Characterization of endoproteolytic processing of dynorphins by proprotein convertases using mouse spinal cord S9 fractions and mass spectrometry

Dynorphins are important neuropeptides with a central role in nociception and pain alleviation. Many mechanisms regulate endogenous dynorphin concentrations, including proteolysis. Proprotein convertases (PCs) are widely expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous big dynorphin (BDyn) and dynorphin A (Dyn A) levels has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild-type (WT), PC1^−/+ and PC2^−/+ animals and mass spectrometry. Our results clearly demonstrate that both PC1 and PC2 are involved in the proteolysis regulation of BDyn and Dyn A with a more important role for PC1. C-terminal processing of BDyn generates specific peptide fragments dynorphin 1–19, dynorphin 1–13, dynorphin 1–11 and dynorphin 1–7, and C-terminal processing of Dyn A generates dynorphin 1–13, dynorphin 1–11 and dynorphin 1–7, all these peptide fragments are associated with PC1 or PC2 processing. Moreover, the proteolysis of BDyn leads to the formation of Dyn A and Leu-Enk, two important opioid peptides. The rate of formation of both is significantly reduced in cellular fractions of spinal cord mutant mice. As a consequence, even the partial inhibition of PC1 or PC2 may impair the endogenous opioid system.     

Characterization and localization of adenosine receptors in rat spinal cord

Adenosine A1 receptors were characterized in membranes from rat dorsal and ventral spinal cord using [3H] cyclohexyladenosine [( 3H]CHA) and compared with those in brain. For determination of anatomical loci of adenosine A1 receptors in the dorsal and ventral spinal cord, various lesions were employed, including kainic acid injections directly into the lumbar dorsal spinal cord, spinal cord hemitransections, dorsal rhizotomies, and neonatal capsaicin treatment. In control animals a single high affinity binding component was observed in dorsal and ventral spinal cord with KD values of 2.3 and 2.6 nM and Bmax values of 170 and 123 fmol/mg of protein, respectively. In comparison, [3H]CHA binding to whole brain membranes exhibited KD and Bmax values of 2.3 nM and 301 fmol/mg of protein, respectively. The IC50 values for CHA, (-)-phenylisopropyl adenosine, adenosine-5'-ethylcarboxamide, 2-chloroadenosine, (+)-phenylisopropyl adenosine, and theophylline to displace [3H]CHA were 3.6, 2.3, 15, 17, 21, and 30,500 nM for dorsal horn and 5.1, 2.7, 9.8, 24, 25, and 21,000 nM for ventral horn. The potencies of the various ligands are similar to those found for brain tissue. Injection of kainic acid directly into the dorsal spinal cord significantly reduced specific [3H]CHA binding by 33% in this tissue when compared to values from saline-injected control animals. This decrease was accompanied histologically by the depletion of intrinsic neuronal cell bodies and extensive gliosis at the injection site. Terminals of descending or primary afferent systems appear not to contain [3H]CHA-binding sites since lesions which interrupt these systems failed to alter the levels of [3H]CHA receptors in denervated spinal cord tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional spinal cord and brain blood flows in the rat

Regional spinal cord blood flow and blood flow in different brain areas were measured in 18 rats by the microsphere technique. By the use of spheres labeled with two different isotopes injected 5 min apart, double determinations were made. The consistency of the results from the two injections was very good. Blood flow was highest in the lumbar part of the spinal cord, somewhat lower in the lower thoracic part, lowest in the upper thoracic part, and again higher in the cervical cord; the mean values of the two determinations were 0.98, 0.74, 0.42, and 0.48 ml X min-1 X g-1 tissue for these regions, respectively. The brain areas investigated were the brain stem, cerebellum, and right and left hemispheres; the respective mean values of the two blood flow determinations in these areas were 0.54, 0.58, 0.33, and 0.38 ml X min-1 X g-1 tissue.     

Immunohistochemical localization of enkephalin in rat brain and spinal cord.

