Spinal Cord Membrane Preparations Research Articles

Time-course changes in ectonucleotidase activities during experimental autoimmune encephalomyelitis

The aim of the present study was to analyze the activities of extracellular purine metabolizing enzymes, CD39 (apyrase, EC 3.6.1.5) and CD73 (ecto-5’ nucleotidase, EC 3.1.3.5) in experimental autoimmune encephalomyelitis (EAE). The levels of ATP, ADP and AMP hydrolysis were analyzed in the blood serum and in the rat spinal cord plasma membrane preparation 8, 15 and 25 days after induction of EAE. The animals were divided in three groups: control (saline), CFA (adjuvant-only) and EAE (CFA and homogenate of spinal cords). Eight days after immunization, ATP, ADP and AMP hydrolysis in the blood serum and spinal cord membrane preparations were unaffected in EAE compared to both, control and CFA group. In the peak of disease, ATP, ADP and AMP hydrolysis in EAE group showed significant decrease in the blood serum and prominent increase in the spinal cord membrane preparation compared to CFA and control group. At the end of illness, as judged by disappearance of clinical manifestation of EAE, ATP, ADP and AMP hydrolysis, although closer to CFA levels, were still significantly different in respect to the CFA group. Modulation of ATP, ADP and AMP hydrolysis suggests that they operate during EAE and might represent the basis of novel therapeutic strategies in immune-mediated diseases, such as MS.

Identification and Functional Characterization of a 5-Transmembrane Domain Variant Isoform of the NTS2 Neurotensin Receptor in Rat Central Nervous System

The present study demonstrated that alternative splicing of the rat nts2 receptor gene generates a 5-transmembrane domain variant isoform (vNTS2) that is co-expressed with the full-length NTS2 receptor throughout the brain and spinal cord, as evidenced by reverse transcription-PCR. The vNTS2 polypeptide is 281 amino acids in length, which is 135 amino acids shorter than the full-length isoform. Immunohistochemical and radioligand binding studies revealed that the HA-tagged recombinant vNTS2 receptor is poorly targeted to plasma membranes in transfected COS-7 cells. Binding studies also showed that the truncated receptor displayed a 5000-fold lower affinity for neurotensin (NT) than its full-length counterpart (IC(50) of 10 mum and 2 nm, respectively). Yet NT binding induced efficient internalization of receptor-ligand complexes in vNTS2-transfected cells. Furthermore, it produced a rapid (<5 min) activation of the mitogen-activated protein kinases (ERK1/2) pathway, indicating functional coupling of the variant receptor. This activation is sustained (>1 h) and is also produced by the NTS2 agonist levocabastine. Western blotting experiments suggested that vNTS2 is not expressed in monomeric form in the rat central nervous system. However, it does appear to form a variety of multimeric complexes, including homodimers and heterodimers, with the full-length NTS2. Indeed, co-immunoprecipitation studies in dually transfected cells demonstrated that the two receptor isoforms can form stable associations. Taken together, the present results indicated that the rat vNTS2 is a functional receptor that may play a role in NT signaling in mammalian central nervous system.

Brain but Not Spinal NR2B Receptor Is Responsible for the Anti-Allodynic Effect of an NR2B Subunit-Selective Antagonist CP-101,606 in a Rat Chronic Constriction Injury Model

In order to examine the site of action of an NR2B subtype-selective NMDA antagonist CP-101,606, we investigated its analgesic effect in a rat model of neuropathic pain at various routes of administration. Mechanical allodynia was induced by chronic constriction injury (CCI) of the sciatic nerve in male Sprague-Dawley rats. Subcutaneous treatment of the animals with CP-101,606 at 10 mg/kg significantly inhibited CCI-induced mechanical allodynia. Intracerebroventricular injection of CP-101,606 at 10, 30 and 100 nmol also inhibited the mechanical allodynia in a dose-dependent manner, the statistically significant effect being achieved at the highest dose tested (100 nmol) without producing any behavioral abnormalities. However, intrathecal injection of CP-101,606 at a dose of 300 nmol failed to inhibit CCI-induced allodynia. A receptor binding assay using rat forebrain and spinal cord membrane preparations demonstrated that [³H]CP-101,606 bound to the brain NR2B receptor with a greater extent compared to the spinal cord one. These findings suggest that the anti-allodynia effect of CP-101,606 is ascribable to blockade of NR2B receptors at the brain, but not at the spinal cord. In contrast, intrathecal injection of a non-selective NMDA antagonist, memantine, significantly inhibited CCI-induced mechanical allodynia at a dose of 300 nmol, indicating the difference in the site of action between the non-selective NMDA antagonist and the NR2B-specific NMDA antagonist.

