Maria Grazia Zizzo

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.

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.

Functional evidence for GABA as modulator of the contractility of the longitudinal muscle in mouse duodenum: Role of GABAA and GABAC receptors

We investigated, in vitro, the effects of gamma-aminobutyric acid (GABA) on the spontaneous mechanical activity of the longitudinal smooth muscle in mouse duodenum. GABA induced an excitatory effect, consisting in an increase in the basal tone, which was antagonized by the GABA(A)-receptor antagonist, bicuculline, potentiated by (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA), a GABA(C)-receptor antagonist and it was not affected by phaclofen, a GABA(B)-receptor antagonist. Muscimol, GABA(A) receptor agonist, induced a contractile effect markedly reduced by bicuculline, tetrodotoxin (TTX), hexamethonium and atropine. Cis-4-aminocrotonic acid (CACA), a specific GABA(C) receptor agonist, induced an inhibitory effect, consisting in the reduction of the amplitude of the spontaneous contractions and muscular relaxation, which was antagonised by TPMPA, GABA(C)-receptor antagonist, TTX or N(omega)-nitro-l-arginine methyl ester (L-NAME), nitric oxide (NO) synthase inhibitor, but not affected by hexamethonium. In conclusion, our study indicates that GABA is a modulator of mechanical activity of longitudinal muscle in mouse duodenum. GABA may act through neuronal presynaptic receptors, namely GABA(A) receptors, leading to the release of ACh from excitatory cholinergic neurons, and GABA(C) receptors increasing the release of NO from non-adrenergic, non-cholinergic inhibitory neurons.

Evidence that ATP or a related purine is an excitatory neurotransmitter in the longitudinal muscle of mouse distal colon

This study analysed the contribution of the purinergic system to enteric neurotransmission in the longitudinal muscle of mouse distal colon.

Interplay between PACAP and NO in mouse ileum

We investigated the possibility that pituitary adenylate cyclase activating peptide (PACAP) has a role in the control of contractility in the mouse ileum. PACAP-(1-27) produced tetrodotoxin (TTX)-insensitive, concentration-dependent reduction of the amplitude of the spontaneous contractions of longitudinal muscle up to their complete disappearance. This effect was inhibited by PACAP-(6-38), PACAP receptor antagonist, and by apamin, blocker of small-conductance Ca2+-activated K+-channels. Nomega-nitro-L-arginine methyl ester (L-NAME), nitric oxide (NO) synthase inhibitor, reduced the PACAP-inhibitory response, and the joint application of apamin plus L-NAME produced additive effects. 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), inhibitor of NO-stimulated soluble guanylate cyclase, significantly reduced the effect of PACAP. Exogenous NO, given as sodium nitroprusside (SNP), induced a concentration-dependent suppression of the phasic contractions, which was unaffected by apamin but reduced by either PACAP-(6-38) or TTX. Neurally evoked muscular relaxation was deeply antagonised by L-NAME. PACAP-(6-38) induced a reduction of the response to EFS only in the absence L-NAME. In conclusion, our results suggest that PACAP controls smooth muscle contractility, acting directly on the muscle cells through PACAP-27 preferring receptors coupled to apamin-sensitive Ca2+-dependent K+-channels and indirectly through the stimulation of NO production. In turn, NO would stimulate the release of PACAP from inhibitory neurones.

Interaction between cannabinoid CB1 receptors and endogenous ATP in the control of spontaneous mechanical activity in mouse ileum

Background and purpose:  Although it is well accepted that cannabinoids modulate intestinal motility by reducing cholinergic neurotransmission mediated by CB1 receptors, it is not known whether the endocannabinoids are involved in more complex circuits and if they interact with other systems. The aim of the present study was to examine possible interactions between cannabinoid CB1 receptors and purines in the control of spontaneous contractility of longitudinal muscle in mouse ileum.

D1 receptors play a major role in the dopamine modulation of mouse ileum contractility.

