Opiate binding to cerebroside sulfate: a model system for opiate-receptor interaction.

Cerebroside sulfate was shown to bind etorphine and levorphanol with high affinity. The relative potency of narcotic analgesics in preventing the binding of levorphanol to cerebroside sulfate correlated well with their reported analgetic activity. The data indicate similarities between cerebroside sulfate and a purified opiate receptor from mouse brain which has been reported to be a proteolipid. Some preliminary animal data also imply the involvement of CS in opiate action We, therefore, propose that CS may serve as a useful “receptor” model for the study of opiate-receptor interaction $\text{in vitro}$.     

Nmr and molecular modeling investigations of the neuropeptide substance P in the presence of 15 mM sodium dodecyl sulfate micelles

To better understand the structural basis of the biological activity of the neuropeptide substance P SP; (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂), two-dimensional nmr spectroscopy experiments and simulated annealing calculations were used to investigate the conformation adopted in the presence of the membrane model system sodium dodecyl sulfate. It was determined that SP in the presence of SDS micelles undergoes a conformational equilibrium between an α- and a 3₁₀-helix involving the midregion (Pro⁴-Gln⁵-Gln⁶-Phe⁷-Phe⁸) of the peptide. The C-terminus adopts an extended conformation while the N-terminus remains quite flexible. The conformation adopted by SP in the presence of SDS micelles yields a structure that is consistent with the model of a neurokinin-1 selective ligand proposed by Convert. © 1994 John Wiley & Sons, Inc.     

Direct neurite-mast cell communication in vitro occurs via the neuropeptide substance P.

Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. However, whether mast cell activation occurs as a direct response to neuronal activation or requires an intermediary cell is unclear. Addressing this issue, we used an in vitro coculture approach comprising cultured murine superior cervical ganglia and rat leukemia basophilic cells (RBLs; possesses properties of mucosal-type mast cells). Following loading with the calcium fluorophore, Fluo-3, neurite-RBL units (separated by <50 nm) were examined by confocal laser scanning microscopy. Addition of bradykinin, or scorpion venom, dose-dependently elicited neurite activation (i.e., Ca2+ mobilization) and, after a lag period, RBL Ca2+ mobilization. Neither bradykinin nor scorpion venom had any direct effect on the RBLs in the absence of neurites. Addition of a neutralizing substance P Ab or a neurokinin (NK)-1 receptor antagonist, but not an NK-2 receptor antagonist, dose-dependently prevented the RBL activation that resulted as a consequence of neural activation by either bradykinin or scorpion venom. These data illustrate that nerve-mast cell cross-talk can occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating via NK-1 receptors, is an important mediator of this communication. Our findings have implications for the neuroimmune signaling cascades that are likely to occur during airways inflammation, intestinal hypersensitivity, and other conditions in which mast cells feature.     

Evidence for the noninvolvement of sulfogalactosylceramide (cerebroside sulfate) in the enkephalin (opiate) receptor.

Mouse neuroblastoma clonal cell line N4TG1 has 20,000 enkephaline (opiate) receptor binding sites/cell, but contains undetectable levels of sulfogalactosylceramide as judged by quantitative and isotope-labeling studies. Mouse glioma cell lines G26-20 and G26-24 contain substantial quantities of sulfogalactosylceramide, but do not bind enkephalins. Preincubation of cells with sulfogalactosylceramide or other anionic glycosphingolipids did not enhance or induce opiate receptors. Thus, sulfogalactosylceramide is not an integral part of the opiate receptor of N4TG1 cells.     

