500 μg/kg. Granisetron, ondansetron and cilansetron were administred at the dose of 20 μg/kg by intraperitoneal (ip), intravenous (iv) or intracolonic (ic) routes. The number of abdominal spike bursts was used as an index of visceral nociception. Intracolonic infusion of glycerol increased significantly (P < 0.05) the number of abdominal spike bursts during the time of infusion compared with saline (30.6 ± 6.6 vs 4.5 ± 3.4 bursts). When administered ip, cilansetron dose‐dependently reduced the frequency of abdominal spike bursts from the dose of 20 μg/kg ip. Administration ip of granisetron and ondansetron at this dose also significantly reduced the number of abdominal spikes (19.0 ± 6.0 and 18.3 ± 6.9 respectively). Cilansetron, ondansetron and granisetron were also effective by iv and ic routes, cilansetron was more active by the ic route. Serotonin, via 5‐HT3 receptors, is involved in the mediation of abdominal contractions induced by intracolonic infusion of glycerol. 5‐HT3 receptor antagonists are also active by ic route suggesting a local site of action.

Intracellular pathways triggered by galanin to induce contraction of pig ileum smooth muscle cells.

1. In order to determine the intracellular mechanisms by which galanin induces contraction of isolated smooth muscle cells from pig ileum, we examined the effects of external Ca2+, relaxing agents, pertussis toxin and forskolin on the galanin‐induced contraction and compared these effects to those observed on the cholecystokinin derivative CCK8‐induced contraction. 2. Galanin induced a concentration‐dependent cell contraction. The maximal contraction (24.5 +/‐ 2.1% of the length of resting cells) was observed at 1 nM of galanin. When cells were incubated in the simultaneous presence of concentrations of galanin (10 fM) and CCK8 (1 pM) which were ineffective alone, or galanin (10 fM) and acetylcholine (100 pM), a synergistic action was observed corresponding to a submaximal contraction. 3. Incubation of cells in Ca(2+)‐free medium caused a significant decrease in galanin‐ but not in CCK‐induced contraction. Nifedipine, a Ca2+ channel blocker, provoked a concentration‐dependent inhibition of galanin‐induced contraction while it had no effect on the contraction induced by CCK8. 4. Vasoactive intestinal polypeptide (VIP) and isoprenaline, known to induce cell relaxation through an increase in intracellular cAMP level, inhibited CCK‐induced cell contraction at concentrations ranging from 1 pM to 1 microM but failed to inhibit cell contraction induced by galanin. 5. When cells were pre‐incubated for 3 h in the presence of 200 ng/ml of pertussis toxin, the contraction induced by galanin was abolished while the CCK‐induced contraction remained unchanged. On the contrary, 10 microM forskolin abolished the contraction induced by 10 nM CCK but had no effect on galanin‐induced contraction. 6. These results indicate that galanin induces a concentration‐dependent contraction of pig ileum smooth muscle by a direct myogenic effect. This effect of galanin involves the activation of a pertussis toxin‐sensitive G protein, which results in an influx of Ca2+ into the cell. This intracellular pathway is insensitive to the relaxing effect of cAMP.

GALANIN INDUCES CONTRACTION OF ISOLATED CELLS FROM CIRCULAR MUSCLE LAYER OF PIG ILEUM

The effects of galanin and its interaction with cholecystokinin and acetylcholine on smooth muscle cells were studied in vitro on isolated cells obtained from pig ileum circular muscle layer. Galanin induced a concentration-dependent cell contraction with a maximal contraction (24.5% decrease in cell length from control) obtained at 1 nM. The concentration of galanin inducing a half-maximal contraction was 3 pM. Tetrodotoxin (10 microM) failed to inhibit cell contraction induced by galanin (1 nM), pentagastrin (10 nM) and acetylcholine (1 microM). Atropine abolished the contraction induced by acetylcholine (1 microM), but had no effect on galanin- and pentagastrin-induced contraction. L 364,718 inhibited the contraction induced by CCK8 but not the galanin-induced contraction. At the uneffective concentration of 10 fM, galanin had a synergistic effect with an uneffective concentration of CCK8 (1 pM). These results suggest that (i) galanin contracts smooth muscle cells from pig ileum by acting on a specific receptor; (ii) galanin and either CCK or acetylcholine may act in a synergistic way to induce cell contraction.

Effect of sorbin derivatives on cholera toxin-induced intestinal secretion in rat in vivo

The effect of synthetic sorbin derivatives was determined on cholera toxin-stimulated jejunal secretion in anesthetized rats in vivo, using both perfused and ligated loop. An inhibitory effect on water secretion induced by cholera toxin was shown with C-terminal sorbin peptides: C20 (YEPGKSSILQHERPVTKPQA-amide), C10 and Dala 7 heptapeptide-amide of sorbin, given by subcutaneous (SC) or intraduodenal administration. When perfused intravenously, C20-sorbin inhibited the cholera-induced stimulation of net flux of water, Na+ and K+, in the jejunum and at the same time the net flux of water and Cl- in the duodenum, which was not in contact with the toxin. 5-hydroxytryptamine was not significantly changed in plasma or fluid. Prostaglandin E2 release in jejunal as well as duodenal fluid was significantly stimulated by cholera toxin, but was not significantly different from basal value after C20 administration.