Studies of G Protein Activation of Orphanin FQ in the Cerebrum, Thalamus and Spinal Cord of Monkeys

Background: The aim of this in vitro study was to investigate []GTPS binding stimulated activation by orphanin FQ in monkey cerebral, thalamic, and spinal membranes. Methods: A rhesus monkey (Macaca mulatta, female, n = 1) was euthanized to obtain cerebral, thalamic, and spinal cord membrane preparations. In the orphanin FQ-stimulated []GTPS binding dose-response curve, (effective concentration 50, nanomolar) and maximum stimulation (% over basal) were determined in the absence or presence of each opioid receptor antagonist, namely, naloxone (20 nM), nor-BNI (3 nM), naltrindole (3 nM), or J-113397 (10 nM) antagonists of the , and nociceptin- opioid receptors respectively. Results: The values of EC50 and maximum stimulation of []GTPS binding were as follows: cortex (5.1 1.4 nM / 55.6 8.2%), thalamus (8.5 [ 1.3 nM / 27.8 4.9%), and spinal cord (11.3 ﾘ 0.2 nM / 15.2 4.5%). Maximum stimulation for these three membranes were significantly different (P ]GTPS binding dose-response curve, but the other opioid receptor antagonists had no effect. Conclusions: Maximum stimulation of []GTPS binding by OFQ in each membrane showed significantly different profiles, suggesting different pharmacologic efficacies by region. The OFQ-stimulated []GTPS bindings in this study were mediated via nociceptin-opioid peptide receptor stimulation.

Epibatidine binding sites and activity in the spinal cord

Epibatidine has been shown to be the most potent nicotinic agonist in several neuronal nicotinic receptor preparations. Similar to other nicotinic agonists, intrathecal (−)-epibatidine elicits dose-dependent increases in pressor, heart rate and pain responses in rats, as well as an increase in latency to withdraw from a noxious thermal stimulus. The latter response requires higher doses and is of shorter duration, suggesting interaction with multiple subtypes of spinal nicotinic receptors. In the present study, we relate the binding properties of (±)-[ 3H]epibatidine in spinal cord membrane preparations to the cardiovascular and behavioral responses. Unlike (−)-[ 3H]cytisine or (−)-[ 3H]nicotine, (±)-[ 3H]epibatidine reveals two sites; the ratio of high affinity to low affinity sites is 2:1. The rank ordering of potencies of the nicotinic agonists in displacing (±)-[ 3H]epibatidine binding from spinal cord membranes correlates with the potencies in eliciting cardiovascular and behavioral responses upon spinal administration. The nicotinic receptor antagonists, α-lobeline, dihydro- β-erythroidine and methyllycaconitine, also displayed similar rank ordering of potencies in displacing (±)-[ 3H]epibatidine, (−)-[ 3H]cytisine or (−)-[ 3H]nicotine binding to spinal nicotinic receptors. Virtually all the nicotinic analogs exhibited a Hill coefficient of less than one in competing with (±)-[ 3H]epibatidine to spinal cord membranes indicating their interaction with at least two classes of binding sites. Intrathecal (−)-epibatidine, in addition to eliciting an initial and subsequently a sustained pressor and tachycardic response, also exhibited a transient intervening bradycardia which coincided temporally with the duration of the analgesia. Repeated administration of (−)-epibatidine desensitized its responses as well as the cardiovascular and behavioral responses to spinal nicotine and cytisine. Intrathecal α-lobeline showed selectivity for blocking the analgesic response, whereas methyllycaconitine exhibited selectivity for the pressor and irritation responses. The NMDA receptor antagonist, AP-5, inhibited the pressor, tachycardic and irritation responses elicited by intrathecal (−)-epibatidine, confirming a role for spinal excitatory amino acids released by the nicotinic agonist. © 1997 Elsevier Science B.V. All rights reserved.

