Automated on-line extraction and profiling of lipoxygenase products of arachidonic acid by high-performance liquid chromatography.
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Animals↗
Biomedical subjects
S Bourgoin
Publications and source records attributed to S Bourgoin.
Subcutaneous formalin induces a segmental release of Met-enkephalin-like material from the rat spinal cord.
The possible influence of a noxious chemical stimulus on the activity of spinal enkephalinergic neurones was examined by measuring the outflow of Met-enkephalin-like material (MELM) in CSF perifusates from the lumbar and cervico-trigeminal zones in halothane-anaesthetized rats. Following subcutaneous injection of 50 microliters of a 10% formalin solution in saline into the muzzle, MELM outflow increased at the cervico-trigeminal level but not at the lumbar level. Conversely, a significant enhancement in MELM outflow occurred at the lumbar but not at the cervico-trigeminal level when formalin was injected into a hind paw. In both cases, the increase in MELM release took place 5-10 min after the treatment and was of short duration (5-10 min). It is concluded that noxious chemical stimuli can induce a transient activation of enkephalinergic neurones within spinal zones receiving the nociceptive inputs. The marked differences in the characteristics of spinal MELM overflow due to noxious chemical, thermal and mechanical stimuli further support the notion that each type of nociceptive stimulus probably activates spinal enkephalinergic systems by triggering distinct neuronal mechanisms.
Animals↗
Regional distribution of calcitonin gene-related peptide-, substance P-, cholecystokinin-, Met5-enkephalin-, and dynorphin A (1-8)-like materials in the spinal cord and dorsal root ganglia of adult rats: effects of dorsal rhizotomy and neonatal capsaicin.
Biochemical mapping of five different peptide-like materials--calcitonin gene-related peptide (CGRP), substance P (SP), Met5-enkephalin (ME), cholecystokinin (CCK), and dynorphin A (1-8) (DYN)--was conducted in the dorsal and ventral zones of the spinal cord at the cervical, thoracic, and lumbar levels in 3-month-old rats 10 days after unilateral dorsal rhizotomy at the cervical level (C4-T2) or after neonatal administration of capsaicin (50 mg/kg s.c.). In control rats, all peptide-like materials were more abundant in the dorsal than in the ventral zone all along the spinal cord. However, in both zones, absolute concentrations of CGRP, SP, ME, and CCK were significantly higher at the lumbar than at the cervical level. Rhizotomy-induced CGRP depletion (-85%) within the ipsilateral dorsal zone of the cervical cord was more pronounced than that due to neonatal capsaicin (-60%), a finding suggesting that this peptide is contained in both capsaicin-sensitive (mostly unmyelinated) and -insensitive (myelinated) primary afferent fibers. In contrast, similar depletions of SP (-50%) were observed after dorsal rhizotomy and neonatal capsaicin treatment, as expected from the presence of SP only in the capsaicin-sensitive small-diameter primary afferent fibers. Although the other three peptides remained unaffected all along the cord by either intervention, evidence for the existence of capsaicin-insensitive CCKergic primary afferent fibers could be inferred from the increased accumulation of CCK (together with SP and CGRP) in dorsal root ganglia ipsilateral to dorsal root sections.
Afferent Pathways↗
Intrathecal porcine calcitonin enhances the release of [Met5]enkephalin-like material from the rat spinal cord.
Perfusion of the intrathecal space of halothane-anaesthetized rats with artificial cerebro-spinal fluid supplemented with porcine calcitonin (1-10 microM) produced a significant increase (+67-110%) in the spinal release of [Met5]enkephalin-like material. The effect of porcine calcitonin was markedly enhanced (+100%) in animals receiving a continuous i.v. infusion of the opioid antagonist naloxone (65 micrograms/kg per min). These data strongly suggest that the antinociceptive effect of intrathecal porcine calcitonin might involve a stimulatory action of the hormone on spinal enkephalinergic interneurones. In addition, presynaptic opioid autoreceptors probably control the activity of these neurones in the rat.
Animals↗
5-HT3 receptor binding sites are on capsaicin-sensitive fibres in the rat spinal cord.
Specific binding sites for [3H]zacopride were found in the dorsal part of the rat spinal cord, particularly in the superficial layers of the dorsal horn. These binding sites had the same pharmacological profile as 5-HT3 receptors in membranes from the rat entorhinal cortex or from NG 108-15 neuroblastoma-glioma cells. Administration of capsaicin (50 mg/kg s.c.) to neonatal rats to induce degeneration of unmyelinated primary sensory fibres resulted in a significant decrease in [3H]zacopride specific binding (-50%) in the dorsal zone of the spinal cord of 4 month-old rats. This decrease was as pronounced as the decrease in [3H]bremazocine and [3H]naloxone binding to opiate receptors. These data support the presynaptic location of 5-HT3 receptors, at least in part, on capsaicin-sensitive primary afferent fibres in the rat spinal cord.