Stimulation of epithelial cell growth by the neuropeptide substance P

The neuropeptide substance P (SP) was found to stimulate DNA synthesis and cell growth for epithelial cells (cornea and lens) in a serum-free environment. The length of treatment time was shown to be important since longer times shifted the dose-response curve to the left. In short-term DNA synthesis studies (40 h) the stimulation with SP (or synergism with insulin) was not apparent until close to 10 μM, however, when DNA synthesis assays were carried out over a long period of time (5 days) stimulation with SP was seen at 1 pM. The stimulation of DNA synthesis by SP was synergistic with insulin for lens epithelial cells, but little synergism was seen with corneal epithelial cells. It cell growth studies on lens epithelial cells SP also showed growth stimulation by itself and synergism with insulin at concentrations of 1–2 pM. The neuropeptide calcitonin gene related peptide (CGRP) showed no DNA synthesis stimulating ability on epithelial cells by itself at concentrations as high as 2.5 μM; however, it was synergistic with SP at a concentration of 0.025 μM. SP pretreatment of epithelial cells for 2 h causes an increase in cellular sensitivity to subsequent addition of either SP or insulin. This increase is consistent with the hypothesis that either the signal from SP persists after its removal from the cell or the dissociation time for SP from its receptor is longer than the wash time.     

Modulation of lymphatic muscle contractility by the neuropeptide substance P

Substance P (SP) is a neuropeptide associated with sensory innervation of lymphoid tissue and a suspected modulator of lymphatic function in inflammation. Only a few studies have examined the effects of SP on lymphatic contraction, and it is not clear to what extent SP acts directly on the lymphatic muscle and/or endothelium or indirectly through changes in intraluminal filling pressure secondary to increases in capillary permeability/filtration. We tested the effects of SP on the spontaneous contractions of rat isolated mesenteric lymphatic vessels under isometric and isobaric conditions, hypothesizing that low concentrations would stimulate lymphatic pumping by enhancing lymphatic muscle contraction in a manner complementary to the effect of increased preload. Under isometric conditions, SP (10 nM) dramatically enhanced lymphatic chronotropy and inotropy. Unlike guinea pig lymphatics, SP actions were not blocked by cyclooxygenase or PLA(2) inhibition. In the absence of SP, ramp increases in isometric preload resulted in x approximately 1.6 increases in contraction amplitude (Amp) and x approximately 1.7 increases in frequency (Freq). SP increased Freq by x approximately 2.4, Amp by x approximately 1.9, and the Amp-Freq product (AFP) by x approximately 3.5. Under isobaric conditions, the pressure elevation from 0.5 to 10 cmH(2)O in the absence of SP decreased Amp by x approximately 0.6 and increased Freq by x approximately 1.8. SP caused a modest increase in Amp, a robust increase in Freq at all pressures, and shifted the AFP-pressure relationship upward and leftward. Therefore, SP has substantial positive inotropic and chronotropic effects on rat lymphatic muscle, improving pump efficiency independent of the effects of preload and broadening of the working range of the lymphatic pump.     

Possible involvement of cerebroside sulfate in opiate receptor binding.

Cerebroside sulfate (CS) appears to fulfill most of the structural requirements of a hypothetical opiate receptor. It possesses many of the properties that are thought to be necessary for the identification of an "opiate receptor," exhibiting high affinity and stereoselective binding to a number of narcotic drugs. Although these properties are insufficient to establish identity of the receptor, it is highly significant that the affinity of this binding can be correlated with the analgetic potency of these drugs in both man and rodents. CS is an endogenous component of brain tissue, and a partially purified opiate receptor from mouse brain has been found to be CS. Other experiments indicate that reduced availability of brain CS decreases the analgetic effects of morphine and this is accompanied by a reduction in number of binding sites, suggesting that the interaction of opiates with CS observed in vitro may also have importance in vivo. CS was also found to be a component of the opiate receptor after marking with 125I-labeled diazosulfanilic acid. The possibility that CS or the SO4-2 group of this lipid may be the "anionic site" of the opiate receptor should be considered.     