Metabolism of galanin and galanin (1-16) in isolated cerebrospinal fluid and spinal cord membranes from rat.

The occurrence of galanin (GAL) in the spinal cord and reports suggesting that it acts as an endogenous inhibitory spinal modulator in sensory/noxious transmission, have focused interest on its metabolism in the spinal cord. The metabolic half-lives and degradation patterns of GAL(1-29) and the high affinity N-terminal fragment GAL(1-16), were determined in isolated cerebrospinal fluid (CSF) from rats, and analysed by reverse phase HPLC. The half-lives for GAL(1-29) and GAL(1-16) in isolated rat CSF at 37 degrees C were 120 min and 60 min, respectively. The first degradation products which we could isolate and identify of GAL(1-16) were: GAL(3-16) and GAL(3-12) and for GAL(1-29): GAL(1-5) and GAL(1-4), all without affinity to spinal galanin receptors. Degradation studies of GAL(1-29) and GAL(1-16) in a spinal cord membrane preparation, in absence or presence of different protease inhibitors: E-64, pepstatin A, 3,4-DCI, bestatin, phosphoramidon, kelatorphan and thiorphan, or metal chelators: EDTA, EGTA and o-phenanthrolin, suggest that a phosphoramidon sensitive zinc-metalloprotease is mainly responsible for the degradation of GAL(1-29) and GAL(1-16), since both o-phenanthrolin (0.3 mM) and phosphoramidon (920 microM) substantially prolong their half-lives.

Structure-activity study of neuropeptide FF: contribution of N-terminal regions to affinity and activity.

Twenty neuropeptide FF (NPFF) analogs having various lengths were synthesized by solid-phase peptide synthesis to gain more information on the role of N-terminal residues for the NPFF receptor affinity. The affinities were evaluated in the rat spinal cord membrane preparations, and the biological activities were measured on morphine analgesia in the mouse tail-flick test. Shortening of the NPFF sequence from the N-terminal produced only a moderate decrease in affinity until NPFF (4-8) was reached. In the same way, NPFF(3-8) significantly decreased morphine analgesia, while NPFF(4-8) had no significant effect at a dose of 22 nmol. The introduction in the N-terminal part of NPFF of a D-enantiomer at positions 2 and 1 or the presence of an N-methyl group on position 3 did not modify affinity and activity. Substitution of proline5 by the D-isomer decreased the affinity of NPFF analogs whatever their length, and [Tyr1,D-Pro5]NPFF(1-8) was 2.5-fold less potent than [Tyr1]NPFF(1-8) in reversing morphine-induced analgesia. In contrast, the presence of a glycine residue in position 5 did not influence the affinity toward NPFF receptors. Data provide evidence that the N-terminal segment of neuropeptide FF is responsible for high-affinity binding.

Role of spinal opioid receptors in the antinociceptive interactions between intrathecal morphine and bupivacaine.

In studies on the clinical management of pain, a combination of morphine and bupivacaine is more effective than either of them alone in producing analgesia. The present study was designed to examine the effect of bupivacaine on morphine-induced antinociception as measured by the tail-flick test in the rat. To understand the basis of this interaction, the effect of bupivacaine on the binding of opioid ligands to their spinal opioid receptors in the rat also was investigated. Intrathecal administration of 5, 20, or 50 micrograms bupivacaine significantly potentiated the antinociception produced by intrathecal administration of 10 micrograms morphine. There was more than a 10-fold increase in the area under the curve (AUC0-60 min) for morphine-induced antinociception in the presence of bupivacaine. At higher doses of morphine (20 micrograms), bupivacaine was not very effective, increased AUC0-60 min for antinociception by only about 25%, and in fact significantly decreased the total duration of morphine-induced antinociception. Radioreceptor assays done with rat spinal cord membrane preparations revealed that bupivacaine (0.1-10 nM) inhibited the binding of specific ligands to mu-receptors but increased the binding to delta- and kappa-receptors. The authors conclude that the facilitation of morphine-induced antinociception by bupivacaine may be associated with a conformational change in the spinal opioid receptors induced by bupivacaine. Although increasing the binding of morphine to kappa-opioid receptors is the most prominent effect, the binding of opioid ligands to all spinal receptors is inhibited at high doses of bupivacaine.

