Rosa Serio

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.

GABA and GABA receptors in the gastrointestinal tract: from motility to inflammation.

Although an extensive body of literature confirmed γ-aminobutyric acid (GABA) as mediator within the enteric nervous system (ENS) controlling gastrointestinal (GI) function, the true significance of GABAergic signalling in the gut is still a matter of debate. GABAergic cells in the bowel include neuronal and endocrine-like cells, suggesting GABA as modulator of both motor and secretory GI activity. GABA effects in the GI tract depend on the activation of ionotropic GABAA and GABAC receptors and metabotropic GABAB receptors, resulting in a potential noteworthy regulation of both the excitatory and inhibitory signalling in the ENS. However, the preservation of GABAergic signalling in the gut could not be limited to the maintenance of physiologic intestinal activity. Indeed, a series of interesting studies have suggested a potential key role of GABA in the promising field of neuroimmune interaction, being involved in the modulation of immune cell activity associated with different systemic and enteric inflammatory conditions. Given the urgency of novel therapeutic strategies against chronic immunity-related pathologies, i.e. multiple sclerosis and Inflammatory Bowel Disease, an in-depth comprehension of the enteric GABAergic system in health and disease could provide the basis for new clinical application of nerve-driven immunity. Hence, in the attempt to drive novel researches addressing both the physiological and pathological importance of the GABAergic signalling in the gut, we summarized current evidence on GABA and GABA receptor function in the different parts of the GI tract, with particular focus on the potential involvement in the modulation of GI motility and inflammation.

Galactosylated polymeric carriers for liver targeting of sorafenib

In this paper, we describe the preparation of liver-targeted polymeric micelles potentially able to carry sorafenib to hepatocytes for treatment of hepatocarcinoma (HCC), exploiting the presence of carbohydrate receptors, ASGPR. These micelles were prepared starting from a galactosylated polylactide-polyaminoacid conjugate. This latter was obtained by chemical reaction of α,β-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-d,l-aspartamide (PHEA-EDA) with polylactic acid (PLA), and subsequent reaction with lactose, leading to PHEA-EDA-PLA-GAL copolymer. Liver-targeted sorafenib-loaded micelles were obtained in aqueous media at low PHEA-EDA-PLA-GAL copolymer concentration value with nanometer size and slightly positive zeta potential. Biodistribution studies on mice demonstrated, after oral administration of sorafenib loaded PHEA-EDA-PLA-GAL micelles, the preferential sorafenib accumulation into the liver. This finding raises hope in terms of future drug delivery strategy of sorafenib-loaded micelles targeted to the liver for the HCC treatment.

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.

Inhibitory responses to exogenous adenosine in murine proximal and distal colon.

1 The aims of the present study were firstly, to characterize pharmacologically the subtypes of P1 purinoreceptors involved in the inhibitory effects induced by exogenous adenosine in longitudinal smooth muscle of mouse colon, and secondly, to examine differences in the function and distribution of these receptors between proximal and distal colon. 2 Adenosine (100 μM–3 mM) caused a concentration‐dependent reduction of the amplitude of spontaneous contractions in the proximal colon, and muscular relaxation in the distal colon. In the proximal colon, adenosine effects were antagonized by a selective A1 receptor antagonist, 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX, 10 nM), but were not modified by 3,7‐dimethyl‐1‐propargylxanthine (DMPX, 10 μM) or by 9‐chloro‐2‐(2‐furanyl)‐5‐((phenylacetyl)amino)‐ [1,2,4]triazolo[1,5‐c]quinazoline (MRS 1220, 0.1 μM), selective A2 and A3 receptor antagonists, respectively. In the distal colon, adenosine effects were antagonized by DPCPX, DMPX, and by a selective A2B receptor antagonist, 8‐[4‐[((4‐cyanophenyl)carbamoylmethyl)oxy]phenyl]‐1,3‐di(n‐propyl) xanthine (MRS 1754, 10 μM), but not by 8‐(3‐chlorostyryl)‐caffeine (CSC, 10 μM), a selective A2A receptor antagonist, or by MRS 1220. 3 Tetrodotoxin (TTX 1 μM), the nitric oxide (NO) synthase inhibitor, N‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM), or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (10 μM), an inhibitor of soluble guanylyl cyclase, reduced adenosine effects only in distal colon. In addition, L‐NAME induced a further reduction of adenosine relaxation in the presence of DPCPX, but not in the presence of MRS 1754. 4 From these results we conclude that, in the murine proximal colon, adenosine induces inhibitory effects via TTX‐insensitive activation of A1 receptor. In the distal colon, adenosine activates both A1 and A2B receptors, the latter located on enteric inhibitory neurons releasing NO.