The distribution of immunoreactive enkephalin in rat brain and spinal cord was studied by immunoperoxidase staining using antiserum to leucine-enkephalin ([Leu5]-enkephalin) or methionine-enkephalin ([Met5]-enkephalin). Immunoreactive staining for both enkephalins was similarly observed in nerve fibers, terminals and cell bodies in many regions of the central nervous system. Staining of perikarya was detected in hypophysectomized rats or colchicine pretreated rats. The regions of localization for enkephalin fibers and terminals include in the forebrain: lateral septum, central nucleus of the amygdala, area CA2 of the hippocampus, certain regions of the cortex, corpus striatum, bed nucleus of the stria terminalis, hypothalamus including median eminence, thalamus and subthalamus; in the midbrain: nucleus interpeduncularis, periaqueductal gray and reticular formation; in the hind brain: nucleus parabrachialis, locus ceruleus, nuclei raphes, nucleus cochlearis, nucleus tractus solitarii, nucleus spinalis nervi trigemini, motor nuclei of certain cranial nerves, nucleus commissuralis and formatio reticularis; and in the spinal cord the substantia gelatinosa. In contrast enkephalin cell bodies appear sparsely distributed in the telencephalon, diencephalon, mesencephalon and rhombencephalon. The results of the histochemical staining show that certain structures which positively stain for enkephalin closely correspond to the distribution of opiate receptors in the brain and thus support the concept that the endogenous opiate peptides are involved in the perception of pain and analgesia. The localization of enkephalin in the preoptic-hypothalamic region together with the presence of enkephalin perikarya in the paraventricular and supraoptic nuclei suggest a role of enkephalin in the regulation of neuroendocrine functions.     

Distribution of adenosine deaminase activity in rat brain and spinal cord

The activity of adenosine deaminase (ADA) was measured in 62 discrete regions of the CNS, and in some autonomic and sensory ganglia, peripheral nerves, and peripheral tissues of the rat using an automated high-pressure liquid chromatography (HPLC) method. The formation of inosine and hypoxanthine as a measure of ADA activity in homogenates of brain was optimal at pH 7.0, linear for up to 60 min at 37 degrees C using 500 microM adenosine as substrate, and linear with protein concentrations ranging from 0.05 to 0.8 mg. The Km and Vmax values for ADA activity in homogenates of whole brain were 47 microM and 107 nmol/mg protein/30 min, respectively. Among the CNS regions examined, the highest activity was found in posterior hypothalamic magnocellular nuclei and the lowest in hippocampus. In general, spinal cord contained relatively low levels of ADA activity, with that in dorsal cord approximately 40% higher than ventral cord. In the periphery, parasympathetic ganglia contained higher levels of ADA than sensory ganglia and brain. Most peripheral tissues--including adrenal gland, lung, liver, and anterior and posterior pituitary--exhibited activity comparable to levels in the posterior hypothalamus. ADA activity in thymus was about 10 times higher than any other tissue examined. The uneven distribution of ADA activity in the rat CNS corresponds well with the immunohistochemical localization of this enzyme in discrete neural systems of this species. Structures that contain high ADA activity exhibit intense ADA immunostaining of neuronal perikarya and/or fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of substance P release from the intermediate area of rat thoracic spinal cord