Animals↗
Segmental release of Met-enkephalin-like material from the spinal cord of rats, elicited by noxious thermal stimuli.
In order to investigate possible changes induced by noxious thermal stimuli in the activity of enkephalinergic neurones at various levels of the spinal cord, either the whole cord, the cervicotrigeminal area or the lumbar area were perifused with artificial CSF at a rate of 0.1 ml/min in halothane anaesthetized rats, and Met-enkephalin-like material (MELM) was measured in 0.5 ml fractions collected from the perifusates. Immersing the muzzle of intact rats in water at 52 degrees C produced a significant enhancement of MELM content in perifusates from both the whole spinal cord and the cervicotrigeminal area but not from the lumbar area. Heating the tail resulted in an increase in MELM release from the whole spinal cord of intact as well as of cervically transected rats. It is concluded that noxious thermal stimuli can induce a segmental release of MELM, i.e., only within spinal zones receiving the nociceptive inputs.
Animals↗
Neonatal capsaicin treatment abolishes the modulations by opioids of substance P release from rat spinal cord slices.
The possible modulation by opioids of substance P (SP) release at the spinal level was studied using slices of the dorsal half of the rat lumbar enlargement superfused with an artificial cerebrospinal fluid. Capsaicin (0.5 microM) selectively evoked a Ca2+-dependent overflow of SP-like material (SPLI) from primary afferent fibers which was enhanced in the presence of mu-opioid agonists (DAGO, FK 33824, sufentanyl, morphine), reduced by the delta-opioid agonist DTLET, and unaltered by the kappa-opioid agonist U 50488 H. Selective antagonists (naloxone, ICI 154129) prevented the effects of mu- and delta-opioid agonists. Neonatal capsaicin (50 mg/kg) abolished the stimulatory effect of in vitro capsaicin (0.5 microM) but not that of 30 mM K+ on SPLI outflow. This K+-induced SPLI release was unaffected by opioids. Presynaptic inhibitory control of SPLI release from capsaicin-sensitive primary afferent fibers might account for the analgesic effect of delta- but not mu- and kappa-opioid agonists at the spinal level.
Animals↗
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and opsonization synergistically enhance leukotriene B4 (LTB4) synthesis induced by phagocytosis in human neutrophils.
Normal human blood neutrophils were studied for their capacity to synthesize leukotriene B4 (LTB4) and its omega-oxidized metabolites after phagocytosis of zymosan. Phagocytosis of serum-opsonized particles led to a higher release of LTs than did unopsonized zymosan. The most striking effect of phagocytosis was observed when neutrophils were primed with granulocyte-macrophage colony-stimulating factor (GM-CSF): opsonization and GM-CSF synergistically increased LTB4 synthesis by neutrophils.
Arachidonate 5-Lipoxygenase↗
Opioid control of the in vitro release of calcitonin gene-related peptide from primary afferent fibres projecting in the rat cervical cord.
In vitro superfusion of slices from the dorsal half of the rat cervical enlargement allowed the measurement of spontaneous, K+ (30 mM)- and capsaicin (0.5 microM)-evoked release of calcitonin gene-related peptide-like immunoreactive material (CGRPLI). The greater part of this immunoreactive material originated in primary afferent fibres since dorsal rhizotomy from C4 to Th2 (8 days before sacrifice) resulted in a 85-90% decrease in CGRPLI release. CGRPLI outflow which persisted after dorsal rhizotomy could still be enhanced by K+-induced depolarization but was no longer sensitive to the stimulatory effect of 0.5 microM capsaicin. Both delta (DTLET, D-Pen2-D-Pen5-enkephalin) and mu (DAGO, PL 017) opioid receptor agonists reduced the K+ evoked release of CGRPLI from the dorsal half of the cervical enlargement. Morphine was also inhibitory but the selective K opioid agonist U 69593 was inactive. As expected from the involvement of delta and mu receptors, the selective opioid antagonist ICI 174864 and naloxone prevented the inhibitory effects of DTLET and DAGO, respectively. These data suggest that opioid-induced presynaptic inhibiton of CGRP-containing primary afferent fibres may be involved in the analgesic effect of intrathecally injected delta and mu opioid agonists in rats.
Afferent Pathways↗
Altered formation of leukotriene B4 in vitro by synovial fluid neutrophils in rheumatoid arthritis.
The synthesis of products of the 5-lipoxygenase pathway by synovial fluid (SF) and peripheral blood neutrophils of 12 patients with rheumatoid arthritis was compared in 3 experimental conditions using high performance liquid chromatographic analysis. Major differences were observed when blood and SF neutrophils were incubated with ionophore A23187, the formation of all 5-lipoxygenase products being lower (p less than 0.0005) in the SF neutrophils. Addition of exogenous arachidonic acid to the A23187 stimulated cells partially overcame the difference in 5-lipoxygenase product synthesis between the 2 neutrophil populations. In contrast, upon incubation with arachidonic acid alone, SF neutrophils produced significantly larger amounts of 5-lipoxygenase products. The increased reactivity of the SF neutrophil 5-lipoxygenase to arachidonic acid and the decreased 5-lipoxygenase product synthesis upon A23187 stimulation may be the consequence of the previous activation of the cells and 5-lipoxygenase product synthesis in situ.
