Origin and modulation of circular smooth muscle layer contractions in the porcine esophagus.

BACKGROUND: The origin and modulation mechanisms controlling timing and amplitude of esophageal body peristalsis are not fully understood. We aimed to characterize the neurotransmitters involved in the origin and modulation of circular smooth muscle esophageal body (EB) contractions. METHODS: Responses of porcine EB strips to electrical stimulation of motor neurons (MNs) were assessed in organ baths and with microelectrodes. The effect of antagonists of inhibitory (L-NAME 1 mmol L(-1) , MRS2179 10 µmol L(-1) ) and excitatory neurotransmitters (atropine 1 µmol L(-1) ; SR140333 1 µmol L(-1) -NK(1) ra-, GR94800 1 µmol L(-1) -NK(2) ra-) and of ganglionic neurotransmitters (hexamethonium 100 µmol L(-1) , ondansetron 1 µmol L(-1) , NF279 10 µmol L(-1) ) were characterized. KEY RESULTS: Electrical field stimulation (EFS) induced a frequency-dependent off-contraction (16.8 ± 0.8 g) following a latency period. Latency was significantly reduced by L-NAME (-66.1 ± 4.1%) and MRS2179 (-25.9 ± 5.6%), and strongly increased by atropine (+36.8 ± 5.8%). Amplitude was reduced by L-NAME (-69.9 ± 10.4%), MRS2179 (-34.1 ± 6.0%), atropine (-42.3 ± 4.7%), hexamethonium (-18.9 ± 3.3%), NF279 (-20.7 ± 3.5%), ondansetron (-16.3 ± 3.2%), GR94800 (-28.0 ± 4.8%) SR140333 (-20.9 ± 7.1%), and α-chymotrypsin (-31.3 ± 7.0%). The EFS induced a monophasic nitrergic inhibitory junction potential. CONCLUSIONS & INFERENCES: Our results suggest that timing (latency) and amplitude of esophageal contractions are determined by a balance of complex interactions between excitatory and inhibitory MNs. Latency depends on the activation of inhibitory MNs releasing NO and a minor purinergic contribution through P2Y(1) receptors, and excitatory MNs releasing ACh. Amplitude depends on a major contribution of excitatory MNs releasing ACh and tachykinins, and also on inhibitory MNs releasing NO, ATP or related purines, and peptidergic neurotransmitters acting as strong modulators of the excitatory neuroeffector transmission.

Regional functional specialization and inhibitory nitrergic and nonnitrergic coneurotransmission in the human esophagus.

The aim of this study was to explore the myenteric mechanisms of control of human esophageal motility and the effect of nitrergic and nonnitrergic neurotransmitters. Human circular esophageal strips were studied in organ baths and with microelectrodes. Responses following electrical field stimulation (EFS) of enteric motoneurons (EMNs) or through nicotinic acetylcholine receptors were compared in the esophageal body (EB) and in clasp and sling regions in the lower esophageal sphincter (LES). In clasp LES strips: 1) sodium nitroprusside (1 nM to 100 µM), adenosine-5'-[ß-thio]diphosphate trilithium salt (1-100 µM), and vasoactive intestinal peptide (1 nM to 1 µM) caused a relaxation; 2) 1 mM N(ω)-nitro-L-arginine (L-NNA) shifted the EFS "on"-relaxation to an "off"-relaxation, partly antagonized by 10 µM 2'-deoxy-N(6)-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS2179) or 10 U/ml α-chymotrypsin; and 3) nicotine-relaxation (100 µM) was mainly antagonized by L-NNA, and only partly by MRS2179 or α-chymotrypsin. In sling LES fibers, EFS and nicotine relaxation was abolished by L-NNA. In the EB, L-NNA blocked the latency period, and MRS2179 reduced "off"-contraction. The amplitude of cholinergic contraction decreased from the EB to both LES sides. EFS induced a monophasic inhibitory junction potential in clasp, sling, and EB fibers abolished by L-NNA. Our study shows a regional specialization to stimulation of EMNs in the human esophagus, with stronger inhibitory responses in clasp LES fibers and stronger cholinergic excitatory responses in the EB. Inhibitory responses are mainly triggered by nitrergic EMNs mediating the inhibitory junction potentials in the LES and EB, EFS on-relaxation in clasp and sling LES sides, and latency in the EB. We also found a minor role for purines (through P2Y(1) receptors) and vasoactive intestinal peptide-mediating part of nonnitrergic clasp LES relaxation.

Specific and complementary roles for nitric oxide and ATP in the inhibitory motor pathways to rat internal anal sphincter.

