Inhibitory purinergic transmission in mouse caecum: role for P2Y1 receptors as prejunctional modulators of ATP release.

Using conventional microelectrode recording techniques, we investigated, in the circular muscle of the mouse caecum, the neurotransmitter(s) involved in the neurally-evoked inhibitory junction potentials (IJPs) and the existence of possible prejunctional mechanisms controlling neurotransmitter release. Electrical field stimulation with single pulses elicited IJPs, consisting only of a "fast" hyperpolarization, while using train stimuli (30-50 Hz) the initial fast hyperpolarization was followed by a slower hyperpolarization. The fast and the slow component were selectively antagonized by apamin, a blocker of calcium-activated potassium channels, and N(omega)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, respectively. Fast IJPs were antagonized also by P2 purinoceptor antagonists, suramin or 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid tetrasodium salt (PPADS), P2Y purinoceptor desensitization by adenosine 5'-O-2-thiodiphosphate (ADPbetaS). 2'-Deoxy-N(6)-methyl ADP diammonium salt (MRS 2179), P2Y1 purinoceptor antagonist, at the concentration of 1 microM increased the amplitude of the fast IJP, while at the concentration of 10 microM induced a reduction. 8,8'-[Carbonylbis[imino-3,1-phenylenecarbonylimino (4-fluoro-3,1-phenylene) carbonylimino]] bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF 157) and 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyl-oxymethyl)-propyl ester (MRS 2395), P2Y11 and P2Y12 purinoceptor antagonist, were without any effect. ATP-induced hyperpolarization was affected by apamin and by P2Y purinoceptor desensitization, but not by MRS 2179. 2-(Methylthio)ATP tetrasodium salt hydrate (2-MeSATP), P2Y1 purinoceptor agonist, at a concentration which did not cause changes in the membrane potential, reduced the amplitude of the fast IJPs. This effect was prevented by MRS 2179. Paired nerve stimulation, either using single pulses or train stimuli, did not cause any alteration of the second-evoked IJP. In conclusion, in the circular muscle of the mouse caecum, ATP is responsible for the fast IJP while nitric oxide is responsible for the slow IJP. ATP-mediated response is dependent on ADPbetaS-sensitive P2Y receptors, which are in part P2Y1, but not P2Y11 or P2Y12 receptor subtypes. In addition, the most substantial finding of this study is the functional demonstration that ATP released by nerve stimulation activates P2Y1 receptors, located prejunctionally, limiting its release by motoneurons.

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

BACKGROUND AND PURPOSE: This study analysed the contribution of the purinergic system to enteric neurotransmission in the longitudinal muscle of mouse distal colon. EXPERIMENTAL APPROACH: Motor responses to exogenous ATP and to nerve stimulation in vitro were assessed as changes in isometric tension. KEY RESULTS: ATP induced a concentration-dependent contraction, reduced by 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzene disulphonic acid (PPADS), suramin, P2Y purinoreceptor desensitisation with adenosine 5'-O-2-thiodiphosphate (ADPbetaS), and atropine, but unaffected by P2X purinoceptor desensitisation with alpha,beta-methylene ATP (alpha,beta-meATP) and by 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl ester (MRS 2395), a P2Y(12) selective antagonist. The response to ATP was increased by 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS 2179), a P2Y(1) selective antagonist, tetrodotoxin (TTX) or N(omega)-nitro-L-arginine methyl ester (L-NAME). ADPbetaS, a P2Y-purinergic agonist, induced muscular contraction, with the same pharmacological profile as the ATP-induced contraction. ADP, a natural ligand for P2Y(1) receptors, induced muscular relaxation, antagonized by MRS 2179 and by TTX or L-NAME. Nerve stimulation elicited a transient nitrergic relaxation, followed by contraction. Contractile responses was reduced by atropine, PPADS, suramin, P2Y purinoceptor desensitisation, but not by P2X purinoceptor desensitisation, MRS 2179 or MRS 2395. None of the purinergic antagonists modified the nerve-evoked relaxation. CONCLUSIONS AND IMPLICATIONS: In the longitudinal muscle of mouse distal colon, ATP, through ADPbetaS-sensitive P2Y purinoceptors, contributed to the excitatory neurotransmission acting directly on smooth muscle and indirectly via activation of cholinergic neurons. Moreover, P2Y1 purinoceptors appear to be located on nitrergic inhibitory neurons. This study provides new insights into the role of purines in the mechanism inducing intestinal transit in mouse colon.

