Postnatal development of the dopaminergic signaling involved in the modulation of intestinal motility in mice.

BACKGROUND: Since antidopaminergic drugs are pharmacological agents employed in the management of gastrointestinal motor disorders at all ages, we investigated whether the enteric dopaminergic system may undergo developmental changes after birth. METHODS: Intestinal mechanical activity was examined in vitro as changes in isometric tension. RESULTS: In 2-d-old (P2) mice, dopamine induced a contractile effect, decreasing in intensity with age, replaced, at the weaning (day 20), by a relaxant response. Both responses were tetrodotoxin (TTX)-insensitive. In P2, dopaminergic contraction was inhibited by D1-like receptor antagonist and mimicked by D1-like receptor agonist. In 90-d-old (P90) mice, the relaxation was reduced by both D1- and D2-like receptor antagonists, and mimicked by D1- and D2-like receptor agonists. In P2, contraction was antagonized by phospholipase C inhibitor, while in P90 relaxation was antagonized by adenylyl cyclase inhibitor and potentiated by phospholipase C inhibitor. The presence of dopamine receptors was assessed by immunofluorescence. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed a significant increase in D1, D2, and D3 receptor expression in proximal intestine with the age. CONCLUSION: In mouse small intestine, the response to dopamine undergoes developmental changes shifting from contraction to relaxation at weaning, as the consequence of D2-like receptor recruitment and increased expression of D1 receptors.

Opposite role played by GABAA and GABAB receptors in the modulation of peristaltic activity in mouse distal colon.

We investigated the role of GABA on intestinal motility using as model the murine distal colon. Effects induced by GABA receptors recruitment were examined in whole colonic segments and isolated circular muscle preparations to analyze their influence on peristaltic reflex and on spontaneous and neurally-evoked contractions. Using a modified Trendelenburg set-up, rhythmic peristaltic contractions were evoked by gradual distension of the colonic segments. Spontaneous and neurally-evoked mechanical activity of circular muscle strips were recorded in vitro as changes in isometric tension. GABA, at low concentrations (10-50 µM), potentiated peristaltic activity and the neural cholinergic contractions, whilst it, at higher concentrations (500 µM-1mM), had inhibitory effects. GABA excitatory effects were mimicked by muscimol, GABAA-receptor agonist, and prevented by bicuculline, GABAA-receptor antagonist, which per se reduced peristaltic activity and the cholinergic contractile responses. Inhibitory effects were mimicked by baclofen, GABAB-receptor agonist, and antagonized by phaclofen, GABAB-receptor antagonist and by hexamethonium, neural nicotinic receptor antagonist. Guanethidine was ineffective on GABA effects. Non-cholinergic responses were not affected by GABA agents. All drugs failed to affect the response to carbachol. Lastly, GABAC receptor agonist/antagonist had any effect on colonic motility. In conclusion, GABA in mouse distal colon is a modulator of peristaltic activity via the regulation of acetylcholine release from cholinergic neurons through interaction with GABAA or GABAB receptors. GABAA receptors are recruited at low GABA concentrations, increasing acetylcholine release and propulsive activity. At high GABA concentrations the activation of GABAB receptors overrides GABAA receptor effects, decreasing acetylcholine release and peristaltic activity.

Arginine vasopressin, via activation of post-junctional V1 receptors, induces contractile effects in mouse distal colon.

The aim of this study was to analyze whether arginine vasopressin (AVP) may be considered a modulator of intestinal motility. In this view, we evaluated, in vitro, the effects induced by exogenous administration of AVP on the contractility of mouse distal colon, the subtype(s) of receptor(s) activated and the action mechanism. Isometric recordings were performed on longitudinal and circular muscle strips of mouse distal colon. AVP (0.001 nM-100 nM) caused concentration-dependent contractile effects only on the longitudinal muscle, antagonized by the V1 receptor antagonist, V-1880. AVP-induced effect was not modified by tetrodotoxin, atropine and indomethacin. Contractile response to AVP was reduced in Ca(2+)-free solution or in the presence of nifedipine, and it was abolished by depletion of calcium intracellular stores after repetitive addition of carbachol in calcium-free medium with addition of cyclopiazonic acid. U-73122, an inhibitor of the phospholipase C, effectively antagonized AVP effects, whilst it was not affected by an adenylyl cyclase inhibitor. Oxytocin induced an excitatory effect in the longitudinal muscle of distal colon at very high concentrations, effect antagonized by V-1880. The results of this study shown that AVP, via activation of V1 receptors, is able to modulate positively contractile activity of longitudinal muscle of mouse distal colon, independently by enteric nerve activation and prostaglandin synthesis. Contractile response is achieved by increase in cytoplasmatic Ca(2+) concentration via extracellular Ca(2+) influx from L-type Ca(2+) channels and via Ca(2+) release from intracellular stores through phospholipase C pathway. No modulation has been observed on the contractility of the circular muscle.

