Inhibitory Neuromuscular Transmission Research Articles

Nitrergic neuro-muscular transmission is up-regulated in patients with diverticulosis.

Neuro-transmission impairment could be associated to motility changes observed in patients with diverticular disease. Therefore, the objective was to characterize the inhibitory neuro-muscular transmission and gene expression changes of the enteric inhibitory pathways in patients with diverticulosis (DS). Circular muscle strips from sigmoid colon of patients with DS and controls were studied using the organ bath technique to evaluate spontaneous contractility and enteric motor neurons stimulated by electrical field and qRT-PCR to assess the expression of nNOS, iNOS, P2Y1 R and PGP9.5. Patients with DS presented decreased spontaneous rhythmic contractions (SRC) that were significantly enhanced after incubation with L-NNA (1mM) and TTX (1μM), and unaffected by the P2Y1 antagonist MRS2500 (1μM). Stimulation on enteric motor neurons caused an increased duration of the latency of OFF-contractions in DS group (p<0.001), antagonized by L-NNA and slightly affected by MRS2500 (1μM). No differences in the IC50 between controls and DS patients were observed on inhibition of SRC for the NO-donor sodium nitroprusside (SNP) and the preferential P2Y agonist ADPβS. Moreover, nNOS relative expression was also up-regulated 2.3-fold in the DS group (p<0.05) whereas there was no significant difference in relative expression of iNOS, P2Y1 R and the neuronal marker PGP9.5 between groups. Patients with DS presented an over-expression of nNOS with increased endogenously NO-mediated responses suggesting enhanced NO-release. Up-regulation in the nitrergic pathway in early stages of the disease might play a role in colonic motor disorders associated to diverticular disease.

Nitrergic and purinergic mechanisms evoke inhibitory neuromuscular transmission in the human small intestine

Inhibitory neuromuscular transmission in the human colon is due to nitrergic and purinergic (P2Y1 -mediated) inputs. The aim of this study was to determine the mechanisms of neuromuscular transmission in different regions of the human small intestine. Ileal (n = 6) and jejunal (n = 6) samples underwent histological examination and were studied using sharp microelectrodes in smooth muscle cells and conventional muscle bath techniques. Electrical field stimulation (EFS) was used to stimulate inhibitory neurons. No histological abnormalities were found. Resting membrane potential was -39.7 ± 1.5 and -45.5 ± 2.1 mV in the jejunum and ileum, respectively. Slow waves and spontaneous contractions were recorded at a frequency of about 8-9 and 6-7 cpm in the jejunum and ileum, respectively. In non-adrenergic, non-cholinergic conditions, EFS caused an inhibitory junction potential and mechanical relaxation. Both responses were blocked by tissue incubation with the nitric oxide synthase inhibitor (Nω-nitro-l-arginine 1 mM) and the P2Y1 receptor blocker 2'-deoxy-N(6) -methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS2179; 10 μM). Both exogenous addition of sodium nitroprusside (1 μM) and the preferential P2Y1 receptor agonist ADPβS (1 μM) hyperpolarized and relaxed smooth muscle cells. MRS2179 (10 μM) blocked ADPβS-induced responses. Similar to colon, inhibitory neurotransmission in the human small intestine is mainly mediated by purinergic (via P2Y1 receptors) and nitrergic inhibitory neurotransmission. Similar mechanisms of inhibitory neurotransmission are present in different regions of the human intestine.

Purinergic signalling in the gastrointestinal tract and related organs in health and disease

Purinergic signalling plays major roles in the physiology and pathophysiology of digestive organs. Adenosine 5'-triphosphate (ATP), together with nitric oxide and vasoactive intestinal peptide, is a cotransmitter in non-adrenergic, non-cholinergic inhibitory neuromuscular transmission. P2X and P2Y receptors are widely expressed in myenteric and submucous enteric plexuses and participate in sympathetic transmission and neuromodulation involved in enteric reflex activities, as well as influencing gastric and intestinal epithelial secretion and vascular activities. Involvement of purinergic signalling has been identified in a variety of diseases, including inflammatory bowel disease, ischaemia, diabetes and cancer. Purinergic mechanosensory transduction forms the basis of enteric nociception, where ATP released from mucosal epithelial cells by distension activates nociceptive subepithelial primary afferent sensory fibres expressing P2X3 receptors to send messages to the pain centres in the central nervous system via interneurons in the spinal cord. Purinergic signalling is also involved in salivary gland and bile duct secretion.