Substance P (SP) nerve terminals innervate the intermediolateral cell column (IML) of the thoracic spinal cord, where SP coexists with serotonin (5-HT), neurokinin A (NKA) and thyrotropin-releasing hormone (TRH). Neither the depolarization-induced release of SP nor the presence of other neurochemicals in the regulation of SP release has been directly studied in this system. In the present study, basal and K⁺-stimulated release of SP from the microdissected intermediate area (including the IML, intercalated nucleus and central autonomic nucleus) of the rat thoracic spinal cord, and the regulation of SP release by presynaptic autoreceptors and by coexisting neurochemicals (5-HT, NKA and TRH) were studied using an in vitro superfusion system. Potassium evoked a concentration- and extracellular Ca²⁺-dependent release of SP. In rats pretreated with the serotoninergic neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), both SP content and the absolute amount of SP released were decreased. However, the fraction of the remaining tissue content of SP released by K⁺ depolarization was not changed subsequent to 5,7-DHT treatment. Moreover, 5-HT, 5-HT_1B agonists (CGS-12066B and RU 24969) and a 5-HT₃ agonist (2-methyl-5-HT) did not alter the K⁺-evoked release of SP. These data demonstrate that SP is released from the intermediate area of the rat thoracic spinal cord and some of the SP released comes from serotoninergic nerve terminals. Although 5-HT coexists with SP in the IML, neither endogenous 5-HT nor 5-HT receptor ligands appear to regulate the release of SP. Other colocalized neuropeptides (NKA and TRH) are not involved in the regulation of SP release because neither NKA, a NK₂ agonist (GR 64349) nor a TRH analog (MK-771) changed the K⁺-evoked release of SP. A neurokinin-1 (NK₁) antagonist (GR 82334) dose-dependently (10^-9-10^-7 M) increased the K⁺ stimulated release of SP. These data suggest the presence of presynaptic inhibitory NK₁ autoreceptors. Whereas, NK₁ agonists, [GR 73632 (10^-9-10^-6 M) and [Sar⁹, Met (O₂)^1]] SP (10^-8-10^-6 M)], increased the basal and K⁺-stimulated release of SP, the excitatory effects of GR 73632 were not blocked by the NK₁ antagonist. Moreover, GR 73632 increased the efflus of SP to a greater extent in the absence of peptidase inhibitors. Thus, the effect of NK₁ agonists on the release of SP may be related to an inhibition of peptide degradation rather than activation of NK₁ autoreceptors. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord

Recent advances in the isolation and characterization of neural precursor cells suggest that they have properties that would make them useful transplants for the treatment of central nervous system disorders. We demonstrate here that spinal cord cells isolated from embryonic day 14 Sprague-Dawley and Fischer 344 rats possess characteristics of precursor cells. They proliferate as undifferentiated neurospheres in the presence of EGF and bFGF and can be maintained in vitro or frozen, expanded and induced to differentiate into both neurons and glia. Exposure of these cells to serum in the absence of EGF and bFGF promotes differentiation into astrocytes; treatment with retinoic acid promotes differentiation into neurons. Spinal cord cells labeled with a nuclear dye or a recombinant adenovirus vector carrying the lacZ gene survive grafting into the injured spinal cord of immunosuppressed Sprague-Dawley rats and non-immunosuppressed Fischer 344 rats for up to 4 months following transplantation. In the presence of exogenously supplied BDNF, the grafted cells differentiate into both neurons and glia. These spinal cord cell grafts are permissive for growth by several populations of host axons, especially when combined with exogenous BDNF administration, as demonstrated by penetration into the graft of axons immunopositive for 5-HT and CGRP. Thus, precursor cells isolated from the embryonic spinal cord of rats, expanded in culture and genetically modified, are a promising type of transplant for repair of the injured spinal cord.     

1H-MRS in spinal cord injury: acute and chronic metabolite alterations in rat brain and lumbar spinal cord

A variety of tests of sensorimotor function are used to characterize outcome after experimental spinal cord injury (SCI). These tests typically do not provide information about chemical and metabolic processes in the injured CNS. Here, we used (1) H-magnetic resonance spectroscopy (MRS) to monitor long-term and short-term chemical changes in the CNS in vivo following SCI. The investigated areas were cortex, thalamus/striatum and the spinal cord distal to injury. In cortex, glutamate (Glu) decreased 1 day after SCI and slowly returned towards normal levels. The combined glutamine (Gln) and Glu signal was similarly decreased in cortex, but increased in the distal spinal cord, suggesting opposite changes of the Glu/Gln metabolites in cortex and distal spinal cord. In lumbar spinal cord, a marked increase of myo-inositol was found 3 days, 14 days and 4 months after SCI. Changes in metabolite concentrations in the spinal cord were also found for choline and N-acetylaspartate. No significant changes in metabolite concentrations were found in thalamus/striatum. Multivariate data analysis allowed separation between rats with SCI and controls for spectra acquired in cortex and spinal cord, but not in thalamus/striatum. Our findings suggest MRS could become a helpful tool to monitor spatial and temporal alterations of metabolic conditions in vivo in the brain and spinal cord after SCI. We provide evidence for dynamic temporal changes at both ends of the neuraxis, cortex cerebri and distal spinal cord, while deep brain areas appear less affected.     