Angiotensin II type II receptors and colonic dysmotility in 2,4-dinitrofluorobenzenesulfonic acid-induced colitis in rats.

BACKGROUND: Angiotensin II (Ang II), the main peptide of the renin-angiotensin system (RAS), has been suggested to be involved in inflammatory bowel diseases. Since RAS has emerged as gut motility regulator, and dysmotility is associated with intestinal inflammation, our objective was to investigate in rat 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis the functionality of RAS and its contribution to colonic motor alterations. METHODS: The effects of Ang II on the longitudinal colonic muscular contractility of control and DNBS-treated rats were characterized in vitro. Transcripts encoding for Ang II receptors were investigated by RT-PCR. KEY RESULTS: Inflamed preparations showed a longitudinal muscle marked hypocontractility. Angiotensin II caused contractile effects in both preparations, but the responses in DNBS preparations were reduced compared to controls. In both preparations, Losartan, AT1 receptor antagonist, reduced Ang II effects. PD123319, AT2 receptor antagonist, enhanced Ang II responses only in DNBS rats, as well as Nω -Nitro-L-arginine (L-NNA), nitric oxide (NO) synthase inhibitor, or tetrodotoxin (TTX), neural toxin. The co-administration of PD123319 and TTX or L-NNA produced no additive effects. PD123319 per se improved colonic contractility in inflamed tissues. The effect was reduced in the presence of L-NNA or TTX. All Ang II receptor subtypes were expressed in both preparations. CONCLUSIONS & INFERENCES: AT1 receptors mediate Ang II contractile responses in rat colon. During inflammation a recruitment of Ang II AT2 receptors would counteract AT1 -contractile activity. A tonic activation of AT2 receptors would contribute to the general reduction in muscle contractility during experimental inflammation. A role for enteric neurons and NO is also suggested.

Evidence for a role of inducible nitric oxide synthase in gastric relaxation of mdx mice.

Alterations of gastric mechanical activity have been reported in mdx mouse, animal model for Duchenne muscular dystrophy. This study examined if alterations in the vasoactive intestinal polypeptide (VIP) system are present in mdx stomach. Gastric mechanical activity was recorded in vitro as changes of endoluminal pressure and neurally or pharmacologically evoked relaxations were analysed in mdxvs normal stomach. Reverse-transcription polymerase chain reaction was used to detect inducible nitric oxide synthase (iNOS) expression. Relaxations to sodium nitroprusside in mdx stomach showed no difference in comparison with normal preparations. In normal stomach, VIP produced relaxation, which was reduced by VIP6-28, antagonist of VIP receptors, but was not modified by Nomega-nitro-L-arginine methyl ester (L-NAME), 1-H-oxodiazol-[1,2,4]-[4,3-a]quinoxaline-1-one (ODQ) or by N-(3-(aminomethyl)-benzyl)acetamidine (1400W) and aminoguanidine, inhibitors of iNOS. In contrast, in mdx stomach VIP responses were antagonized not only by VIP6-28, but also by L-NAME, ODQ, 1400W or aminoguanidine. In normal stomach, the slow relaxation evoked by stimulation at high frequency was reduced by VIP6-28, but it was unaffected by 1400W or aminoguanidine. In mdx stomach, it was reduced by VIP6-28 or 1400W, which did not show additive effects. iNOS mRNA was expressed only in mdx stomach. The results suggest that in mdx gastric preparations, iNOS is functionally expressed, being involved in the slow relaxation induced by VIP.

Activation of angiotensin II type 1 receptors and contractile activity in human sigmoid colon in vitro.

