Flavia Mulè

Peripheral motor action of glucagon-like peptide-1 through enteric neuronal receptors

Background  Glucagon‐like peptide‐1 (GLP‐1) is a proglucagon‐derived peptide expressed in the enteroendocrine‐L cells of small and large intestine and released in response to meal ingestion. Glucagon‐like peptide‐1 exerts inhibitory effects on gastrointestinal motility through vagal afferents and central nervous mechanisms; however, no data is available about a direct influence on the gastrointestinal wall. Our aim was to investigate the effects of GLP‐1 on the spontaneous and evoked mechanical activity of mouse duodenum and colon and to identify the presence and distribution of GLP‐1 receptors (GLP‐1R) in the muscle coat.

NANC inhibitory neurotransmission in mouse isolated stomach: involvement of nitric oxide, ATP and vasoactive intestinal polypeptide

The neurotransmitters involved in NANC relaxation and their possible interactions were investigated in mouse isolated stomach, recording the motor responses as changes of endoluminal pressure from whole organ. Field stimulation produced tetrodotoxin‐sensitive, frequency‐dependent, biphasic responses: rapid transient relaxation followed by a delayed inhibitory component. The inhibitor of the synthesis of nitric oxide (NO), L‐NAME, abolished the rapid relaxation and significantly reduced the slow relaxation. Apamin, blocker of Ca2+‐dependent K+ channels, or ADPβS, which desensitises P2y purinoceptors, reduced the slow relaxation to 2–8 Hz, without affecting that to 16–32 Hz or the fast relaxation. α‐Chymotrypsin or vasoactive intestinal polypeptide 6–28 (VIP6–28), antagonist of VIP receptors, failed to affect the fast component or the delayed relaxation to 2–4 Hz, but antagonised the slow component to 8–32 Hz. Relaxation to sodium nitroprusside was not affected by L‐NAME, apamin or ADPβS, but was reduced by α‐chymotrypsin or VIP6–28. Relaxation to VIP was abolished by α‐chymotrypsin, antagonised by VIP6–28, but was not affected by L‐NAME, apamin or ADPβS. Relaxation to ATP was abolished by apamin, antagonised by ADPβS, but was not affected by L‐NAME or α‐chymotrypsin. The present results suggest that NO is responsible for the rapid relaxation and partly for the slow relaxation. ATP is involved in the slow relaxation evoked by low frequencies of stimulation. VIP is responsible for the slow relaxation evoked by high frequencies of stimulation. The different neurotransmitters appear to work in parallel, although NO could serve also as a neuromodulator that facilitates release of VIP.

Glucagon-like peptide-2 relaxes mouse stomach through vasoactive intestinal peptide release

Glucagon-like peptide-2 (GLP-2) influences different aspects of the gastrointestinal function, including epithelial growth, digestion, absorption, motility, and blood flow. Intraluminal pressure from isolated mouse stomach was recorded to investigate whether GLP-2 affects gastric tone and to analyze its mechanism of action. Regional differences between diverse parts of the stomach were also examined using circular muscular strips from fundus and antrum. In the whole stomach, GLP-2 (0.3-100 nM) produced concentration-dependent relaxation with a maximum that was about 75% of relaxation to 1 microM isoproterenol (IC50=2.5 nM). This effect was virtually abolished by desensitization of GLP-2 receptors or by alpha-chymotrypsin. The relaxant response to GLP-2 was not affected by tetrodotoxin, a blocker of neuronal voltage-dependent Na+ channels, but it was significantly reduced by omega-conotoxin GVIA, a blocker of neuronal N-type voltage-operated Ca2+ channels. Nomega-nitro-L-arginine methyl ester, a blocker of nitric oxide synthase, or apamin, a blocker of Ca2+-dependent potassium channels, failed to affect the gastric response to the peptide. However, the relaxation was significantly antagonized by [Lys1,Pro2,5,Arg3,4,Tyr6]VIP7-28, a vasoactive intestinal peptide (VIP) receptor antagonist (GLP-2 maximum effect=45% of relaxation to 1 microM isoproterenol), and virtually abolished by desensitization of the VIP receptors. GLP-2 induced concentration-dependent relaxation in carbachol-precontracted fundic strips but not in antral strips. These results provide the first experimental evidence that GLP-2 is able to induce gastric relaxation acting peripherally on the mouse stomach. The effect appears to be mediated by prejunctional neural release of VIP and confined to fundic region.