Clonidine reduces substance P binding in rat spinal cord membrane preparation.

The effects of clonidine on substance P (SP) binding was investigated using rat brain and spinal cord membrane preparations preincubated with various concentrations of clonidine. [3H]SP specific binding in the spinal cord was significantly decreased with 10(-4) M clonidine, but no effect on binding was seen in the brain. Scatchard analysis of SP binding indicated that Bmax was significantly depressed without changing the affinity. The mechanism of clonidine-induced analgesia includes a spinal neural component and action on the SP receptors.

Clonidine Reduces Substance P Binding in Rat Spinal Cord Membrane Preparation

The effects of clonidine on substance P (SP) binding was investigated using rat brain and spinal cord membrane preparations preincubated with various concentrations of clonidine. [3H]SP specific binding in the spinal cord was significantly decreased with 10-4 M clonidine, but no effect on binding was seen in the brain. Scatchard analysis of SP binding indicated that Bmax was significantly depressed without changing the affinity. The mechanism of clonidine-induced analgesia includes a spinal neural component and action on the SP receptors.

P-464 - Clonidine reduces substance P binding in rat spinal cord membrane preparation.

P-464 - Clonidine reduces substance P binding in rat spinal cord membrane preparation.

Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study

An in vitro binding assay, using 125I-YLFQPQRFamide, a newly synthetized iodinated analog of FLFQPQRFamide, in which Phe 1 (F) has been substituted by a Tyr (Y), was developed to demonstrate and characterize putative binding sites of this brain morphine modulating peptide. This radioligand bound in a time-dependent manner to rat spinal cord membrane preparation. This binding was dose-dependent, saturable and reversible. Both kinetic data and saturation measured at equilibrium lead to the existence of a homogenous population of high affinity binding sites with a K d value of 0.09–0.1 nM and a maximal capacity B max of 14.5 ± 2fmol/mg protein. Results of competition experiments show that both FLFQPQRFamide and its analog YLFQPQRFamide had a similar capacity to inhibit the 125I-YLFQPQRFamide binding, suggesting that this radioiodinated analog is a good tool to study binding characteristics of FLFQPQRFamide receptors. The related octadecapeptide AGEGLSSPFWSLAAPQRFamide, another mammalian morphine modulating peptide competes for radioligand binding with similar potency. Our results also show that μ, δ and κ opiate receptor agonists as well as the antagonist naloxone were not able to affect binding either in presence or in absence of 120 mM NaCl. Together, these data demonstrate that FLFQPQRFamide does not function as an endogenous opiate receptor antagonist and that its capacity to reduce opiate-induced analgesia is supported by specific binding sites.

Functional coupling of GABA A receptors and benzodiazepine recognition site subtypes in the spinal cord of the rat

The interaction between GABA A receptors and benzodiazepine (BZD) recognition site subtypes in the spinal cord of the rat was investigated. Computer analysis of displacement curves for [ 3H]flunitrazepam ([ 3H]FNT) binding by 2-oxo-quazepam (2OXOQ) indicated the presence of two subtypes of BZD recognition sites in this region. Type I sites accounted for approximately 25% of the total number of BZD recognition sites, the remainder being Type II sites. A similar proportion of Type I and Type II sites was obtained by Scatchard analysis of the saturation curves for [ 3H]FNT, [ 3H]2OXOQ and [ 3H]ethyl-β-carboline-3-carboxylate ([ 3H]βCCE) binding. The in vitro addition of GABA (10 −8-10 −4 M) to spinal cord membrane preparations produced an increase in the binding of [ 3H]FNT and [ 3H]2OXOQ. The maximal enhancement produced by GABA was 50 and 82% above control values for [ 3H]FNT and [ 3H]2OXOQ, respectively. In contrast, GABA stimulated both [ 3H]FNT and [ 3H]2OXOQ binding in the cerebellum to a similar extent. We also evaluated the effects of different ligands for BZD recognition sites on the binding of [ 3H]GABA to spinal cord membranes, as compared with brain areas containing a higher proportion (> than 30%) of Type I sites. Diazepam, quazepam and the β-carboline, ZK 93423, enhanced the specific binding of [ 3H]GABA in a concentration-dependent manner (10 −7-10 −5 M) in the cerebral cortex and hippocampus but not in the spinal cord and cerebellum. These results indicate that there is a regional variation in the interaction between GABA and BZD recognition sites in the central nervous system.