Comparison of the immunogenicity of botulinum toxin type A and the efficacy of A1 and A2 neurotoxins in animals with A1 toxin antibodies

One issue with botulinum toxin type A products is a reduced therapeutic response in patients that have been injected with frequent dosing over a prolonged period. A possible cause of this is hemagglutinin, found in progenitor toxins, displaying adjuvant activity, enhancing antibody production against the toxin. We investigated whether there is any difference in immunogenicity between the LL toxin-derived subtype A1 (A1LL) and the neurotoxin-derived subtypes A1 and A2 (A1NTX and A2NTX, respectively), and investigated whether A2NTX is effective in animals which produce antibodies against A1LL.Neutralizing antibodies were detected in the A1LL-administered group; however, they were not detected in swine and rabbits administered multiple doses of A2NTX. These results indicate that A2NTX has a lower immunogenicity than A1LL. In rats with neutralizing antibodies, produced by the administration of A1LL, that were administered either A1NTX or A2NTX, A2NTX showed more potent inhibitory neuromuscular transmission than A1NTX. In human sera immunized with the botulinum toxoid vaccine (containing LL, L, and M toxoid derived subtype A1) reacted with either A1NTX or A2NTX, A2NTX showed more potent inhibitory neuromuscular transmission than A1NTX. This suggests that A2NTX has a greater therapeutic value in humans who have neutralizing antibodies against the A1 toxin.

Autoantibodies to low-density lipoprotein receptor-related protein 4 that is essential for the neuromuscular junction formation

Myasthenia gravis (MG) is an autoimmune disorder characterized by skeletal muscle fatigability and muscle weakness resulting from inhibition of neuromuscular transmission by autoantibodies against synaptic apparatus in the neuromuscular junction (NMJ). MG is probably classified into three groups: anti-acetylcholine receptor (AChR) antibody-positive MG; antimuscle-specific kinase (MuSK) antibody-positive MG; and seronegative MG, which is negative for both antibodies. In patients with seronegative MG, pathogenic factors remain elusive. Recently, a new autoantibody against low-density lipoprotein receptor-related protein 4 (Lrp4), which is one of the postsynaptic apparatus and indispensable for the formation of the NMJ, has been identified and has attracted attention as a third MG-related autoantibody. In the present review, we discuss the role of Lrp4 in the formation of NMJ, and the molecular mechanism(s) of pathogenesis for anti-Lrp4 antibody-positive MG.

R‐type Ca2+ channels and inhibitory neuromuscular transmission in the gastrointestinal tract

Enteric inhibitory neuromuscular transmission relaxes gut smooth muscle via nitric oxide (NO) and purinergic mechanisms. In the guinea pig ileum, R‐type Ca2+ channels couple to release of NO while P/Q‐ and N‐type Ca2+ channels couple to release of the purine. In the current studies we tested the hypothesis that R‐type Ca2+ channels also couple selectively to NO release in the mouse colon by studying relaxations and inhibitory junction potentials (IJPs).MethodsLongitudinal muscle relaxations were studied using an in vitro organ bath system and intracellular recordings were conducted to study IJPs. Both responses were evoked using electrical field stimulation. The effects of drugs applied to the organ bath solution were investigated.ResultsThe R‐type Ca2+ channel blocker, NiCl2 (1–50 micromolar) did not alter neurogenic relaxation (10 Hz, 1s stimulus train) of the mouse distal colon. However, NiCl2 (50 micromolar) reduced the peak response and integrated areal of the IJP (1–60 Hz, 200 ms stimulus train).ConclusionR‐type Ca2+ channels couple to release of inhibitory neurotransmitters in the guinea pig and mouse gastrointestinal tract. The relationship between IJPs and relaxation of the mouse colon is not clear.