Glucagon-like peptide-2 modulates neurally evoked mucosal chloride secretion in guinea pig small intestine in vitro

Glucagon-like peptide-2 (GLP-2) is an important neuroendocrine peptide in intestinal physiology. It influences digestion, absorption, epithelial growth, motility, and blood flow. We studied involvement of GLP-2 in intestinal mucosal secretory behavior. Submucosal-mucosal preparations from guinea pig ileum were mounted in Ussing chambers for measurement of short-circuit current (I(sc)) as a surrogate for chloride secretion. GLP-2 action on neuronal release of acetylcholine was determined with ELISA. Enteric neuronal expression of the GLP-2 receptor (GLP-2R) was studied with immunohistochemical methods. Application of GLP-2 (0.1-100 nM) to the serosal or mucosal side of the preparations evoked no change in baseline I(sc) and did not alter transepithelial ionic conductance. Transmural electrical field stimulation (EFS) evoked characteristic biphasic increases in I(sc), with an initially rapid rising phase followed by a sustained phase. Application of GLP-2 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-2R antagonist GLP-2-(3-33) significantly reversed suppression of the EFS-evoked responses by GLP-2. Tetrodotoxin, scopolamine, and hexamethonium, but not vasoactive intestinal peptide type 1 receptor (VPAC1) antagonist abolished or reduced to near zero the EFS-evoked responses. GLP-2 suppressed EFS-evoked acetylcholine release as measured by ELISA. Pretreatment with GLP-2-(3-33) offset this action of GLP-2. In the submucosal plexus, GLP-2R immunoreactivity (-IR) was expressed in choline acetyltransferase-IR neurons, somatostatin-IR neurons, neuropeptide Y-IR neurons, and vasoactive intestinal peptide-IR neurons. We conclude that submucosal neurons in the guinea pig ileum express GLP-2R. Activation of GLP-2R decreases neuronally evoked epithelial chloride secretion by suppressing acetylcholine release from secretomotor neurons.

Dual effect mediated by protease‐activated receptors on the mechanical activity of rat colon

The present study examined the mechanical effects of agonist enzymes and receptor‐activating peptides for protease‐activated receptor (PAR)‐1 and PAR‐2 on longitudinal and circular muscle of rat isolated colonic segments in the attempt to clarify the PAR functional role in intestinal motility. The responses to PAR‐1 and PAR‐2 activation were examined in vitro by recording simultaneously the changes of endoluminal pressure (index of circular muscle activity) and of isometric tension (index of longitudinal muscle activity). Both PAR‐1 agonists, thrombin (0.1 nM – 3 μM) and SFLLRN‐NH2 (1 nM – 3 μM), and PAR‐2 agonists, trypsin (0.1 nM – 10 μM) and SLIGRL‐NH2 (1 nM – 10 μM), induced different effects in the two muscular layers: a reduction of the spontaneous contractions in the circular muscle and a contractile effect or biphasic, relaxation followed by contraction, depending on the concentration, in the longitudinal muscle. The inhibitory effects were greatly reduced or abolished by apamin (0.1 μM) indicating that they mainly occur via activation of Ca2+‐dependent small conductance, K+‐channels. The responses to PAR‐1 and PAR‐2 were unaffected by tetrodotoxin (1 μM) or indomethacin (1 μM) suggesting that are independent by products of cyclooxygenase or by neural action potentials. These findings indicate that both PAR‐1 and PAR‐2 are functionally expressed in rat colon. PARs mediate changes of the mechanical activity of longitudinal and circular muscle which might explain the alterations of colonic motility observed during inflammatory conditions.

Glucagon-like peptide-2 and mouse intestinal adaptation to a high-fat diet.