Embryonic brain precursors transplanted into kainate lesioned rat spinal cord

Embryonic day 14 rat cerebral cortex-derived precursors were expanded with FGF2 and labeled with BrdU prior to being transplanted into the kainic acid-lesioned adult rat spinal cord. While these precursors give rise to cells with neuronal, astrocytic and oligodendroglial phenotypes vitro, they remained largely undifferentiated up to 12 weeks in vivo. Numerous BrdU-labeled cells were found in injured gray matter, and also lining the dilated central canal that sometimes accompanies these lesions. BrdU-labeled cells never co-expressed Map2ab, rarely co-expressed GFAP but often co-expressed nestin, even after 12 weeks in vivo. These observations suggest that the environment of the kainic acid-injured spinal cord is not hostile to transplanted embryonic cerebral cortex-derived precursors, but also is not conducive to their neuronal differentation.     

Immunofluorescent labeling of tight junctions in the rat brain and spinal cord

Tight junctions may play an important role in maintaining the integrity of the blood-brain barrier. These junctions can be individually visualized using electron microscopy but no current technique is able to provide a more global picture of the presence and density of tight junctions in central nervous system tissue. We used an antibody that recognizes a high molecular weight protein (ZO-1) associated with tight junctions, to identify these specialized junctions within the rat brain and spinal cord. Immunofluorescent labeling showed a network of tight junctions between cells in the brain vasculature, leptomeninges and choroid plexus, and between tanycytes lining the floor of the third ventricle and the central canal of the spinal cord. Anti-ZO-1 labeled the majority of cells associated with the blood-brain barrier and may prove a useful marker, possibly in conjunction with functional dye studies, in evaluating the anatomical and functional integrity of the blood-brain barrier.     

Transient expression of NGF-receptor-like immunoreactivity in postnatal rat brain and spinal cord

The pattern of expression of nerve growth factor (NGF)-receptor-like immunoreactivity during postnatal development in rat central nervous system (CNS) was analyzed using immunohistochemical localization of the receptor. Interestingly, in addition to the expected staining in basal forebrain, several structures were strongly labelled only during specific postnatal developmental stages. These structures included fibers in the thalamus, the external granule cell layer of the cerebellum and motor neurons, indicating that specific neurotrophic mechanisms might play an important role for the labelled cells during a precisely defined period.     

The opioid octapeptide Met-enkephalin-Arg-Gly-Leu: Characterization and distribution in rat spinal cord

The regional quantitation, immunohistochemical localization and molecular heterogeneity of Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ were examined in rat spinal cord with a specific radioimmunoassay. A rostrocaudal gradient in Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ content was observed; the highest levels occurred in sacral cord. Dorsal cord content was higher than that of ventral cord at all spinal segments. Immunohistochemical staining supported and refined the latter observation: a dense network of perikarya and fibers was found in Laminae I and II of the dorsal horn. Cell bodies were frequently observed in lamina IV. Additional terminals were seen around the central canal and in the ventral gray matter, often outlining perikarya of motor neurons. Total Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ immunoreactivity could be fractionated into two main components using gel filtration chromatography. Nearly half of the total immunoreactivity eluted as a high molecular weight peptide; the other half which co-eluted with Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ was further identified to be authentic Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ on reverse phase high pressure liquid chromatography. The present data, in conjunction with our previous study of Met⁵-enkephaline and Met⁵-enkephalin-Arg⁶-Phe⁷ indicates that all opioid peptides derived from preproenkephalin A are present in spinal cord and most likely are stored in the same neurons. Immunohistochemical localization of Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ in dorsal and ventral cord suggest a role for this peptide in both sensory and motor integration.     