Inhibitory neuromuscular transmission in the mouse distal colon is mediated by SK and calcium activated chloride channels

Neurogenic colonic smooth muscle relaxation is caused by inhibitory junction potentials (IJPs). We attempted to identify neurotransmitter, receptor and ion channel mechanisms that mediate IJPs in the mouse distal colon.MethodsIntracellular recordings were obtained from circular smooth muscle cells in vitro. Electrical field stimulation was used to evoke IJPs. Drugs were applied in the organ bath solution.ResultsThe P2Y1 receptor antagonist MRS‐2179 (10 μM) reduced IJP amplitude (66 ± 4%) and area (24 ± 11%). The nitric oxide synthase (NOS) inhibitor L‐NNA (100 μM) reduced IJP area (33 ± 8%) but not amplitude. Co‐application of MRS‐2179 and L‐NNA blocked IJPs. The SK channel blocker, apamin (0.1 μM) reduced IJP amplitude (36 ± 5%) and area 41 ± 12%. The Ca2+‐activated Cl‐channel blocker, niflumic acid (50 μM) reduced IJP amplitude (52 ± 6%) and area (44 ± 6%). Co‐application of niflumic acid and apamin blocked IJPs. ATP (5 mM) produced a larger hyperpolarization (20 ± 2 mV) compared to β‐NAD (10 ± 3 mV). MRS‐2179 and apamin reduced the ATP response amplitude by 34 ± 8% and 31 ± 1% respectively. MRS‐2179 and apamin reduced the β‐NAD (5 mM) response by 71 ± 14% and 61 ± 5% respectively.ConclusionIJPs are mediated via P2Y1 and NOS mediated pathways, which involve SK and Ca2+‐activated Cl‐channels. Responses caused by β‐NAD more closely mimic the IJP compared to ATP. Supported by NIH (DK094932).

Functional role of vasoactive intestinal polypeptide in inhibitory motor innervation in the mouse internal anal sphincter

There is evidence that vasoactive intestinal polypeptide (VIP) participates in inhibitory neuromuscular transmission (NMT) in the internal anal sphincter (IAS). However, specific details concerning VIP-ergic NMT are limited, largely because of difficulties in selectively blocking other inhibitory neural pathways. The present study used the selective P2Y1 receptor antagonist MRS2500 (1 μm) and the nitric oxide synthase inhibitor N(G)-nitro-l-arginine (l-NNA; 100 μm) to block purinergic and nitrergic NMT to characterize non-purinergic, non-nitrergic (NNNP) inhibitory NMT and the role of VIP in this response. Nerves were stimulated with electrical field stimulation (0.1-20 Hz, 4-60 s) and the associated changes in contractile and electrical activity measured in non-adrenergic, non-cholinergic conditions in the IAS of wild-type and VIP(-/-) mice. Electrical field stimulation gave rise to frequency-dependent relaxation and hyperpolarization that was blocked by tetrodotoxin. Responses during brief trains of stimuli (4 s) were mediated by purinergic and nitrergic NMT. During longer stimulus trains, an NNNP relaxation and hyperpolarization developed slowly and persisted for several minutes beyond the end of the stimulus train. The NNNP NMT was abolished by VIP6-28 (30 μm), absent in the VIP(-/-) mouse and mimicked by exogenous VIP (1-100 nm). Immunoreactivity for VIP was co-localized with neuronal nitric oxide synthase in varicose intramuscular fibres but was not detected in the VIP(-/-) mouse IAS. In conclusion, this study identified an ultraslow component of inhibitory NMT in the IAS mediated by VIP. In vivo, this pathway may be activated with larger rectal distensions, leading to a more prolonged period of anal relaxation.

The roles of purinergic signaling during gastrointestinal inflammation

Extracellular purines play important roles as neurotransmitters and paracrine mediators in the gastrointestinal (GI) tract. Inflammation of the GI tract causes marked changes in the release and extracellular catabolism of purines, and can modulate purinoceptor expression and/or signaling. The functional consequences of this include suppression of the purinergic component of inhibitory neuromuscular and neurovascular transmission, increased release of purines from immune and epithelial cells, loss of enteric neurons to damage through P2X(7) purinoceptors, and enhanced activation of pain fibres. The purinergic system represents an important target for drug therapies that may improve GI inflammation and its consequences.

Relative contribution of SKCa and TREK1 channels in purinergic and nitrergic neuromuscular transmission in the rat colon

Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 μM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 μM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 μM), ODQ (10 μM), and N(ω)-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 μM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 μM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 μM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.