Natural killer cell functions mediated by the neuropeptide substance P

The neuropeptide substance P (SP) can modulate a number of immunological functions in vitro and in vivo. Here, we investigated if SP boosts migration and cytotoxicity of natural killer cells, thus providing a further link between "innate immunity" and neurogenic inflammatory processes like asthma bronchiale. We demonstrate a dose-dependent effect of SP on natural killer cell migration with a maximal response at 10(-8) M SP. SP was shown to stimulate unstimulated as well as interleukin-2 (IL-2)-activated natural killer cells. Stimulation of natural killer cell migration was neurokinin-1 receptor dependent. Furthermore, mRNA encoding the neurokinin-1 receptor was demonstrated as being present in natural killer cells using RT-PCR while mRNA of the neurokinin-2 receptor was not detectable. Additionally, SP seems to influence specific cytotoxicity against Raji and K567 effector cells by a receptor-independent mechanism. In conclusion, our data indicate that functionally active neurokinin-1 receptors can be expressed by human natural killer cells. Substance P might therefore be a novel link between neural structures and innate immunity.     

Modulation of colonic motility by substance P, cholecystokinin and neuropeptide Y.

The effects of substance P, cholecystokinin and neuropeptide Y were examined on rabbit distal colonic motility. All three agents produced increased contractile activity but the mechanisms responsible differed depending on the agent tested. In the intact animal, peptide effects were measured under basal conditions and following exposure to atropine, tetrodotoxin and the alpha-adrenergic antagonist phentolamine. Administration of all three peptides resulted in a stimulation of colonic motility. Phentolamine did not significantly effect substance P-, cholecystokinin- or neuropeptide Y-induced activity. By contrast, the in vivo activity induced by cholecystokinin and neuropeptide Y, but not substance P, was nearly eliminated by tetrodotoxin. Only the neuropeptide Y response was partially atropine sensitive. In isolated colonic strips, cholecystokinin-induced activity, but not that produced by neuropeptide Y or substance P, was blocked by tetrodotoxin. Atropine did not significantly inhibit any of the hormone-induced contractions.     

On the nature of interaction of dodecyl sulfate with proteins. Evidence from uncharged polypeptides

Circular dichroism and equilibrium dialysis measurements in aqueous solution reveal no strong interaction between dodecyl sulfate and the unionized polypeptides poly(N⁵-ω-hydroxyethyl-L-glutamine), poly(N⁵-ω-hydroxypropyl-L-glutamine) and poly(N⁵-bis(ω-hydroxyethyl)-L-glutamine). Dodecyl sulfate does not affect the stability of the helical forms of poly(N⁵-ω-hydroxypropyl-L-glutamine) and poly(N⁵-bis(ω-hydroxyethyl)-L-glutamine) in water.     

Content of phospholipid, cerebroside and cerebroside sulfate in the central nervous system of mice with acute experimental viral demyelination

A study was made of the content of phospholipids, cerebrosides and cerebroside sulfates in the central nervous system of mice with experimental acute viral encephalomyelitis. No considerable changes in phospholipid content were revealed. A significant drop in the content of cerebrosides and cerebroside sulfates was defected in the CNS, being more pronounced in the spinal cord of sick animals. The reduction in the content of glycolipids can be explained by myelin disintegration and by the effect of viruses on the olygodendrocytes in which cerebrosides and cerebroside sulfates are synthesized.     

Distribution and origin of substance P and neuropeptide Y-immunoreactive nerves in the guinea-pig heart

Summary The localization and origin of substance P (SP)-, neuropeptide Y (NPY)-, and noradrenaline/tyrosine hydroxylase (NA/TH)-immunoreactive (IR) nerves in the guinea-pig heart were investigated by means of immunohistochemistry; quantitative analysis was performed by radioimmunoassay (NPY) and high performance liquid chromatography (NA). Both untreated animals and animals subjected to stellatectomy, combined stellatectomy and local capsaicin pretreatment of the vagal nerves or systemic application of capsaicin were studied. A dense network of SP-IR nerves was observed in the right atrium in different locations: (1) around local cardiac ganglion cells, (2) close to blood vessels, (3) within the myocardium, and (4) close to and within peri and endocardium.A moderately dense SP-innervation, mainly related to blood vessels, was found in the ventricles. Very dense networks of NPY and TH-IR nerve fibers with an overlapping distributional pattern around blood vessels and in the myocardium were seen in both the atria and the ventricles. In addition, some cell bodies in local cardiac ganglia were NPY-IR. Bilateral stellatectomy resulted in a reduction of SP-IR in the right atrium (55% of control), which was more pronounced after additional capsaicin pretreatment of the vagal nerves (44% of control).In the left ventricle no significant depletion of SP-IR was seen by either stellatectomy or combined stellatectomy and capsaicin treatment of the vagal nerves. It was not possible to establish any defined target areas within the heart for vagal or spinal SP-IR afferents by use of immunohistochemical methods. Systemic capsaicin treatment caused a total loss of SP-IR nerves in the heart. After bilateral stellatectomy the levels of NPY-IR and NA were reduced to about 10% of control in both the right atrium and left ventricle. In accordance, NPY and TH-IR nerves were also almost totally absent in the heart after bilateral stellatectomy.     