Simultaneous functional magnetic resonance imaging in the rat spinal cord and brain

Functional magnetic resonance imaging (fMRI) method was developed to investigate the pattern and temporal relationship in neuronal pathways of brain and spinal cord. Signal intensity changes correlating with stimulation patterns were observed simultaneously in the rat spinal cord and brain using fMRI at 9.4 T. Electrical stimulation of the forepaw was used to elicit activity. A quadrature volume RF coil covering both brain and the cervical spinal cord was used. Sets of fast spin echo (FSE) images were acquire simultaneously for both brain and spinal cord fMRI. Experiments were repeated in single animal and across animals. Activities within the dorsal horn of the spinal cord and within the somatosensory cortex were observed consistently within each animal as well as across animals.     

Amino acid uptake in the spinal cord and brain of the rat after short-term spinal hemisection

The uptake of [³H]lysine was used to study the amino acid incorporation into trichloracetic acid-precipitable protein and soluble fractions of the spinal cord of rats subjected to a left hemisection at T2. Groups of animals were studied 3, 6, and 12h and 1, 3, 7, and 14 days after hemisection. Each group consisted of two sham-operated, and five spinal hemisected animals; five intact normal animals were also studied. Samples were taken from left and right somatomotor and occipital cortex and from both sides of the spinal cord as far as 16 mm rostral and caudal to the site of hemisection. Amino acid uptake into the protein and acid-soluble fractions exhibited significant alteration in [³H]lysine incorporation with time in both sham-operated and operated animals. Radioactivity of the trichloracetic acid-soluble fraction was elevated above normal in both groups at 3 h postoperative in the spinal cord and by 6 h in the brain. However, overall increases in acid-precipitable protein radio-activity did not appear until 6 h after hemisection and 6 h after the sham operation. These increases appeared to be associated with operative stress and/or trauma. At the site of lesion, local elevations in the soluble-fraction radioactivity were noted as early as 3 h after hemisection and as far as 4 mm rostral. Elevations in the acid-precipitable protein at the lesion site were not seen until 3 days postoperative and were generally restricted to the left (hemisected) side of the spinal cord. The local increases were attributed to increases in neuronal, neuroglial, and infiltrating cell metabolism possibly associated with regenerative changes.     

Characterization of tissue-derived macromolecules affecting transmitter synthesis in rat spinal cord neurons

Rat spinal cord cells maintained in neuron-rich cultures were exposed to extracts of skeletal muscle or to medium conditioned by non-neuronal cells. The conditioned media enhanced neuronal acetylcholine (ACh) synthesis, choline acetyltransferase activity, and protein synthesis, and decreased gamma-aminobutyric acid (GABA) synthesis. Muscle extract prepared from newborn rats produced similar enhancements but did not depress GABA synthesis. Muscle extracts prepared from normal and denervated adult rat limbs contained relatively little activity. These results suggest that different molecular factors might mediate the effects on GABA and ACh synthesis. Gel filtration of conditioned media and muscle extracts revealed that all of these activities were confined to a macromolecular fraction with an apparent Mr of 40,000. These tissue-derived factors affecting neuronal protein and transmitter synthesis are in turn distinct from a neuronal survival-promoting factor obtained from serum (Kaufman, L. M., and J. N. Barrett (1983) Science 220: 1394-1396).     

Characterization of sodium-dependent, high-affinity serotonin uptake in rat spinal cord synaptosomes