AIM: To analyse the effects of angiotensin II (Ang II) on the contractility of human sigmoid colon, and to characterize the subtype(s) of receptor(s) involved and the related action mechanism. METHODS: The contractility of sigmoid colon circular muscle strips was recorded isometrically. RT-PCR and immunohistochemistry were used to reveal the eventual existence of a local renin-angiotensin system (RAS) and the distribution of Ang II receptors. RESULTS: Transcripts encoding for the Ang II type 1 (AT1 ) and the Ang II type 2 (AT2 ) receptor subtypes and for the angiotensin-converting enzyme in the whole-thickness muscular wall were observed. Ang II caused a concentration-dependent contractile response, which is antagonized by losartan, AT1 receptor antagonist, but not by PD123319, AT2 receptor antagonist. The joint application of losartan and PD123319 did not produce any additive effect. The contractile response to Ang II was partially reduced by tetrodotoxin, Na(+) voltage-gated neural channel blocker, and to some extent by SR48968, tachykinin NK2 receptor antagonist. However, hexamethonium, nicotinic receptor antagonist, atropine, cholinergic muscarinic receptor antagonist and SR140333, tachykinin NK1 receptor antagonist, were ineffective. Immunohistochemical analysis showed that AT1 receptors were expressed on the smooth muscle layers and myenteric plexus. CONCLUSION: Ang II positively modulates the spontaneous contractile activity of human sigmoid colon via activation of post-junctional and pre-junctional AT1 receptors, the latter located on the enteric nerves that modulate the release of tachykinins. The presence of the components of RAS in the human colon suggests that Ang II can be also locally generated to control colonic motility.

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. N omega-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 alpha-chymotrypsin. In normals, alpha-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.

Prevalence of asthma and respiratory symptoms in childhood in an urban area of north-east Italy.

In the present study we have assessed the prevalence of asthma-related symptoms in school children resident in the urban area of Verona, in the north east of Italy, as part of the International Study of Asthma and Allergies in Childhood (ISAAC). The entirely of the population of children aged 6-7 yrs (total 2,350, from 64 schools) and 13-14 years (total 2,500 from 42 schools) living in the area was selected. A questionnaire was distributed at school. This was addressed to the parents of the younger population and distributed directly to the older children. In the two groups 2,091 (89%) and 2,179 (87%) questionnaires were returned, respectively. The results obtained show a more frequent history of "wheeling ever" in the young population compared to the older group (23.2 versus 7.9%, respectively) (p < 0.001). There was no difference in the prevalence of wheezing in the last 12 months (7.3 and 7.4%, respectively), and of asthma (4.6 and 3.5%, respectively). In the older group there was a higher incidence of exercise-induced wheezing (12.0 versus 3.2%), which was particularly significant in the females compared to males (14.2 versus 10.0, respectively; p < 0.01). Our data confirm that there is a marked variation in the prevalence of asthma and asthma-related respiratory symptoms in different centres throughout the world, since we obtained a lower percentage of reported symptoms in comparison to other ISAAC centres.

Angiotensin II contractile effects in mouse colon: role for pre- and post-junctional AT(1A) receptors.

AIM: This study investigates whether a local renin-angiotensin system (RAS) exists in mouse colon and whether angiotensin II (Ang II) may play a role in the regulation of the contractile activity. METHODS: Isometric recordings were performed in vitro on the longitudinal muscle of mouse proximal and distal colon. Transcripts encoding for RAS components were investigated by RT-PCR. RESULTS: Ang II caused, in both preparations, a concentration-dependent contractile effect, antagonized by losartan, AT(1) receptor antagonist, but not by PD123319, AT(2) receptor antagonist. The combination of losartan plus PD123319 caused no change on the Ang II-induced contraction than losartan alone. Tetrodotoxin, neural blocker, reduced the contractile response to Ang II in the proximal colon, whilst the response was abolished in the distal colon. In both preparations, atropine, muscarinic receptor antagonist, or SR140333, NK(1) receptor antagonist, reduced the Ang II responses. Ondansetron, 5-HT(3) receptor antagonist, SR48968, NK(2) receptor antagonist, or hexamethonium, nicotinic receptor antagonist, were ineffective. The joint application of atropine and SR140333 produced no additive effect. Atropine reduced NK(1) -induced contraction. Transcripts encoding RAS components were detected in the colon samples. However, just AT(1A) mRNA was expressed in both preparations, and AT(2) mRNA was expressed only in the distal colon. CONCLUSION: In the murine colon, local RAS may play a significant role in the control of contractile activity. Ang II positively modulates the spontaneous contractile activity via activation of post-junctional and pre-junctional AT(1A) receptors, the latter located on the enteric neurones, modulating the release of tachykinins and acetylcholine.