Altered electrical activity in colonic smooth muscle cells from dystrophic (mdx) mice

Because the colon from dystrophic (mdx) mice shows an altered motor pattern, probably due to neural disorders, our aim was to examine the electrophysiological properties of muscle cells and the functionality of nitrergic transmission in circular muscle from normal and mdx colon. Normal colonic cells (resting membrane potential [RMP] about −50 mV) showed spontaneous hyperpolarizations (inhibitory junction potentials; IJPs) and cyclic slow depolarizations were sometimes recorded. Mdx colon had a depolarized RMP (about –36 mV) and spontaneous IJPs, but the cyclic activity was never observed. In the normal colon, Nω‐nitro‐ L‐arginine methyl ester ( L‐NAME) induced depolarization and abolished the cyclic activity. In the mdx colon, L‐NAME caused a slight depolarization. Both preparations displayed the same value of RMP in the presence of L‐NAME. In normals, neural stimulation induced nonadrenergic, noncholinergic IJPs composed of fast hyperpolarizations followed by a nitrergic slow hyperpolarization, selectively abolished by L‐NAME. In the mdx colon the evoked IJPs were composed only of the initial fast hyperpolarization, the nitrergic component being absent. The hyperpolarization to sodium nitroprusside was not significantly different in both preparations. We conclude that the colon from animals lacking in dystrophin displays different electrophysiological features because of an impairment of nitric oxide function.

Exogenous glucagon-like peptide 1 reduces contractions in human colon circular muscle.

Glucagon-like peptide 1 (GLP1) is a naturally occurring peptide secreted by intestinal L-cells. Though its primary function is to serve as an incretin, GLP1 reduces gastrointestinal motility. However, only a handful of animal studies have specifically evaluated the influence of GLP1 on colonic motility. Consequently, the aims of this study were to investigate the effects induced by exogenous GLP1, to analyze the mechanism of action, and to verify the presence of GLP1 receptors (GLP1Rs) in human colon circular muscular strips. Organ bath technique, RT-PCR, western blotting, and immunofluorescence were used. In human colon, exogenous GLP1 reduced, in a concentration-dependent manner, the amplitude of the spontaneous contractions without affecting the frequency and the resting basal tone. This inhibitory effect was significantly reduced by exendin (9-39), a GLP1R antagonist, which per se significantly increased the spontaneous mechanical activity. Moreover, it was abolished by tetrodotoxin, a neural blocker, or Nω-nitro-l-arginine - a blocker of neuronal nitric oxide synthase (nNOS). The biomolecular analysis revealed a genic and protein expression of the GLP1R in the human colon. The double-labeling experiments with anti-neurofilament or anti-nNOS showed, for the first time, that immunoreactivity for the GLP1R was expressed in nitrergic neurons of the myenteric plexus. In conclusion, the results of this study suggest that GLP1R is expressed in the human colon and, once activated by exogenous GLP1, mediates an inhibitory effect on large intestine motility through NO neural release.

Food intake in lean and obese mice after peripheral administration of glucagon-like peptide-2

We investigated the potential anorectic action of peripherally administered glucagon-like peptide 2 (GLP2) in lean and diet-induced obese (DIO) mice. Mice, fasted for 16 h, were injected i.p. with native GLP2 or [Gly2]GLP2, stable analog of GLP2, before or after GLP2 (3-33), a GLP2 receptor (GLP2R) antagonist, or exendin (9-39), a GLP1R antagonist. Food intake was measured at intervals 1, 2, 4, 8, and 24 h postinjection. In addition, we tested in lean mice the influence of [Gly2]GLP2 on gastric emptying and the effects of GLP1 alone or in combination with [Gly2]GLP2 on food intake. [Gly2]GLP2 dose dependently and significantly inhibited food intake in lean and DIO mice. The reduction of food intake occurred in the first hour postinjection and it was sustained until 4 h postinjection in lean mice while it was sustained until 2 h postinjection in DIO mice. GLP2 significantly inhibited food intake in both lean and DIO mice but only in the first hour postinjection. The efficiency of [Gly2]GLP2 or GLP2 in suppressing food intake was significantly weaker in DIO mice compared with lean animals. The [Gly2]GLP2 anorectic actions were blocked by the GLP2R antagonist GLP2 (3-33) or by the GLP1R antagonist exendin (9-39). The coadministration of [Gly2]GLP2 and GLP1 did not cause additive effects. [Gly2]GLP2 decreased the gastric emptying rate. Results suggest that GLP2 can reduce food intake in mice in the short term, likely acting at a peripheral level. DIO mice are less sensitive to the anorectic effect of the peptide.