Endogenous glucagon-like peptide-2 (GLP2) is a key mediator of refeeding-induced and resection-induced intestinal adaptive growth. This study investigated the potential role of GLP2 in mediating the mucosal responses to a chronic high-fat diet (HFD). In this view, the murine small intestine adaptive response to a HFD was analyzed and a possible involvement of endogenous GLP2 was verified using GLP2 (3-33) as GLP2 receptor (GLP2R) antagonist. In comparison with animals fed a standard diet, mice fed a HFD for 14 weeks exhibited an increase in crypt-villus mean height (duodenum, 27.5±3.0%; jejunum, 36.5±2.9%; P<0.01), in the cell number per villus (duodenum, 28.4±2.2%; jejunum, 32.0±2.9%; P<0.01), and in Ki67-positive cell number per crypt. No change in the percent of caspase-3-positive cell in the villus-crypt was observed. The chronic exposure to a HFD also caused a significant increase in GLP2 plasma levels and in GLP2R intestinal expression. Daily administration of GLP2 (3-33) (30-60  ng) for 4 weeks did not modify the crypt-villus height in control mice. In HFD-fed mice, chronic treatment with GLP2 (3-33) reduced the increase in crypt-villus height and in the cell number per villus through reduction of cell proliferation and increase in apoptosis. This study provides the first experimental evidence for a role of endogenous GLP2 in the intestinal adaptation to HFD in obese mice and for a dysregulation of the GLP2/GLP2R system after a prolonged HFD.

Relaxant effects of flavonoids on the mouse isolated stomach: Structure-activity relationships

Flavonoids are a large heterogeneous group of benzo-gamma-pyrone derivatives, which are abundantly present in our diet. In this study we investigated the effects of six flavonoids (apigenin, genistein, quercetin, rutin, naringenin and catechin) on the gastric tone in mouse isolated stomach. The mechanical activity was recorded as changes of intraluminal pressure. All flavonoids tested produced a concentration-dependent relaxation, which was reversible after washout. The relative order of potency of the flavonoids was apigenin> or =genistein>quercetin>naringenin> or =rutin>catechin. Analysis of the chemical structure showed that the relaxant activity was progressively diminished by the presence of hydroxyl group at C-3, saturation of the C-2, C-3 double bound, saturation of the C-2, C-3 double bound coupled with lack of the C-4 carbonyl and glycosylation. The flavonoid-induced relaxations were not modified in the presence of tetrodotoxin, a voltage-dependent Na(+)-channel blocker, N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, indomethacin, an inhibitor of cycloxygenase or tetraethylammonium, a non-selective blocker of potassium channels. In conclusion, this study provides the first experimental evidence for gastric relaxant activity of flavonoids. This action is influenced to a great extent by the structure of the molecules and it seems not to be dependent on neural action potentials, NO/prostaglandin production or activation of K(+) channels.

Signal Transduction Pathways Involved in the Mechanical Responses to Protease-Activated Receptors in Rat Colon

Recording simultaneously in vitro the changes of endoluminal pressure (index of circular muscle activity) and isometric tension (index of longitudinal muscle activity), we examined the mechanisms responsible for the apamin-sensitive relaxant and contractile responses induced by protease-activated receptor (PAR)-1 and PAR-2 activating peptides, SFLLRN-NH2 and SLIGRL-NH2, respectively, in rat colon. In the circular muscle, the inhibitory effects of SFLLRN-NH2 and SLIGRL-NH2 were significantly reduced by ryanodine, an inhibitor of Ca2+release from the sarcoplasmic reticulum, but unaffected by 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X), a protein kinase C (PKC) inhibitor, or genistein, a tyrosine kinase inhibitor. In the longitudinal muscle, the contractile responses to SFLLRN-NH2 and SLIGRL-NH2 were significantly reduced by nifedipine, an L-type calcium channel blocker, ryanodine, GF109203X, genistein, and abolished by U73122. The effects of genistein were additive with GF109203X but not with nifedipine. In the longitudinal muscle, the relaxant responses to the highest concentrations of SFLLRN-NH2 and SLIGRL-NH2were abolished by nifedipine, reduced by genistein, and unaffected by ryanodine or GF109203X. In conclusion, influx of extracellular Ca2+ through L-type voltage-dependent channels or release of Ca2+ from intracellular stores are determining for the opening of the apamin-sensitive K+ channels responsible for longitudinal muscle relaxation or circular muscle inhibitory response, respectively, in rat colon. The longitudinal muscle contraction is mediated by activation of PLC; PKC and tyrosine kinase are involved in the cascade process, playing a parallel role. Indeed, tyrosine kinase and L-type Ca2+ channels would act sequentially. The influx of Ca2+ in turn would cause release of Ca2+from sarcoplasmic reticulum.