Characteristics and specific localization of receptors for atrial natriuretic peptides at non-neuronal cells in cultured mouse spinal cord cells

Characteristics of atrial natriuretic peptide receptors were determined in cultured mouse spinal cord cells. Saturation and competition experiments demonstrated the presence of a single class of atrial natriuretic peptide binding sites with high affinity (K D = 0.054 nM) and a density of 1.92 fmoles/10 6 cells. A similar affinity (K D = 0.070 nM) was observed in rat spinal cord membrane preparations. These atrial natriuretic peptide binding sites were functional receptors since the treatment of cells with atrial natriuretic peptide increased cyclic guanosine monophosphate levels within these cells in a classical time-dependent manner. When atrial natriuretic peptide was applied onto the cell body of intracellularly recorded spinal cord neurons, this peptide did not evoke a change of the input resistance or of the resting membrane potential value. Light-microscopic autoradiography studies showed that no atrial natriuretic peptide binding could be detected on typical birefringent neurons but it could be located on astroglial and epithelial cells as identified by immunocytochemical markers. These results show that functional atrial natriuretic peptide receptors with high affinity exist in cultured mouse spinal cord cells and are not located on neurons. The presence of atrial natriuretic peptide receptors on astrocytes suggests that this neuropeptide might be a good candidate for neuron-glial communication. As the atrial natriuretic peptide binding sites previously shown in epithelia responsible for maintaining fluid and electrolyte gradients, the atrial natriuretic peptide receptors on epithelial cells in these spinal cord cultures may be involved in vivo in the control of water balance in the central nervous system.

A quantitative study of the developmental expression of nerve growth factor (NGF) receptor in rats

The developmental expression of nerve growth factor (NGF) receptor was quantitated in either homogenates or plasma membrane-enriched preparations from whole rat embryos or from isolated tissues. The assay involved crosslinking 125I-NGF to receptors followed by immunoprecipitation with a monoclonal antibidy to rat NGF receptor. In some cases, the pellet was resuspended and subjected to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiographic analysis. The NGF receptor was found in whole embryo homogenates as early as embryonic Day 10 (E10) (earliest age examined). The NGF receptor content in whole embryos per milligram protein increased about 3-fold from E11 to E18 and decreased slightly at E20. SDS-PAGE autoradiography showed that the molecular weights of 125I-NGF-bound receptors did not vary with age. The NGF receptor content in sciatic nerve homogenates decreased 23-fold from newborn to adulthood. The change of NFG receptor level in hindleg muscle had a profile similar to that seen in sciatic nerve. The NGF receptor content in superior cervical ganglion (SCG) or dorsal root ganglion (DRG) homogenate preparations was expressed in two ways. (1) On a per milligram protein basis, in SCG, the receptor density was decreased slightly from E20 to adulthood; in DRG, it was relatively constant from E15 through postnatal Day 0 (PND-0) and then dropped 6.7-fold in adults. (2) On a per ganglion basis, in SCG, it increased 4.4-fold from E20 to adult; in DRG, it increased 9-fold from E15 to PND-0 and then stayed constant through adulthood. In brain membrane preparations, the NGF receptor level decreased 11-fold from E15 to adulthood. In spinal cord membrane preparations, it decreased 7-fold from E18 to adulthood. Levels of receptor in cord were always greater than in brain. These data suggest that alterations in the NGF receptor density may have a role in changes in tissue responsiveness to NGF during development.