BACKGROUND: The neurotransmitters mediating inhibitory pathways to internal anal sphincter (IAS) have not been fully characterized. Our aim was to assess the putative release of nitric oxide, purines and vasoactive intestinal peptide (VIP) from inhibitory motor neurons (MNs) and their role in the myogenic tone, resting membrane potential (RMP) of smooth muscle cells (SMC), spontaneous inhibitory junction potentials (sIJP), mechanical relaxation, and IJP induced by electrical field stimulation (EFS) or nicotine. METHODS: Rat IAS strips were studied using organ baths, microelectrodes, and immunohistochemistry. KEY RESULTS: Internal anal sphincter strips developed active myogenic tone (0.31 g), enhanced and stabilized by prostaglandin F(2α) (PGF2α). L-NNA (1 mmol L(-1)) depolarized SMC and increased tone but did not modify sIJP. In contrast, the specific P2Y(1) receptor antagonist MRS2500 (1 µmol L(-1)) did not modify the RMP or the basal tone but abolished sIJP. Electrical field stimulation and nicotine (10 µmol L(-1)) caused IAS relaxation (-45.9%VS-52.2%), partially antagonized by L-NNA (35%-45%, P ≤ 0.05) and fully abolished by MRS2500 (P ≤ 0.001). Electrical field stimulation induced a biphasic inhibitory junction potential (IJP), the initial fast component was selectively blocked by MRS2500 and the sustained slow component was blocked by L-NNA. Vasoactive intestinal peptide 6-28 (0.1 µmol L(-1)) or α-chymotrypsin (10 U mL(-1)) did not modify the RMP, sIJP, EFS-induced IJP, or relaxation. P2Y(1) receptors were immunolocalized in the circular SMC of IAS. CONCLUSIONS & INFERENCES: The effects of inhibitory MNs on rat IAS are mediated by a functional co-transmission process involving nitrergic and purinergic pathways through P2Y(1) receptors with specific and complementary roles on the control of tone, sIJP, and hyperpolarization and relaxation of IAS following stimulation of inhibitory MNs.

Selective stimulation of intrinsic excitatory and inhibitory motor pathways in porcine lower oesophageal sphincter.

The mechanisms of stimulation of inhibitory and excitatory motor neurons (MNs) in the lower oesophageal sphincter (LOS) are not fully understood. The aim of this study was to assess the effect of selective stimulation of inhibitory and excitatory MNs in porcine LOS through nicotinic acetylcholine receptors (nAChRs), 5-HT(3) and P2X receptors. Circular LOS strips from adult pigs were studied in organ baths. We compared the effects of stimulation of MNs by electrical field stimulation (26 V, 0.3-20 Hz); nicotine (1-300 micromol L(-1)); 5-HT and 2-Me-5-HT (1 nmol(-1)-30 micromol L(-1)); and alpha,beta-methylene ATP (alpha,beta-meATP 1-100 micromol L(-1)); in standard Krebs solution; a non-adrenergic non-nitrergic non-purinergic (NANNNP) solution; and a non-adrenergic non-cholinergic (NANC) solution. Electrical stimulation of inhibitory MNs caused an intense LOS relaxation (-78.94 +/- 4.50% of LOS tone); and of excitatory MNs, a strong contraction (17.89 +/- 1.96 g). Nicotine 100 micromol L(-1) relaxed LOS (-84.67 +/- 3.98%) in standard Krebs solution, an effect reduced by Tetrodotoxin (TTX) 1 micromol L(-1). Nicotine induced a weak TTX-sensitive contraction (1.64 +/- 0.4 g) in NANNNP solution. 5-HT 10 micromol L(-1) and 2-Me-5-HT 30 micromol L(-1) contracted LOS in standard, NANC and NANNNP conditions, maximal responses (7.30 +/- 1.52 g, 3.50 +/- 0.18 g respectively) being reduced by TTX. alpha,beta-meATP 100 micromol L(-1) caused a LOS relaxation (-17.45 +/- 6.62%) unaffected by TTX in NANC solution, and a contraction (6.7 +/- 0.85 g) antagonized by TTX in NANNNP solution. Our results suggest selective mechanisms for stimulation of intrinsic excitatory and inhibitory motor pathways in porcine LOS. Inhibitory MNs are strongly stimulated by nAChRs and do not respond to stimulation of 5-HT(3) and P2X receptors. By contrast, excitatory MNs are stimulated through 5-HT(3) and P2X receptors, stimulation through nACRs being difficult and causing a weak response.

Mechanisms controlling function in the clasp and sling regions of porcine lower oesophageal sphincter.