Pharmacological characterization of uracil nucleotide-preferring P2Y receptors modulating intestinal motility: a study on mouse ileum.

We investigated the possible modulation of the intestinal contractility by uracil nucleotides (UTP and UDP), using as model the murine small intestine. Contractile activity of a mouse ileum longitudinal muscle was examined in vitro as changes in isometric tension. Transcripts encoding for uracil-sensitive receptors was investigated by RT-PCR. UDP induced muscular contractions, sensitive to PPADS, suramin, or MRS 2578, P2Y(6) receptor antagonist, and mimicked by PSB 0474, P2Y(6)-receptor agonist. UTP induced biphasic effects characterized by an early inhibition of the spontaneous contractile activity followed by muscular contraction. UTP excitatory effects were antagonized by PPADS, suramin, but not by MRS 2578, whilst the inhibitory effects were antagonized by PPADS but not by suramin or MRS 2578. UTPγS, P2Y(2)/(4) receptor agonist but not 2-thio-UTP, P2Y(2) receptor agonist, mimicked UTP effects. The inhibitory effects induced by UTP was abolished by ATP desensitization and increased by extracellular acidification. UDP or UTP responses were insensitive to TTX, atropine, or L-NAME antagonized by U-73122, inhibitor of phospholipase C (PLC) and preserved in the presence of nifedipine or low Ca(2+) solution. Transcripts encoding the uracil nucleotide-preferring receptors were expressed in mouse ileum. Functional postjunctional uracil-sensitive receptors are present in the longitudinal muscle of the mouse ileum. Activation of P2Y(6) receptors induces muscular contraction, whilst activation of P2Y(4) receptors leads to inhibition of the contractile activity. Indeed, the presence of atypical UTP-sensitive receptors leading to muscular contraction is suggested. All uracil-sensitive receptors are linked to the PLC pathway.

Can guanine-based purines be considered modulators of intestinal motility in rodents?

Adenine-based purines play a pivotal role in the control of gastrointestinal motility in rodents. Recently, guanine-based purines have been also shown to exert extracellular effects in the central nervous system raising the possibility of the existence of distinct receptors for guanine-based purines. Thus, it seems likely to speculate that also guanine-based purines may play a role in the modulation of the intestinal contractility. Spontaneous and neurally-evoked mechanical activity was recorded in vitro as changes in isometric tension in circular muscle strips from mouse distal colon. Guanosine up to 3mM or guanine up to 1mM failed to affect the spontaneous mechanical activity, but reduced the amplitude of the electrical field stimulation (EFS)-induced cholinergic contractions, without affecting the early nitrergic relaxation. Both compounds failed to affect the direct contractile responses evoked by carbachol. No desensitization of the response was observed. Guanine-based purine effects were not altered by theophylline, P1 purinoceptor antagonist, by PPADS or suramin, P2 purinoceptor antagonists, by ODQ, guanilyl cyclase inhibitor, or by DDA, adenylyl cyclase inhibitor. Nucleoside uptake inhibitors, dipyridamole or 6-[(4-Nitrobenzyl)thio]-9-ß-D-ribofuranosylpurine (NBTI), antagonized the inhibitory effects induced by guanosine without interfering with guanine. On the contrary, adenine, a competitive inhibitor of nucleobase uptake, antagonized guanine-induced effects. In conclusion, our data indicate that guanosine and guanine are able to modulate negatively the excitatory cholinergic neurotransmission in the circular muscle layer of mouse colon. Guanine-based purines appear to interfere with prejunctional acethylcoline release. Their effects are dependent by their cellular uptake, and independent by adenine-based purine receptors.

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.