Effect of the neuropeptide substance P on the rat bone marrow-derived osteogenic cells in vitro

Substance P containing, thin, sensory nerve fibres have been demonstrated in bone and bone marrow. However the role of substance P in bone tissue is not fully understood. We investigated the effects of substance P on the growth and development of rat bone marrow-derived osteogenic cells in vitro. To examine this, the marrow-derived osteogenic cells were treated from 3rd to 6th day of subculture with substance P at concentrations 10(-10), 10(-9) and 10(-8)M. [(3)H]-thymidine, L-2,3-[(3)H]-proline incorporation, protein accumulation, alkaline phosphatase activity, and calcium deposition were measured in cultures. Substance P slightly stimulated [(3)H]-thymidine incorporation at 10(-10) M. Protein accumulation and L-2,3-[(3)H]-proline incorporation were enhanced in a dose dependent manner. Simultaneous application of spantide, a substance P receptor antagonist, could not block substance P-induced L-2,3-[(3)H]-proline incorporation probably because of statistically significant effect of spantide itself. Calcium deposits were significantly lower (about 30%) in cultures treated with SP. This effect was probably due in part by the fall in alkaline phosphatase activity which in substance P treated cultures was decreased about 17%. Our results indicate that substance P could be one of the factors modulating bone metabolism.     

Antinociceptive effect of spinal cholinergic stimulation: interaction with substance P

Activation of central muscarinic receptors results in an antinociceptive response in experimental animals. Employing intrathecal (i.t.) injection and radiant heat applied to a rat's tail as the experimental paradigm, a spinally-mediated antinociceptive response was obtained following injection of cholinergic agonists. Since "cholinergic' analgesia is mediated independently of the opiate system, the possibility was considered that this response was mediated through inhibition of the local release of substance P. Rats were prepared with indwelling i.t. catheters which terminated in the L2-L3 region of the spinal cord. I.t. injection of carbachol (0.05-5 micrograms) or neostigmine (1-10 micrograms), but not nicotine (0.5-10 micrograms) produced dose-related increases in tail flick latencies. Pretreatment with i.t. injection of atropine or hemicholinium-3 significantly inhibited the antinociceptive response to neostigmine. Spinal substance P levels were measured 30 min following 0.5 micrograms carbachol. Levels in the dorsal horn were reduced by 30% compared with saline controls. Levels in the ventral horn were unchanged by carbachol. These results support the role of endogenous spinal acetylcholine in pain modification and suggest an interaction with substance P neurons of the dorsal spinal cord.     

Improved version of the Kean partition assay for cerebroside sulfate

In the Kean method for the colorimetric determination of sulfatide (1968. J. Lipid Res. 9: 319-327), the lipid is partitioned together with a blue cationic compound between two phases formed from chloroform, methanol, and water. The blue cation enters the chloroform-rich phase only as an ion pair with the lipid. This method has been improved by the use of a new mixture of solvents in which the desired layer floats above the excess dye. The lower volatility of the new solvent system improves the reproducibility of the technique.     