Galanin induces opposite effects via different intracellular pathways in smooth muscle cells from dog colon

Smooth muscle cells isolated by enzymatic digestion were used to determine the direct effects of galanin on circular and longitudinal muscle layers from dog proximal colon and to investigate the intracellular pathways involved in these effects. Effects of galanin were compared to those observed with other contracting [cholecystokinin octapeptide (CCK8)] and relaxing [vasoactive intestinal peptide (VIP)] agents. In longitudinal cells, galanin and CCK8 induced a contraction that was maximal at 1 nM galanin and 1 nM CCK8 and was 23.9 +/- 4.5% and 23.4 +/- 3.4%, respectively, of the length of resting cells. Incubation of cells in Ca(2+)-free medium or in the presence of nifedipine caused an inhibition of galanin-induced contraction whereas it had no effect on the contraction induced by CCK8. Vasoactive intestinal peptide, forskolin, and 8 bromo cAMP inhibited CCK-induced contraction but failed to inhibit contraction induced by galanin. The contraction induced by galanin was abolished; the CCK-induced contraction was unchanged by pertussis toxin. In circular cells, CCK8 induced a contraction that was maximal at 10 nM and was 24.2 +/- 2.6%. Galanin had no effect by itself. When cells were preincubated (1 min) with galanin (10 fM-1 microM), the CCK8-induced contraction was inhibited, with a maximal effect at 10 nM galanin. Likewise, VIP inhibited the CCK8-induced contraction with a maximal effect at 1 microM. Preincubation of cells with somatostatin, N-ethylmaleimide, and (R)-p-cAMPS inhibited galanin- and VIP-induced relaxation. In conclusion, galanin induces a contraction of longitudinal smooth muscle cells that is dependent on an influx of extracellular calcium and an activation of pertussis toxin G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo inhibitory effect of lanreotide (BIM 23014), a new somatostatin analog, on prostaglandin- and cholera toxin-stimulated intestinal fluid in the rat

The antisecretory action of subcutaneously (SC) administered somatostatin(1-14), octreotide, and lanreotide on jejunal net flux of water under basal, prostaglandin E1 (PGE1)- and cholera toxin (CT)-stimulated secretory conditions was determined in vivo on isolated intestinal loop in anesthetized rats. Both PGE1 and CT induced intestinal hypersecretion in the rats. This secretory effect was not affected by SC administration of saline. Lanreotide (1, 10, and 100 micrograms/kg) reduced the maximal PGE1-induced secretion, while 200 micrograms/kg had no effect. Similarly, octreotide (1 and 10 micrograms/kg) and somatostatin (1-14) (0.1 and 1 microgram/kg) reduced the increase of net water flux induced by PGE1. However, higher doses of octreotide (100 and 200 micrograms/kg) and somatostatin(1-14) (10 and 100 micrograms/kg) had no effect on PGE1-induced secretion. Lanreotide, octreotide, and somatostatin(1-14) (1 and 10 micrograms/kg) abolished the maximal secretion induced by cholera toxin. However, 100 micrograms/kg of lanreotide, octreotide, and somatostatin(1-14) had no effect on cholera toxin-induced secretion. The present study shows that lanreotide, octreotide, and somatostatin(1-14) reduce the secretion induced by PGE1 and abolish that induced by CT. These effects were obtained with doses of less than 100 micrograms/kg of the products, higher doses being ineffective. The higher efficacy against CT-induced hypersecretion as compared to PGE1-induced hypersecretion suggests a direct antisecretory effect at the enterocyte level and indicates the usefulness of these products as antidiarrheal agents in nonhormonally mediated diarrhea.

Iloprost: Intracellular Ca2+-dependent Contractile Effect on Isolated Smooth Muscle Cells from Guinea-pig Ileum

Prostaglandin E2 (PGE2) and iloprost induced a concentration‐dependent contraction of smooth muscle cells isolated from the circular layer of guinea‐pig ileum. PGE2‐ and iloprost‐induced contractions were inhibited by the selective EP1‐receptor antagonist, SCI9220 (1‐acetyl‐2‐(8‐chloro‐10, 11‐dihydrodibenz (b,f) (1,4) oxazepine‐10‐carbonyl)‐hydrazine), indicating the involvement of the EP1 subtype of the PGE2 receptor. When cells were incubated for 10 min in the presence of strontium (4 mm L−1), an inhibitor of the release of Ca2+ from intracellular store, the contractile effect of PGE2 and iloprost was inhibited. In contrast, incubation of cells in Ca2+‐free medium, Ca2+‐free medium plus EGTA, or in the presence of nifedipine, an organic Ca2+‐channel blocker, did not alter the PGE2‐ and iloprost‐induced contraction. These observations suggest that the myogenic effect of PGE2 and iloprost on intestinal smooth muscle is dependent on the release of intracellular calcium.