Synaptosomal accumulation of [3H]serotonin was used to determine if the rat spinal cord possesses a high-affinity neuronal uptake system for serotonin. Two temperature-dependent accumulation processes were found, one sodium-dependent, the second sodium-independent. Sodium-dependent [3H]serotonin accumulation was linear with sodium concentrations up to 143 mM, was associated with the purified synaptosomal fraction (P2B), and decreased 76% by osmotic lysis, 88% by sonication, and 96% by 0.1% Triton X-100. Drug inhibition studies demonstrated fluoxetine to be the most potent inhibitor of this system (IC50 0.075 microM) while desipramine (IC50 0.43 microM) and nomifensine (IC50 0.95 microM) were less potent. Kinetic analysis revealed that sodium-dependent accumulation in purified synaptosomes was saturable at low [3H]serotonin concentrations (Ku = 50 nM, Vmax = 4 pmol/mg protein/min). Sodium-independent [3H]5-HT accumulation was substantially less sensitive to fluoxetine, desipramine and nomifensine. While sodium-independent accumulation was not significantly affected by osmotic lysis, it was markedly increased by prior sonication of tissue. Also, in contrast to sodium-dependent accumulation, sodium-independent accumulation was evenly distributed in all tissue fractions, and was not saturable at low [3H]serotonin concentrations. It is concluded that sodium-dependent [3H]serotonin accumulation reflects uptake into spinal serotonergic nerve terminals while sodium-independent accumulation probably reflects a temperature-sensitive binding to membrane fragments. Comparison to brain uptake of serotonin and the necessity for using 37 degrees C sodium-free blanks rather than 0 degree C blanks in spinal cord homogenates is discussed.     

Characterization of nerve growth factor binding to embryonic rat spinal cord neurons

The binding of iodinated beta-nerve growth factor, [125I]-NGF, to embryonic (E16) rat spinal cord cells, was investigated to characterize the binding properties and cellular distribution of nerve growth factor receptors. Spinal cord cells prepared without trypsin yielded two classes of NGF binding sites with Kd's of 3 x 10(-11) M and 4 x 10(-9) M. Fractionation of the cells by discontinuous gradients composed of 8%, 12%, and 17% metrizamide was used to separate motoneurons from other cell types. The motoneuron enriched fraction (8% metrizamide) contained approximately 10% of the cells and 64% of the choline acetyltransferase (ChAT) activity. In contrast, the 12% metrizamide fraction contained most (51%) of the cells and 36% of the ChAT activity, while the 17% metrizamide fraction contained the remainder of the cells and negligible amounts of ChAT activity. Characterization of [125I]-NGF binding to each metrizamide fraction showed that the motoneuron-enriched fraction exhibited both high and low affinity binding sites, while the other metrizamide fractions exhibited only the low affinity binding sites. These findings indicate that although low affinity NGF receptors appear to be relatively evenly distributed amongst embryonic rat spinal cord cells, high affinity NGF receptors are found primarily on motoneurons.     

Solubilization of the glycine receptor from rat spinal cord

Glycine receptors, as detected by glycine-displaceable [3H]strychnine binding, were solubilized from a membrane fraction of rat spinal cord by the non-ionic detergent Triton X-100. The solubilized material retained its high affinity for [3H]strychnine and exhibited the typical pharmacological properties of the membrane-bound glycine receptor. On sucrose density gradients, the solubilized receptor had a sedimentation coefficient of 8.3 +/- 0.4 S. Gel exclusion chromatography on Sepharose 6 B in the presence of phosphatidylcholine gave a Stokes radius of 7.3 +/- 0.3 nm.     

Intramedullary spinal cord metastasis and multiple brain metastases from urothelial carcinoma

Intramedullary spinal cord metastases (ISCM) are rare spinal cord neoplasms associated with severe neurological deterioration and poor life expectancy. However, their incidence is expected to increase as a result of advances in diagnostic techniques and longer survival of patients with cancer due to improvements in cancer therapy. Reports on ISCM from primary urothelial carcinoma are virtually non existent. We report a 74-year-old male patient with a significant history of a high-grade urothelial carcinoma who presented with progressive back pain and concomitant weakness, grade 3–4/5 proximally and 0–1/5 distally, and distal hyperesthesia and hyperalgesia, particularly of the left lower limb. MRI revealed a contrast-enhancing intramedullary lesion at Th11/Th12. Laminectomies of Th11/Th12 and lesion resection were performed. Postoperative histopathological examinations confirmed the metastatic nature of the lesion. Subsequently the patient developed multiple brain metastases. Radiation therapy was refused by the patient. We conclude that ISCM are devastating complications of systemic cancer. Early and thorough diagnosis, as well as carefully considered and prompt therapy, is important for minimizing the patient’s functional deficit, thus improving quality of life.