Tonic inhibitory action by nitric oxide on spontaneous mechanical activity in rat proximal colon: involvement of cyclic GMP and apamin-sensitive K+ channels

The cellular mechanisms by which endogenous nitric oxide (NO) modulates spontaneous motility were investigated in rat isolated proximal colon. The mechanical activity was detected as changes in intraluminal pressure. Apamin (1–100 nM) produced a concentration‐dependent increase in the amplitude of the spontaneous pressure waves. The maximal contractile effect was of the same degree as that produced by Nω‐nitro‐L‐arginine methyl ester (L‐NAME) (100 μM) and the joint application of apamin plus L‐NAME had no additive effects. Apamin (0.1 μM) reduced the inhibitory effects (i.e. reduction in the amplitude of the pressure waves) induced by sodium nitroprusside (SNP) (1 nM–10 μM) or 8‐Br‐cyclic GMP (1–100 μM). 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) (0.1–5 μM), inhibitor of NO‐stimulated guanylate cyclase, produced a concentration‐dependent increase of the spontaneous contractions. ODQ (1 μM) in the presence of apamin (0.1 μM) did not produce any further increase in the contraction amplitude, whereas after L‐NAME (100 μM) it decreased the spontaneous contractions. ODQ (1 μM) reduced the SNP inhibitory effects. Zaprinast (1–50 μM), inhibitor of cyclic GMP phosphodiesterase, produced a concentration‐dependent decrease of the spontaneous contractions. The effects of zaprinast were significantly reduced in the presence of apamin (0.1 μM) or L‐NAME (100 μM). These results suggest that small conductance Ca2+‐dependent K+ channels and cyclic GMP are involved in the modulation of the spontaneous contractile activity in rat proximal colon. Cyclic GMP production system and opening of apamin‐sensitive K+ channels appear to work sequentially in transducing an endogenous NO signal.

Glucagon-like peptide-1 relaxes gastric antrum through nitric oxide in mice.

Glucagon-like-peptide-1 (GLP-1) is a proglucagon-derived peptide expressed in the intestinal enteroendocrine-L cells and released after meal ingestion. GLP-1 reduces postprandial glycemia not only by its hormonal effects, but also by its inhibitory effects on gastrointestinal motility. Recently, we showed that GLP-1 acts in the enteric nervous system of mouse intestine. Therefore our working hypothesis was that GLP-1 may have also a direct influence on the gastric mechanical activity since the major part of experimental studies about its involvement in the regulation of gastric motility have been conducted in in vivo conditions. The purposes of this study were (i) to examine exogenous GLP-1 effects on mouse gastric mechanical activity using isolated whole stomach; (ii) to clarify the regional activity of GLP-1 using circular muscular strips from gastric fundus or antrum; (iii) to analyze the mechanism of action underlying the observed effects; (iv) to verify regional differences of GLP-1 receptors (GLP-1R) expression by RT-PCR. In the whole stomach GLP-1 caused concentration-dependent relaxation significantly anatagonized by exendin (9-39), an antagonist of GLP-1R and abolished by tetrodotoxin (TTX) or N(ω)-nitro-l-arginine methyl ester (l-NAME), inhibitor of nitric oxide (NO) synthase. GLP-1 was without any effect in fundic strips, but it induced concentration-dependent relaxation in carbachol-precontracted antral strips. The effect was abolished by TTX or l-NAME. RT-PCR analysis revealed a higher expression of GLP-1R mRNA in antrum than in fundus. These results suggest that exogenous GLP-1 is able to reduce mouse gastric motility by acting peripherally in the antral region, through neural NO release.