Activity-dependent metaplasticity of inhibitory and excitatory synaptic transmission in the lamprey spinal cord locomotor network.
Paired intracellular recordings have been used to examine the activity-dependent plasticity and neuromodulator-induced metaplasticity of synaptic inputs from identified inhibitory and excitatory interneurons in the lamprey spinal cord. Trains of spikes at 5-20 Hz were used to mimic the frequency of spiking that occurs in network interneurons during NMDA or brainstem-evoked locomotor activity. Inputs from inhibitory and excitatory interneurons exhibited similar activity-dependent changes, with synaptic depression developing during the spike train. The level of depression reached was greater with lower stimulation frequencies. Significant activity-dependent depression of inputs from excitatory interneurons and inhibitory crossed caudal interneurons, which are central elements in the patterning of network activity, usually developed between the fifth and tenth spikes in the train. Because these interneurons typically fire bursts of up to five spikes during locomotor activity, this activity-dependent plasticity will presumably not contribute to the patterning of network activity. However, in the presence of the neuromodulators substance P and 5-HT, significant activity-dependent metaplasticity of these inputs developed over the first five spikes in the train. Substance P induced significant activity-dependent depression of inhibitory but potentiation of excitatory interneuron inputs, whereas 5-HT induced significant activity-dependent potentiation of both inhibitory and excitatory interneuron inputs. Because these metaplastic effects are consistent with the substance P and 5-HT-induced modulation of the network output, activity-dependent metaplasticity could be a potential mechanism underlying the coordination and modulation of rhythmic network activity. (+info)
Molecular dynamics study of substance P peptides in a biphasic membrane mimic.
Two neuropeptides, substance P (SP) and SP-tyrosine-8 (SP-Y8), have been studied by molecular dynamics (MD) simulation in a TIP3P water/CCl4 biphasic solvent system as a mimic for the water-membrane system. Initially, distance restraints derived from NMR nuclear Overhauser enhancements (NOE) were incorporated in the restrained MD (RMD) in the equilibration stage of the simulation. The starting orientation/position of the peptides for the MD simulation was either parallel to the water/CCl4 interface or in a perpendicular/insertion mode. In both cases the peptides equilibrated and adopted a near-parallel orientation within approximately 250 ps. After equilibration, the conformation and orientation of the peptides, the solvation of both the backbone and the side chain of the residues, hydrogen bonding, and the dynamics of the peptides were analyzed from trajectories obtained in the RMD or the subsequent free MD (where the NOE restraints were removed). These analyses showed that the peptide backbone of nearly all residues are either solvated by water or are hydrogen-bonded. This is seen to be an important factor against the insertion mode of interaction. Most of the interactions with the hydrophobic phase come from the hydrophobic interactions of the side chains of Pro-4, Phe-7, Phe-8, Leu-10, and Met-11 for SP, and Phe-7, Leu-10, Met-11 and, to a lesser extent, Tyr-8 in SP-Y8. Concerted conformational transitions took place in the time frame of hundreds of picoseconds. The concertedness of the transition was due to the tendency of the peptide to maintain the necessary secondary structure to position the peptide properly with respect to the water/CCl4 interface. (+info)
Molecular dynamics study of substance P peptides partitioned in a sodium dodecylsulfate micelle.
Two neuropeptides, substance P (SP) and SP-tyrosine-8 (SP-Y8), have been studied by molecular dynamics (MD) simulation in an explicit sodium dodecylsulfate (SDS) micelle. Initially, distance restraints derived from NMR nuclear Overhauser enhancements (NOE) were incorporated in the restrained MD (RMD) during the equilibration stage of the simulation. It was shown that when SP-Y8 was initially placed in an insertion (perpendicular) configuration, the peptide equilibrated to a surface-bound (parallel) configuration in approximately 450 ps. After equilibration, the conformation and orientation of the peptides, the solvation of both the backbone and the side chain of the residues, hydrogen bonding, and the dynamics of the peptides were analyzed from trajectories obtained from the RMD or the subsequent free MD (where the NOE restraints were removed). These analyses showed that the peptide backbones of all residues are either solvated by water or are hydrogen-bonded. This is seen to be an important factor against the insertion mode of interaction. Most of the interactions come from the hydrophobic interaction between the side chains of Lys-3, Pro-4, Phe-7, Phe-8, Leu-10, and Met-11 for SP, from Lys-3, Phe-7, Leu-10, and Met-11 in SP-Y8, and the micellar interior. Significant interactions, electrostatic and hydrogen bonding, between the N-terminal residues, Arg-Pro-Lys, and the micellar headgroups were observed. These latter interactions served to affect both the structure and, especially, the flexibility, of the N-terminus. The results from simulation of the same peptides in a water/CCl4 biphasic cell were compared with the results of the present study, and the validity of using the biphasic system as an approximation for peptide-micelle or peptide-bilayer systems is discussed. (+info)
Two affinities for a single antagonist at the neuronal NK1 tachykinin receptor: evidence from quantitation of receptor endocytosis.
1. In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide-preferring receptor related to the NK1r. 2. We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist-induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea-pig ileum. 3. SP and septide gave sigmoid log concentration-response curves and were equipotent in inducing NK1r endocytosis. 4. The NK1r antagonists, CP-99994 (2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine dihydrochloride and MEN-10581, cyclo(Leu,[CH2NH]Lys(benzyloxycarbonyl)-Gln-Trp-Phe-betaAla) were both more potent in inhibiting endocytosis (50 x and 8 x greater respectively) against septide than against SP. 5. The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide-preferring tachykinin receptor. (+info)
Capsaicin-sensitive C-fiber-mediated protective responses in ozone inhalation in rats.
