Xiaochun Bian

P2X2 subunits contribute to fast synaptic excitation in myenteric neurons of the mouse small intestine

P2X receptors are ATP‐gated cation channels composed of one or more of seven different subunits. ATP acts at P2X receptors to contribute to fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons but the subunit composition of enteric P2X receptors is unknown. These studies used tissues from P2X2 wild‐type (P2X2+/+) and P2X2 gene knockout (P2X2‐/‐) mice to investigate the role of this subunit in enteric neurotransmission. Intracellular electrophysiological methods were used to record synaptic and drug‐induced responses from ileal myenteric neurons in vitro. Drug‐induced longitudinal muscle contractions and peristaltic contractions of ileal segments were also studied in vitro. Gastrointestinal transit was measured as the progression in 30 min of a liquid radioactive marker administered by gavage to fasted mice. RT‐PCR analysis of mRNA from intestinal tissues and data from immunohistochemical studies verified P2X2 gene deletion. The fEPSPs recorded from S neurons in tissues from P2X2+/+ mice were reduced by mecamylamine (nicotinic cholinergic receptor antagonist) and PPADS (P2X receptor antagonist). The fEPSPs recorded from S neurons from P2X2−/− mice were unaffected by PPADS but were blocked by mecamylamine. ATP depolarized S and AH neurons from P2X2+/+ mice. ATP depolarized AH but not S neurons from P2X2‐/‐ mice. α,β‐Methylene ATP (α,β‐mATP)(an agonist at P2X3 subunit‐containing receptors) did not depolarize S neurons but it did depolarize AH neurons in P2X2+/+ and P2X2‐/‐ mice. Peristalsis was inhibited in ileal segments from P2X2‐/‐ mice but longitudinal muscle contractions caused by nicotine and bethanechol were similar in segments from P2X2+/+ and P2X2‐/‐ mice. Gastrointestinal transit was similar in P2X2+/+ and P2X2‐/‐ mice. It is concluded that P2X2 homomeric receptors contribute to fEPSPs in neural pathways underlying peristalsis studied in vitro.

Peristalsis is impaired in the small intestine of mice lacking the P2X3 subunit

P2X receptors are ATP‐gated cation channels composed of one or more of seven different subunits. P2X receptors participate in intestinal neurotransmission but the subunit composition of enteric P2X receptors is unknown. In this study, we used tissues from P2X3 wild‐type (P2X3+/+) mice and mice in which the P2X3 subunit gene had been deleted (P2X3−/−) to investigate the role of this subunit in neurotransmission in the intestine. RT‐PCR analysis of mRNA from intestinal tissues verified P2X3 gene deletion. Intracellular electrophysiological methods were used to record synaptic and drug‐induced responses from myenteric neurons in vitro. Drug‐induced longitudinal muscle contractions were studied in vitro. Intraluminal pressure‐induced reflex contractions (peristalsis) of ileal segments were studied in vitro using a modified Trendelenburg preparation. Gastrointestinal transit was measured as the progression in 30 min of a liquid radioactive marker administered by gavage to fasted mice. Fast excitatory postsynaptic potentials recorded from S neurons (motoneurons and interneurons) were similar in tissues from P2X3+/+ and P2X3−/− mice. S neurons from P2X3+/+ and P2X3−/− mice were depolarized by application of ATP but not α,β‐methylene ATP, an agonist of P2X3 subunit‐containing receptors. ATP and α,β‐methylene ATP induced depolarization of AH (sensory) neurons from P2X3+/+ mice. ATP, but not α,β‐methylene ATP, caused depolarization of AH neurons from P2X3−/− mice. Peristalsis was inhibited in ileal segments from P2X3−/− mice but longitudinal muscle contractions caused by nicotine and bethanechol were similar in segments from P2X3+/+ and P2X3−/− mice. Gastrointestinal transit was similar in P2X3+/+ and P2X3−/− mice. It is concluded that P2X3 subunit‐containing receptors participate in neural pathways underlying peristalsis in the mouse intestine in vitro. P2X3 subunits are localized to AH (sensory) but not S neurons. P2X3 receptors may contribute to detection of distention or intraluminal pressure increases and initiation of reflex contractions.

Descending inhibitory reflexes involve P2X receptor-mediated transmission from interneurons to motor neurons in guinea-pig ileum.