Changes in neuromuscular transmission in the VIP−/− mouse internal anal sphincter

Vasoactive intestinal polypeptide (VIP) has long been proposed as an inhibitory neurotransmitter in the internal anal sphincter (IAS) but direct evidence for this hypothesis is limited. The present study examined the role of VIP in inhibitory neuromuscular transmission (NMT) in the mouse IAS by comparing contractile and membrane potential responses to electrical field stimulation (EFS) of nerves in wild type (WT) and VIP−/− mice. EFS with short trains of stimuli (4s) gave rise to frequency (0.1–20 Hz) dependent relaxation and hyperpolarization in WT and VIP−/−mice. Peak EFS‐induced responses were not different between mice under control conditions and in the presence of the P2Y1 receptor antagonist MRS2500 (1 μM) while they were abolished with combined MRS2500 plus the nNOS inhibitor L‐NNA (100 μM) indicating that VIP is not required for nitrergic NMT. With longer trains of EFS (5Hz, 30–60s; 20 Hz 10–30s) a more slowly developing nitrergic and purinergic‐independent response was observed in WT but not VIP−/−mice suggesting that this component is due to VIP. In WT mice this component was blocked by the VIP receptor antagonist VIP6‐28 (10 μM) further supporting this hypothesis. In conclusion, these data provide evidence that the third inhibitory neural component in the mouse IAS is due to VIP. This pathway requires higher levels of EFS and persists well beyond the period of nerve stimulation. Grant support: DK078736.

Pharmacological characterization of purinergic inhibitory neuromuscular transmission in the human colon

In the present study, we further characterize the purinergic receptors mediating the inhibitory junction potential (IJP) and smooth muscle relaxation in the human colon using a new, potent and selective agonist (MRS2365), and antagonists (MR2279 and MRS2500) of the P2Y(1) receptor. The P2Y(12) antagonist AR-C66096 was tested as well. Using this pharmacological approach, we tested whether β-nicotinamide adenine dinucleotide (β-NAD) fulfilled the criteria to be considered an inhibitory neurotransmitter in the human colon. We carried out muscle bath and microelectrode experiments on circular strips from the human colon and calcium imaging recordings on HEK293 cells, which constitutively express the human P2Y(1) receptor. Both the fast component of IJP and non-nitrergic relaxation was concentration-dependently inhibited by MRS2279 and MRS2500. This antagonism was confirmed in HEK293 cells. However, AR-C66096 did not modify either inhibitory response. Adenosine 5'-Ο-2-thiodiphosphate and MRS2365 caused a smooth muscle hyperpolarization and transient inhibition of spontaneous motility that was antagonized by MRS2279 and MRS2500. β-Nicotinamide adenine dinucleotide inhibited the spontaneous motility (IC(50) = 3.3 mmol L(-1) ). Nevertheless, this effect was not antagonized by high concentrations of P2Y(1) antagonists. Inhibitory purinergic neuromuscular transmission in the human colon was pharmacologically assessed by the use of new P2Y(1) receptor antagonists MRS2179, MRS2279, and MRS2500. The rank order of potency of the P2Y(1) antagonists is MRS2500 > MRS2279 > MRS2179. We found that β-NAD partially fulfills the criteria to be considered an inhibitory neurotransmitter in the human colon, but the relative contribution of each purine (ATP/ADP vsβ-NAD) requires further studies.

Inhibition of neuromuscular transmission in the intact rat by emetine.

Inhibition of neuromuscular transmission in the intact rat by emetine.

In-vitro and in-vivo antivenin activity of 2-[2-(5,5,8a-trimethyl-2-methylene-decahydro-naphthalen-1-yl)-ethylidene]-succinaldehyde against Ophiophagus hannah venom

Curcuma zedoaroides A. Chaveerach & T. Tanee, locally known as Wan-Paya-Ngoo-Tua-Mia, is commonly used in the North-Eastern part of Thailand as a 'snakebite antidote'. The aim of this study was to isolate the active compound from the rhizome of C. zedoaroides, to determine its structure and to assess its antagonistic activity in vitro and in vivo against King cobra venom. The active compound was obtained from C. zedoaroides by extraction with acetone followed by purification using column chromatography; its X-ray structure was determined. Its inhibition of venom lethality was studied in vitro in rat phrenic nerve-hemidiaphragms and in vivo in mice. The acetone extract of the Curcuma rhizomes contained a C20 dialdehyde, [2-(5,5,8a-trimethyl-2-methylene-decahydro-naphthalen-1-yl)-ethylidene]-succinaldehyde, as the major component. The isolated curcuma dialdehyde was found active in vitro and in vivo for antivenin activity against the King cobra venom. Using isolated rat phrenic nerve-hemidiaphragm preparations, a significant antagonistic effect on the inhibition of neuromuscular transmission was observed in vitro. Inhibition on muscle contraction, produced by the 4 microg/ml venom, was reversed by 2-16 microg/ml of Curcuma dialdehyde in organ bath preparations over a period of 2 h. Mice intraperitoneally injected with 0.75 mg/kg venom and dialdehyde at 100 mg/kg had a significantly increased survival time. Injection of Curcuma dialdehyde (100 mg/kg) 30 min before the subcutaneous injection of the venom resulted in a 100% survival time after 2 h compared with 0% for the control group. The in vitro and in vivo evaluation confirmed the medicinal use of traditional snake plants against snakebites. The bioactivity is linked to an isolated molecule and not a result of synergistic effects of a mixture. The active compound was isolated and the structure fully elucidated, including its stereochemistry. This dialdehyde is a versatile chemical building block and can be easily obtained from this plant source.