Adenosine negatively regulates duodenal motility in mice: role of A(1) and A(2A) receptors.

BACKGROUND AND PURPOSE: Adenosine is considered to be an important modulator of intestinal motility. This study was undertaken to investigate the role of adenosine in the modulation of contractility in the mouse duodenum and to characterize the adenosine receptor subtypes involved. EXPERIMENTAL APPROACH: RT-PCR was used to investigate the expression of mRNA encoding for A(1), A(2A), A(2B) and A(3) receptors. Contractile activity was examined in vitro as changes in isometric tension. KEY RESULTS: In mouse duodenum, all four classes of adenosine receptors were expressed, with the A(2B) receptor subtype being confined to the mucosal layer. Adenosine caused relaxation of mouse longitudinal duodenal muscle; this was antagonized by the A(1) receptor antagonist and mimicked by N(6) -cyclopentyladenosine (CPA), selective A(1) agonist. The relaxation induced by A(1) receptor activation was insensitive to tetrodotoxin (TTX) or N(ω) -nitro-l-arginine methyl ester (l-NAME). Adenosine also inhibited cholinergic contractions evoked by neural stimulation, effect reversed by the A(1) receptor antagonist, but not myogenic contractions induced by carbachol. CPA and 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680), A(2A) receptor agonist, both inhibited the nerve-evoked cholinergic contractions. l-NAME prevented only the CGS-21680-induced effects. S-(4-Nitrobenzyl)-6-thioinosine, a nucleoside uptake inhibitor, reduced the amplitude of nerve-evoked cholinergic contractions, an effect reversed by an A(2A) receptor antagonist or l-NAME. CONCLUSIONS AND IMPLICATIONS: Adenosine can negatively regulate mouse duodenal motility either by activating A(1) inhibitory receptors located post-junctionally or controlling neurotransmitter release via A(1) or A(2A) receptors. Both receptors are available for pharmacological recruitment, even if only A(2A) receptors appear to be preferentially stimulated by endogenous adenosine.

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 CB(1) 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 CB(1) receptors and purines in the control of spontaneous contractility of longitudinal muscle in mouse ileum. EXPERIMENTAL APPROACH: The mechanical activity of longitudinally oriented ileal segments from mice was recorded as isometric contractions. KEY RESULTS: The selective CB(1) receptor agonist, N-(2-chloroethyl)5,8,11,14-eicosaetraenamide (ACEA) reduced, concentration dependently, spontaneous contractions in mouse ileum. This effect was almost abolished by tetrodotoxin (TTX) or atropine. Inhibition by ACEA was not affected by theophylline (P1 receptor antagonist) or by P2Y receptor desensitization with adenosine 5'[beta-thio]diphosphate trilithium salt, but was significantly reversed by pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (P2 receptor antagonist), by P2X receptor desensitization with alpha,beta-methyleneadenosine 5'-triphosphate lithium salt (alpha,beta-MeATP) or by 8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino) bis(1,3,5-naphthalenetrisulphonic acid)] (P2X receptor antagonist). Contractile responses to alpha,beta-MeATP (P2X receptor agonist) were virtually abolished by TTX or atropine, suggesting that they were mediated by acetylcholine released from neurones, and significantly reduced by ACEA. CONCLUSION AND IMPLICATIONS: In mouse ileum, activation of CB(1) receptors, apart from reducing acetylcholine release from cholinergic nerves, was able to modulate negatively, endogenous purinergic effects, mediated by P2X receptors, on cholinergic neurons. Our study provides evidence for a role of cannabinoids in the modulation of interneuronal purinergic transmission.