1 The role of P2X receptors in descending inhibitory reflexes evoked by distension or mucosal distortion in the guinea‐pig ileum was studied using intracellular recording from the circular muscle in a two‐chambered organ bath. This allowed separate superfusion of the sites of reflex stimulation and recording, thereby allowing drugs to be selectively applied to different parts of the reflex pathway. 2 Inhibitory junction potentials (IJPs) evoked by electrical field stimulation (EFS) in the recording chamber were compared with those evoked during reflexes to control for effects of P2 receptor antagonists on neuromuscular transmission. 3 The P2 receptor antagonists suramin (100 μm) and pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (10 and 60 μm; PPADS), when added to the recording chamber, depressed reflexly evoked IJPs significantly more than those evoked by EFS. In particular, 10 μm PPADS depressed IJPs evoked by distension or mucosal distortion by about 50 %, but had little effect on IJPs evoked by EFS. 4 Blockade of synaptic transmission in the stimulation chamber with a low Ca2+‐high Mg2+ solution depressed, but did not abolish, IJPs evoked by distension. The residual reflex IJPs were unaffected by PPADS (10 μm), hyoscine (1 μm), hyoscine plus hexamethonium (200 μm), or hysocine plus hexamethonium plus PPADS in the recording chamber. 5 We conclude that P2X receptors are important for synaptic transmission from descending interneurons to inhibitory motor neurons in descending inhibitory reflex pathways of guinea‐pig ileum. Transmission from anally directed axons of distension‐sensitive intrinsic sensory neurons to inhibitory motor neurons is unlikely to involve P2X, muscarinic or nicotinic receptors.

In vitro continuous amperometric monitoring of 5-hydroxytryptamine release from enterochromaffin cells of the guinea pig ileum

A diamond microelectrode was used to sensitively, reproducibly and stably record overflow of 5-hydroxytryptamine (5-HT, serotonin) from enterochromaffin cells (EC) of the intenstinal mucosal layer. 5-HT is an important neurotransmitter and paracrine signalling molecule in the gastrointestinal tract. The diamond microelectrode was formed by overcoating a sharpened 76 microm diameter Pt wire with a thin layer of conducting diamond. After insulation with polypropylene, the conically-shaped microelectrode had a diameter of about 10 microm at the tip and 80 microm at the cylindrical portion. The exposed length was 100-200 microm. Continuous amperometry with the microelectrode poised at a detection potential of 700 mV vs. Ag|AgCl was used to measure 5-HT overflow as an oxidation current. 5-HT overflow was elicited by both mechanical and electrical stimulation. Some minor electrode fouling, a common problem with the oxidative detection of 5-HT, was seen for diamond but the response stabilized enabling recording in vitro. Both 5-HT and the paracrine hormone, melatonin, were detected in the extracellular solution. The 5-HT oxidation current increased in the presence of the serotonin transporter (SERT) inhibitor, fluoxetine (1 microM), providing evidence that the oxidation current was associated with 5-HT.

Diamond microelectrodes for in vitro electroanalytical measurements: current status and remaining challenges.

An emerging research field in electrochemistry today is the preparation, characterization and application of diamond microelectrodes for electroanalytical measurements in biological media. Interest in this new electrode material stems from its outstanding properties: (i) hardness, (ii) low, stable and pH-independent background current, (iii) morphological and microstructural stability over a wide range of potentials, (iv) good electrochemical responsiveness for multiple redox analytes without any conventional pre-treatment and (v) weak molecular adsorption of polar molecules that leads to a high level of resistance to response deactivation and electrode fouling. Diamond electrodes have advanced in recent years from being simply a scientific curiosity into a viable material for electroanalysis. In this article, we highlight the current state of progress by our laboratory and others on the preparation, study of the basic electrochemical properties, and application of this new type of microelectrode for in vitro electroanalytical measurements, and discuss some of the remaining challenges.

Activation of Vascular BK Channel by Tempol in DOCA-Salt Hypertensive Rats

Large-conductance Ca2+-activated potassium (BK) channels modulate vascular smooth muscle tone. Tempol, a superoxide dismutase (SOD) mimetic, lowers blood pressure and inhibits sympathetic nerve activity in normotensive and hypertensive rats. In the present study, we tested the hypotheses depressor responses caused by tempol are partly mediated by vasodilation. It was found that tempol, but not tiron (a superoxide scavenger), dose-dependently relaxed mesenteric arteries (MA) in anesthetized sham and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Tempol also reduced perfusion pressure in isolated, norepinephrine (NE) preconstricted MA from sham and DOCA-salt hypertensive rats. Maximal responses in DOCA-salt rats were twice as large as those in sham rats. The vasodilation caused by tempol was blocked by iberiotoxin (IBTX, BK channel antagonist, 0.1 &mgr;mol/L) and tetraethylammonium chloride (TEA) (1 mmol/L). Tempol did not relax KCl preconstricted arteries in sham or DOCA-salt rats, and N&ohgr;-nitro-l-arginine methyl ester (l-NAME), apamin, or glibenclamide did not alter tempol-induced vasodilation. IBTX constricted MA and this response was larger in DOCA-salt compared with sham rats. Western blots and immunohistochemical analysis revealed increased expression of BK channel &agr; subunit protein in DOCA-salt arteries compared with sham arteries. Whole-cell patch clamp studies revealed that tempol enhanced BK channel currents in HEK-293 cells transiently transfected with mslo, the murine BK channel a subunit. These currents were blocked by IBTX. The data indicate that tempol activates BK channels and this effect contributes to depressor responses caused by tempol. Upregulation of the BK channel &agr; subunit contributes to the enhanced depressor response caused by tempol in DOCA-salt hypertension.

Nicotinic transmission at functionally distinct synapses in descending reflex pathways of the rat colon.