BACKGROUND: Characterization of functional differences between lower oesophageal sphincter (LOS) clasp and sling muscles might aid the development of more specific pharmacological and surgical approaches for the treatment of motility disorders. METHODS: Circular LOS strips from 25 adult pigs were studied in organ baths to compare the physiology of clasp and sling fibres. RESULTS: Sling strips developed greater tone than clasp fibres (mean(s.e.m.) 7.59(0.89) versus 4.72(0.67) g; P = 0.017). LOS tone was more dependent on extracellular calcium in clasp strips and on the activity of cholinergic enteric motor neurones (EMNs) in sling strips. The amplitude of maximal relaxation caused by electrical field stimulation (EFS, 3Hz) of EMNs was greater in clasp strips (mean(s.e.m.) 74.5(2.3) versus 58.1(2.2) per cent of tone; P < 0.001). EFS-induced relaxation was reduced in clasp fibres and fully blocked in sling fibres by nitrergic blockade with 10 micromol/l 1H-[1,2,4]oxadiazole-[4,3-alpha]quinoxalin-1-one (ODQ). The amplitude of EFS cholinergic responses was significantly greater in sling fibres. In the clasp region, relaxation caused by stimulation of EMNs with 100 micromol/l nicotine was reduced by ODQ. In sling fibres, nicotine induced relaxation at rest and cholinergic contraction following ODQ. CONCLUSION: Clasp and sling fibres of the porcine LOS show marked intrinsic functional differences. This should be considered when developing more specific approaches to human LOS motility disorders.

On the mechanism of conversion of N-acyl-4-acyloxy-beta-lactams into 2-substituted 1,3-oxazin-6-ones. Can a low-barrier transition state be antiaromatic?

The mechanism of the conversion of N-acyl-4-acyloxy-beta-lactams into 1,3-oxazin-6-ones has been investigated using ab initio and density functional theories. It has been found that two pseudopericyclic reactions are involved in the whole process. The first key reaction is a retro-[4-exo-dig] cyclization instead of a thermal conrotatory electrocyclic ring opening. Magnetic characterization of the corresponding transition structure shows antiaromatic character, despite the low activation energy associated with this process. The second step is very exothermic and has no activation barrier. It corresponds to another pseudopericyclic reaction instead of a six-electron disrotatory electrocyclization. These results confirm that there is no correlation between aromaticity and pseudopericyclic reactions. In contrast, thermal-symmetry-allowed pericyclic reactions are always aromatic. Therefore, magnetic analysis of the corresponding transition structures constitutes a useful tool to distinguish between both kinds of processes.

New stereoselective intramolecular

Efficient 1,4-asymmetric induction has been achieved in the highly stereocontrolled intramolecular [2 + 2] cycloadditions between ketenimines and imines, leading to 1,2-dihydroazeto[2, 1-b]quinazolines. The chiral methine carbon adjacent to the iminic nitrogen controls the exclusive formation of the cycloadducts with relative trans configuration at C2 and C8. The stepwise mechanistic model, based on theoretical calculations, fully supports the stereochemical outcome of these cycloadditions.

Highly efficient induction of chirality in intramolecular

Highly stereocontrolled, intramolecular [2 + 2] cycloadditions between ketenimines and imines leading to 1,2-dihydroazeto[2, 1-b]quinazolines have been achieved. The source of stereocontrol is a chiral carbon atom adjacent either to the iminic carbon or nitrogen atom. In the first case, the stereocontrol stems from the preference for the axial conformer in the first transition structure. In the second case, the origin of the stereocontrol lies on the two-electron stabilizing interaction between the C-C bond being formed and the sigma orbital corresponding to the polar C-X bond, X being an electronegative atom. These models can be extended to other related systems for predicting the stereochemical outcome in this intramolecular reaction.

Modification of regioselectivity in cycloadditions to C70 under microwave irradiation

The regioselectivity of the cycloaddition of N-methylazomethine ylide to C70 can be modified by using microwave irradiation as the source of energy. Under microwave irradiation and by choosing the appropriate solvent and irradiation power, the 5-6 isomer is the major product, a situation that is in contrast to conventional heating where the 1-2 isomer predominates. Moreover, isomer 7-21, which represents 13% of monoadducts under classical heating, is not formed under microwave irradiation and with ODCB as solvent. Theoretical calculations predict an asynchronous mechanism and suggest that the modification of the regiochemical outcome is related to the relative energies and hardnesses of the transition structures involved.

New insights on the origins of the stereocontrol of the staudinger reaction: [2 + 2] cycloaddition between ketenes and N-silylimines.

Density-functional theory studies on the [2 + 2] reaction between N-silylimines and ketenes to form the corresponding 2-azetidinones (beta-lactams) help to clarify several aspects on the mechanism of the Staudinger reaction. This reaction has been studied experimentally by Panunzio et al. It is shown that the formation of the 2-azetidinone ring takes place via two consecutive reactions. The first reaction consists of the nucleophilic addition of the iminic nitrogen to the sp-hybridized carbon atom of the ketene, with simultaneous migration of the silyl group from the imine to the oxygen atom of the ketene. This leads to silyl enol intermediates, in good agreement with the experimental results. Formation of the N-silylated beta-lactam takes place via a domino reaction consisting of a conrotatory thermal electrocyclization followed by a new silatropic rearrangement. It is also found that isomerization of the starting N-silylimine has a lower activation barrier than that associated with the formation of the C-N bond, which explains the stereochemical outcome experimentally observed. Further considerations on the asymmetric torquoelectronic effects involved in this reaction are also reported.