P2Y1 Receptors Mediate Apamin-Sensitive and -Insensitive Inhibitory Junction Potentials in Murine Colonic Circular Smooth Muscle

Purinergic inhibitory neuromuscular transmission plays an important role in the control of intestinal motility. In most tissues this neurotransmission is apamin-sensitive, but recent studies in human colonic circular smooth muscle (CSM) suggest the presence of apamin-insensitive purinergic inhibitory junction potentials (IJPs). The current studies used conventional intracellular recordings on colonic CSM strips to characterize the purinergic IJPs in murine colonic CSM. P2Y1 receptor expression was examined by using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The IJP induced by nerve stimulation (NS) of one and four pulses in neuronal nitric-oxide synthase knockout mice consists of an apamin-sensitive and a dominant apamin-resistant component. These are identical to the IJPs in wild-type and CD1 mice in the presence of N(omega)-nitro-l-arginine methyl ester (200 microM) and were significantly inhibited by alpha,beta-methylene ATP (50 microM), an analog of ATP. IJPs were not affected by the P2X receptor antagonist 2',3'-o-(2,4,6-trinitrophenyl)-ATP (10 microM). Furthermore, apamin-resistant IJPs induced by single-pulse NS were abolished by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (100 microM), a P2 receptor antagonist; 2'-deoxy-N6-methyl adenosine 3,5-diphosphate (MRS-2179; 10 microM), a selective P2Y1 receptor antagonist; and tetrodotoxin (1 microM). Aboral NS induced apamin-sensitive purinergic IJPs, whereas oral and circumferential NS produced apamin-sensitive and -resistant IJPs, with the latter predominating. RT-PCR and immunohistochemistry confirmed the presence of P2Y1 receptors on smooth muscle and in the myenteric plexus. These data suggest that, depending on stimulus location, activation of P2Y1 receptors produces both apamin-sensitive and apamin-resistant IJPs in murine colonic CSM.

Investigation of 'CRATKML' derived peptides as antidotes for the in vivo and in vitro paralytic effect of botulinum neurotoxin A

CRATKML-derived peptides (Cp) prevent Botulinum neurotoxin A (BoNT/A) cleavage of purified SNAP25. We tested the effects of four cell membrane permeable Cp analogs on BoNT/A poisoned neuromuscular junctions. In vitro pretreatment with these Cps did not prevent BoNT/A inhibition of neuromuscular transmission (NMT). In vivo pretreatment with these Cps did not protect NMT against BoNT/A. The lack of prophylactic effect paralleled a lack of therapeutic effect on BoNT/A intoxicated mice. These data demonstrate that although Cps are effective BoNT/A antagonists in cell free systems, they are not effective against in vitro or in vivo BoNT/A actions of motor nerve endings.

Deletion of P2X2 and P2X3 receptor subunits does not alter motility of the mouse colon

Purinergic P2X receptors contribute to neurotransmission in the gut. P2X receptors are ligand-gated cation channels that mediate synaptic excitation in subsets of enteric neurons. The present study evaluated colonic motility in vitro and in vivo in wild type (WT) and P2X2 and P2X3 subunit knockout (KO) mice. The muscarinic receptor agonist, bethanechol (0.3–3 μM), caused similar contractions of the longitudinal muscle in colon segments from WT, P2X2 and P2X3 subunit KO mice. Nicotine (1–300 μM), acting at neuronal nicotinic receptors, caused similar longitudinal muscle relaxations in colonic segments from WT and P2X2 and P2X3 subunit KO mice. Nicotine-induced relaxations were inhibited by nitro-l-arginine (NLA, 100 μM) and apamin (0.1 μM) which block inhibitory neuromuscular transmission. ATP (1–1000 μM) caused contractions only in the presence of NLA and apamin. ATP-induced contractions were similar in colon segments from WT, P2X2 and P2X3 KO mice. The mouse colon generates spontaneous migrating motor complexes (MMCs) in vitro. The MMC frequency was higher in P2X2 KO compared to WT tissues; other parameters of the MMC were similar in colon segments from WT, P2X2 and P2X3 KO mice. 5-Hydroxytryptophan-induced fecal output was similar in WT, P2X2 and P2X3 KO mice. These data indicate that nicotinic receptors are located predominately on inhibitory motor neurons supplying the longitudinal muscle in the mouse colon. P2X2 or P2X3 subunit containing receptors are not localized to motor neurons supplying the longitudinal muscle. Synaptic transmission mediated by P2X2 or P2X3 subunit containing receptors is not required for propulsive motility in the mouse colon.