Abstract  We examined descending reflex pathways in the rat colon using intracellular recording techniques. Inhibitory junction potentials (IJPs) were recorded from circular smooth muscle when descending pathways were excited by combined mucosal compression and distension. IJPs were reduced to 71% of control when synaptic transmission was blocked in the oral stimulation chamber of a divided organ bath suggesting that two reflex pathways exist, the one involving descending sensory neurones and the other involving descending interneurones. Hexamethonium (200 μmol L−1) in the recording chamber abolished reflexly evoked IJPs, while in the stimulation chamber, it was as effective as synaptic blockade. When hexamethonium was added to a chamber lying between the stimulation and recording chambers, it again sharply depressed IJPs to 27% of control; an extent similar to synaptic blockade. A P2 receptor antagonist did not reveal any purinergic neurotransmission. Either granisetron (5‐HT3 receptor antagonist, 1 μmol L−1) or SB204070 (5‐HT4 receptor antagonist, 1 μmol L−1) in the stimulation chamber significantly decreased IJPs; these decreases were not additive. We conclude that some sensory neurones and interneurones in rat colon have long anally projecting axons and that acetylcholine, acting via nicotinic receptors, is the primary neurotransmitter from sensory neurones, to inhibitory motor neurones and between interneurones.

Electrochemical monitoring of nitric oxide released by myenteric neurons of the guinea pig ileum

Abstract  Nitric oxide (NO) released by myenteric neurons in isolated segments of guinea pig ileum was monitored in vitro using continuous amperometry. NO was detected as an oxidation current recorded with a boron‐doped diamond microelectrode held at 1 V vs a Ag|AgCl reference electrode. This potential was sufficient to oxidize NO. Longitudinal muscle‐myenteric plexus (LMMP) and circular muscle strip preparations were used. In the LMMP preparation, NO release was evoked by superfusion of 1 μmol L−1 nicotine, which activates nicotinic acetylcholine receptors expressed by myenteric neurons and myenteric nerve endings. The oxidation current was ascribed to NO based on the following observations: (i) no response was detected at less positive potentials (0.75 V) at which only catecholamines and biogenic amines are oxidized, (ii) the current was abolished in the presence of the nitric oxide synthase antagonist, N‐nitro‐l‐arginine (l‐NNA) and (iii) oxidation currents were attenuated by addition of the NO scavenger, myoglobin, to the superfusing solution. In the LMMP preparation, stimulated release produced a maximum current that corresponded nominally to 46 nmol L−1 of NO. The oxidation currents decreased to 10 and 2 nmol L−1, respectively, when the tissue was perfused with tetrodotoxin and l‐NNA. Oxidation currents recorded from circular muscle strips (stimulated using nicotine) were threefold larger than those recorded from the LMMP. This study shows that NO release can be detected from various in vitro preparations of the guinea pig ileum using real‐time electroanalytical techniques.

Inhibitory neuromuscular transmission to ileal longitudinal muscle predominates in neonatal guinea pigs

Background  Inhibitory neurotransmission to the longitudinal muscle is more prominent in the neonatal than in the adult guinea pig ileum.

Postnatal downregulation of inhibitory neuromuscular transmission to the longitudinal muscle of the guinea pig ileum

Abstract  Neuromuscular transmission is crucial for normal gut motility but little is known about its postnatal maturation. This study investigated excitatory/inhibitory neuromuscular transmission in vitro using ileal nerve‐muscle preparations made from neonatal (≤48 h postnatal) and adult (∼4 months postnatal) guinea pigs. In tissues from neonates and adults, nicotine (0.3–30 μmol L−1) contracted longitudinal muscle preparations in a tetrodotoxin (TTX) (0.3 μmol L−1)‐sensitive manner. The muscarinic receptor antagonist, scopolamine (1 μmol L−1), reduced substantially nicotine‐induced contractions in neonatal tissues but not adult tissues. In the presence of Nω‐nitro‐l‐arginine (NLA, 100 μmol L−1) to block nitric oxide (NO) mediated inhibitory neuromuscular transmission, scopolamine‐resistant nicotine‐induced contractions were revealed in neonatal tissues. NLA enhanced the nicotine‐induced contractions in neonatal but not in adult tissues. Electrical field stimulation (20 V; 0.3 ms; 5–25 Hz, scopolamine 1 μmol L−1 present) caused NLA and TTX‐sensitive longitudinal muscle relaxations. Frequency–response curves in neonatal tissues were left‐shifted compared with those obtained in adult tissues. Immunohistochemical studies revealed that NO synthase (NOS)‐immunoreactivity (ir) was present in nerve fibres supplying the longitudinal muscle in neonatal and adult tissues. However, quantitative studies demonstrated that fluorescence intensity of NOS‐ir nerve fibres was higher in neonatal than adult tissues. Nerve fibres containing substance P were abundant in longitudinal muscle in adult but not in neonatal tissues. Inhibitory neuromuscular transmission is relatively more effective in the neonatal guinea pig small intestine. Delayed maturation of excitatory motor pathways might contribute to paediatric motility disturbances.