Delay of neutrophil apoptosis by the neuropeptide substance P: involvement of caspase cascade

Neutrophil apoptosis is an important event in the resolution of inflammation. The role of substance P (SP) in neutrophil apoptosis has not been previously investigated. We found that substance P delays apoptosis in neutrophils. Human neutrophils were isolated and cultured up to 24 hours. Apoptosis was detected by light and electron microscopy, as well as DNA-fragmentation assays. Substance P delayed the spontaneous apoptosis of neutrophils at 6, 12, 18 and 24 hours in a dose-dependent fashion in the range of 10-100 microM. Whereas the both peptide neurokinin-1 (NK-1) receptor antagonists [D-Pro(2), D-Trp(7,9)]-SP and GR 82334 inhibited the substance P effect on neutrophils, the nonpeptide NK(1) receptor antagonist L-703.606 itself, an analogue of CP-96,345, induced apoptosis of neutrophils. Surprisingly, the effect of L-703.606 could be prevented by substance P. Western blotting results showed that the neuropeptide substance P inhibited the spontaneous apoptosis-associated caspase-3 activation in the same concentration range as described above. Parallel the inhibition of cleavage of focal adhesion kinase (FAK), a substrate of caspases could be observed by substance P. In conclusion, our results extend the range of biological effects of the neuropeptide substance P and provide new insight to the role of this tachykinin in the modulation of the inflammatory response by the nervous system.     

Evidence for the existence of substance P in the prepubertal rat ovary. II. Immunocytochemical localization

Nerve fibers containing substance P (SP) were localized in ovaries from juvenile and peripubertal rats by immunofluorescence. These fibers were closely associated with the theca externa of antral follicles, as well as being in the interstitial tissue and within the tunica adventitia of small blood vessels, mostly arterioles. Consistently, the greatest amount of SP immunoreactivity was observed surrounding the ovarian vasculature. Substance P was not detected in cells or within the corpora lutea (CL). Additionally, the peripubertal animals seemed to have a greater concentration of ovarian SP than the juvenile animals. Possible functional roles for this peptide in the ovary are discussed.     

Fast atom bombardment-collision activated dissociation-linked field scanning mass spectrometry of the neuropeptide substance P

Amino acid sequence-determining information is obtained from nanomole amounts of the underivatized, biologically important peptide substance P by combining fast atom bombardment, collision activated dissociation, and linked field scanning mass spectrometry. Protonated molecular ions of substance P are produced by fast atom bombardment mass spectrometry, accelerated to high translational energy (8 kV), and transit a collision chamber. Collision activated dissociations occur in the first field-free region. Amino acid sequence-determining ions are collected by scanning the magnetic and electric fields, keeping their ratio constant. In this manner, the precursor-product relationship among ions produced during fragmentation of the protonated molecular ion is firmly established.     

The neuropeptide substance P is a critical mediator of burn-induced acute lung injury

The classical tachykinin substance P (SP) has numerous potent neuroimmunomodulatory effects on all kinds of airway functions. Belonging to a class of neuromediators targeting not only residential cells but also inflammatory cells, studying SP provides important information on the bidirectional linkage between how neural function affects inflammatory events and, in turn, how inflammatory responses alter neural activity. Therefore, this study aimed to investigate the effect of local burn injury on inducing distant organ pulmonary SP release and its relevance to lung injury. Our results show that burn injury in male BALB/c mice subjected to 30% total body surface area full thickness burn augments significant production of SP, preprotachykinin-A gene expression, which encodes for SP, and biological activity of SP-neurokinin-1 receptor (NK1R) signaling. Furthermore, the enhanced SP-NK1R response correlates with exacerbated lung damage after burn as evidenced by increased microvascular permeability, edema, and neutrophil accumulation. The development of heightened inflammation and lung damage was observed along with increased proinflammatory IL-1beta, TNF-alpha, and IL-6 mRNA and protein production after injury in lung. Chemokines MIP-2 and MIP-1alpha were markedly increased, suggesting the active role of SP-induced chemoattractants production in trafficking inflammatory cells. More importantly, administration of L703606, a specific NK1R antagonist, 1 h before burn injury significantly disrupted the SP-NK1R signaling and reversed pulmonary inflammation and injury. The present findings show for the first time the role of SP in contributing to exaggerated pulmonary inflammatory damage after burn injury via activation of NK1R signaling.