P2Y1 receptors mediate inhibitory neuromuscular transmission in the rat colon

Inhibitory junction potentials (IJP) are responsible for smooth muscle relaxation in the gastrointestinal tract. The aim of this study was to pharmacologically characterize the neurotransmitters [nitric oxide (NO) and adenosine triphosphate (ATP)] and receptors involved at the inhibitory neuromuscular junctions in the rat colon using newly available P2Y(1) antagonists. Organ bath and microelectrode recordings were used to evaluate the effect of drugs on spontaneous mechanical activity and resting membrane potential. IJP and mechanical relaxation were studied using electrical field stimulation (EFS). N(omega)-nitro-L-arginine (L-NNA) inhibited the slow component of the IJP and partially inhibited the mechanical relaxation induced by EFS. MRS2179, MRS2500 and MRS2279, all selective P2Y(1) receptor antagonists, inhibited the fast component of the IJP without having a major effect on the relaxation induced by EFS. The combination of both L-NNA and P2Y(1) antagonists inhibited the fast and the slow components of the IJP and completely blocked the mechanical relaxation induced by EFS. Sodium nitroprusside caused smooth muscle hyperpolarization and cessation of spontaneous motility that was prevented by oxadiazolo[4,3-alpha]quinoxalin-1-one. Adenosine 5'-O-2-thiodiphosphate, a preferential P2Y agonist, hyperpolarized smooth muscle cells and decreased spontaneous motility. This effect was inhibited by P2Y(1) antagonists. The co-transmission process in the rat colon involves ATP and NO. P2Y(1) receptors mediate the fast IJP and NO the slow IJP. The rank order of potency of the P2Y(1) receptor antagonists is MRS2500 greater than MRS2279 greater than MRS2179. P2Y(1) receptors might be potential pharmacological targets for the regulation of gastrointestinal motility.

Quantitative determination of biological activity of botulinum toxins utilizing compound muscle action potentials (CMAP), and comparison of neuromuscular transmission blockage and muscle flaccidity among toxins

The biological activity of various types of botulinum toxin has been evaluated using the mouse intraperitoneal LD 50 test (ip LD 50). This method requires a large number of mice to precisely determine toxin activity, and so has posed a problem with regard to animal welfare. We have used a direct measure of neuromuscular transmission, the compound muscle action potential (CMAP), to evaluate the effect of different types of botulinum neurotoxin (NTX), and we compared the effects of these toxins to evaluate muscle relaxation by employing the digit abduction scoring (DAS) assay. This method can be used to measure a broad range of toxin activities the day after administration. Types A, C, C/D, and E NTX reduced the CMAP amplitude one day after administration at below 1 ip LD 50, an effect that cannot be detected using the mouse ip LD 50 assay. The method is useful not only for measuring toxin activity, but also for evaluating the characteristics of different types of NTX. The rat CMAP test is straightforward, highly reproducible, and can directly determine the efficacy of toxin preparations through their inhibition of neuromuscular transmission. Thus, this method may be suitable for pharmacology studies and the quality control of toxin preparations.

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

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 (< or =48 h postnatal) and adult ( approximately 4 months postnatal) guinea pigs. In tissues from neonates and adults, nicotine (0.3-30 micromol L(-1)) contracted longitudinal muscle preparations in a tetrodotoxin (TTX) (0.3 micromol L(-1))-sensitive manner. The muscarinic receptor antagonist, scopolamine (1 micromol L(-1)), reduced substantially nicotine-induced contractions in neonatal tissues but not adult tissues. In the presence of N(omega)-nitro-l-arginine (NLA, 100 micromol 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 micromol 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.