To assess the role of lung sensory C fibers during and after inhalation of 1 part/million ozone for 8 h, we compared breathing pattern responses and epithelial injury-inflammation-repair in rats depleted of C fibers by systemic administration of capsaicin as neonates and in vehicle-treated control animals. Capsaicin-treated rats did not develop ozone-induced rapid, shallow breathing. Capsaicin-treated rats showed more severe necrosis in the nasal cavity and greater inflammation throughout the respiratory tract than did control rats exposed to ozone. Incorporation of 5-bromo-2'-deoxyuridine (a marker of DNA synthesis associated with proliferation) into terminal bronchiolar epithelial cells was not significantly affected by capsaicin treatment in rats exposed to ozone. However, when normalized to the degree of epithelial necrosis present in each rat studied, there was less 5-bromo-2'-deoxyuridine labeling in the terminal bronchioles of capsaicin-treated rats. These observations suggest that the ozone-induced release of neuropeptides does not measurably contribute to airway inflammation but may play a role in modulating basal and reparative airway epithelial cell proliferation. (+info)
Real-time visualization of the cellular redistribution of G protein-coupled receptor kinase 2 and beta-arrestin 2 during homologous desensitization of the substance P receptor.
The substance P receptor (SPR) is a G protein-coupled receptor (GPCR) that plays a key role in pain regulation. The SPR desensitizes in the continued presence of agonist, presumably via mechanisms that implicate G protein-coupled receptor kinases (GRKs) and beta-arrestins. The temporal relationship of these proposed biochemical events has never been established for any GPCR other than rhodopsin beyond the resolution provided by biochemical assays. We investigate the real-time activation and desensitization of the human SPR in live HEK293 cells using green fluorescent protein conjugates of protein kinase C, GRK2, and beta-arrestin 2. The translocation of protein kinase C betaII-green fluorescent protein to and from the plasma membrane in response to substance P indicates that the human SPR becomes activated within seconds of agonist exposure, and the response desensitizes within 30 s. This desensitization process coincides with a redistribution of GRK2 from the cytosol to the plasma membrane, followed by a robust redistribution of beta-arrestin 2 and a profound change in cell morphology that occurs after 1 min of SPR stimulation. These data establish a role for GRKs and beta-arrestins in homologous desensitization of the SPR and provide the first visual and temporal resolution of the sequence of events underlying homologous desensitization of a GPCR in living cells. (+info)
Neurogenic plasma leakage in mouse airways.
1. This study sought to determine whether neurogenic inflammation occurs in the airways by examining the effects of capsaicin or substance P on microvascular plasma leakage in the trachea and lungs of male pathogen-free C57BL/6 mice. 2. Single bolus intravenous injections of capsaicin (0.5 and 1 micromol kg(-1), i.v.) or substance P (1, 10 and 37 nmol kg(-10, i.v.) failed to induce significant leakage in the trachea, assessed as extravasation of Evans blue dye, but did induce leakage in the urinary bladder and skin. 3. Pretreatment with captopril (2.5 mg kg(-1), i.v.), a selective inhibitor of angiotensin converting enzyme (ACE), either alone or in combination with phosphoramidon (2.5 mg kg(-1), i.v.), a selective inhibitor of neutral endopeptidase (NEP), increased baseline leakage of Evans blue in the absence of any exogenous inflammatory mediator. The increase was reversed by the bradykinin B2 receptor antagonist Hoe 140 (0.1 mg kg(-1), i.v.). 4. After pretreatment with phosphoramidon and captopril, capsaicin increased the Evans blue leakage above the baseline in the trachea, but not in the lung. This increase was reversed by the tachykinin (NK1) receptor antagonist SR 140333 (0.7 mg kg(-1), i.v.), but not by the NK2 receptor antagonist SR 48968 (1 mg kg(-1), i.v.). 5. Experiments using Monastral blue pigment as a tracer localized the leakage to postcapillary venules in the trachea and intrapulmonary bronchi, although the labelled vessels were less numerous in mice than in comparably treated rats. Blood vessels of the pulmonary circulation were not labelled. 6. We conclude that neurogenic inflammation can occur in airways of pathogen-free mice, but only after the inhibition of enzymes that normally degrade inflammatory peptides. Neurogenic inflammation does not involve the pulmonary microvasculature. (+info)
The novel analgesic compound OT-7100 (5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimid ine) attenuates mechanical nociceptive responses in animal models of acute and peripheral neuropathic hyperalgesia.
We investigated the effects of OT-7100, a novel analgesic compound (5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidi ne), on prostaglandin E2 biosynthesis in vitro, acute hyperalgesia induced by yeast and substance P in rats and hyperalgesia in rats with a chronic constriction injury to the sciatic nerve (Bennett model), which is a model for peripheral neuropathic pain. OT-7100 did not inhibit prostaglandin E2 biosynthesis at 10(-8)-10(-4) M. Single oral doses of 3 and 10 mg/kg OT-7100 were effective on the hyperalgesia induced by yeast. Single oral doses of 0.1, 0.3, 1 and 3 mg/kg OT-7100 were effective on the hyperalgesia induced by substance P in which indomethacin had no effect. Repeated oral administration of OT-7100 (10 and 30 mg/kg) was effective in normalizing the mechanical nociceptive threshold in the injured paw without affecting the nociceptive threshold in the uninjured paw in the Bennett model. Indomethacin had no effect in this model. While amitriptyline (10 and 30 mg/kg) and clonazepam (3 and 10 mg/kg) significantly normalized the nociceptive threshold in the injured paw, they also increased the nociceptive threshold in the uninjured paw. These results suggest that OT-7100 is a new type of analgesic with the effect of normalizing the nociceptive threshold in peripheral neuropathic hyperalgesia. (+info)