Since the role of dopamine in the bowel motility is far from being clear, our aim was to analyse pharmacologically the effects of dopamine on mouse ileum contractility. Contractile activity of mouse ileum was examined in vitro as changes in isometric tension. Dopamine caused a concentration-dependent reduction of the spontaneous contraction amplitude of ileal muscle up to their complete disappearance. SCH-23390, D1 receptor antagonist, which per se increased basal tone and amplitude of spontaneous contractions, antagonized the responses to dopamine, whilst sulpiride or domperidone, D2 receptor antagonists, were without effects. The application of both D1 and D2 antagonists had additive effects. SKF-38393, D1 receptor agonist, mimicked dopamine-induced effects. Dopamine responses were insensitive to tetrodotoxin, atropine, nitric oxide synthase inhibitor or adenosine receptor antagonists, but they were reduced by adenylyl cyclase inhibition or apamin. Dopamine at a concentration which did not cause a significant reduction of phasic contractions inhibited the cholinergic contractions in response to field stimulation. SCH-23390 per se induced an increase of the neural cholinergic contraction and antagonized the dopamine effects, whilst sulpiride or domperidone did not. The application of D1 and D2 antagonists had additive effects. In conclusion, mouse ileum is under basal inhibitory control by dopamine, through D1 receptor activation, linked to adenylyl cyclase and activation of apamin-sensitive potassium channels. An agonistic interaction of the dopamine receptor subtypes in the regulation intestinal contractility has being also highlighted. This study would provide new insight on the pharmacology of the modulation of the gastrointestinal contractility by dopamine.

A1 receptors mediate adenosine inhibitory effects in mouse ileum via activation of potassium channels.

AIMS We investigated the effects induced by exogenous adenosine on the spontaneous contractile activity of the longitudinal muscle of a mouse ileum, the receptor subtypes activated, the involvement of enteric nerves and whether opening of K+ channels was a downstream event leading to the observed effects. MAIN METHODS Mechanical responses of the mouse ileal longitudinal muscle to adenosine were examined in vitro as changes in isometric tension. KEY FINDINGS Adenosine caused a concentration-dependent reduction of the spontaneous contraction amplitude of the ileal longitudinal muscle up to its complete disappearance. This effect induced was markedly reduced by an A1 receptor antagonist, but not by A2 and A3 receptor antagonists and mimicked only by the A1 receptor agonist. Adenosine uptake inhibitors did not change adenosine potency. A1 receptor expression was detected at the smooth muscle level. Adenosine responses were insensitive to tetrodotoxin, atropine or nitric oxide synthase inhibitor. Tetraethylammonium and iberiotoxin, BK(Ca) channel blockers, significantly reduced adenosine effects, whilst 4-aminopyridine, a K(v) blocker, apamin, a small conductance Ca2+-activated K+ (SK(Ca)) channel blocker, charybdotoxin, an intermediate conductance Ca2+-activated K+ (IK(Ca)) and BK(Ca) channel blocker, or glibenclamide, an ATP-sensitive K+ channel blocker, had no effects. The combination of apamin plus iberiotoxin caused a reduction of the purinergic effects greater than iberiotoxin alone. SIGNIFICANCE Adenosine acts as an inhibitory modulator of the contractility of mouse ileal longitudinal muscle through postjunctional A1 receptors, which in turn would induce opening of BK(Ca) and SK(Ca) potassium channels. This study would provide new insight in the pharmacology of purinergic receptors involved in the modulation of the gastrointestinal contractility.

Duodenal contractile activity in dystrophic (mdx) mice: reduction of nitric oxide influence

The present study was undertaken to analyse duodenal contractility in adult dystrophic (mdx) mice. The spontaneous changes of the isometric tension and the responses of longitudinal duodenal muscle to nonadrenergic, noncholinergic (NANC) nerve stimulation and to exogenous drugs were compared between normal and mdx mice. Duodenal segments from mdx mice displayed spontaneous contractions with higher frequency than normals. Nω‐nitro‐l‐arginine methyl ester (l‐NAME) increased the frequency of contractions in normals without affecting that in mdx mice. In normals, NANC nerve stimulation elicited a transient relaxation abolished by l‐NAME. In mdx mice a frank relaxation was not observed, the inhibitory response consisted just in the suppression of the phasic activity. This response was reduced by l‐NAME and abolished by the subsequent addition of α‐chymotrypsin. In normals, α‐chymotrypsin hardly affected NANC relaxation, whilst it significantly antagonised that in mdx mice. Mdx duodenal muscle also showed a reduced responsiveness to sodium nitroprusside, and to 8‐bromoguanosine 3′, 5′‐cyclic monophosphate in comparison with normal preparations. The results indicate that mdx mice experience duodenal contractile disturbances due to an impairment of NO function with defective responsiveness of the muscle to NO. The reduction in NO influence is functionally compensated by the peptidergic system.