Adult↗
Direct stimulatory effect of calcitonin on [3H]5-hydroxytryptamine release from the rat spinal cord.
The in vitro effects of porcine, salmon and human calcitonin on the K+-evoked overflow of [Met5]enkephalin, substance P and [3H]5-HT (previously taken up) were investigated in superfusion experiments with spinal cord slices. Porcine and salmon calcitonin did not affect the release of [Met5]enkephalin and substance P but enhanced that of [3H]5-HT. In contrast, human calcitonin was inactive. The stimulatory effect of porcine and salmon calcitonin on K+-evoked [3H]5-HT overflow was found with slices from the dorsal or the ventral half of the lumbar enlargement but not with hippocampal or hypothalamic slices. The calcitonin effect on [3H]5-HT outflow persisted in the absence of extracellular Ca2+ but was totally suppressed by 5-HT uptake inhibitors such as citalopram and chlorimipramine and by the 5-HT-releasing agent, p-chloroamphetamine. Direct investigation of the possible action of porcine calcitonin on [3H]5-HT uptake and release demonstrated that the enhanced [3H]5-HT overflow resulted from a p-chloramphetamine-like 5-HT-releasing effect of the hormone at the spinal level. This action might be involved in the potent analgesic effect of intrathecal calcitonin.
Animals↗
Studies on the effects of disease-modifying antirheumatic drugs on lipoxygenase product synthesis in human neutrophils in vitro.
The disease-modifying antirheumatic drugs sodium aurothiomalate, D-penicillamine and chloroquine phosphate were tested on leukotriene (LT) synthesis in human blood polymorphonuclear leukocytes stimulated with either the ionophore A23187, zymosan or f-Met-Leu-Phe. Lipoxygenase products were analyzed by reversed-phase high-performance liquid chromatography. The study demonstrated that the drugs can either inhibit or enhance 5-lipoxygenase product synthesis in human leukocytes, depending on the stimulus used to activate the cells and the drug concentration. The data also suggested that increased substrate availability accounted for the stimulatory effects of the drugs on leukotriene synthesis.
Anti-Inflammatory Agents, Non-Steroidal↗
Spontaneous and evoked release of met-enkephalin-like material from the spinal cord of arthritic rats in vivo.
Perfusion of the intrathecal space with artificial CSF was achieved in control and arthritic rats under halothane anaesthesia in order to collect the met-enkephalin-like material (MELM) released from the whole spinal cord. On the fourth week following the intradermal injection of Freund's adjuvant to induce arthritis, a marked reduction (-56%) in the spontaneous outflow of MELM was noted in arthritic rats. This effect did not involve changes in the degradation process of MELM, since it persisted when kelatorphan was added to the perfusing fluid in order to inhibit completely the peptidases acting on met-enkephalin. Raising the K+ concentration in the perfusing fluid from 2.4 to 40 mM, as well as moving the hind paws, produced a significant enhancement of MELM release which was (at least) as pronounced in arthritic as in control rats. These results suggest that the basal activity of spinal enkephalinergic neurones, but not that triggered by various stimuli, is reduced in arthritic rats.
Animals↗
In vivo and in vitro release of central neurotransmitters in relation to pain and analgesia.
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Analgesia↗
Autoradiographic evidence of serotonin1 binding sites on primary afferent fibres in the dorsal horn of the rat spinal cord.
Spinal serotonin1 (5-HT1)(labelled by [3H]5-HT), 5-HT1A (labelled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT)), mu- (labelled by [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAGO) and [3H]naloxone) and delta-opiate (labelled by [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr [( 3H]DSTLE] receptor binding sites were studied in adult rats using quantitative autoradiography after either neonatal treatment with capsaicin or unilateral cervical dorsal rhizotomy. Both treatments produced a significant loss of 5-HT (-20 to -30%) and opiate (-30 to -45%) binding sites within the superficial layers of the dorsal horn, suggesting they are partly located presynaptically on primary afferent fibres. Thus, 5-HT, as well as opiates, might generate analgesia by acting--at least partly--on primary afferent nociceptive fibres at the spinal level.
Afferent Pathways↗
Involvement of the dorsolateral funiculi in the spinal release of Met-enkephalin-like material triggered by heterosegmental noxious mechanical stimuli.
The lumbar spinal cord was superfused with artificial CSF at a rate of 0.1 ml/min in halothane anaesthetized rats. Under resting conditions, Met-enkephalin-like material (MELM) found in superfusates corresponded to a spinal release of 4.2 +/- 1.4 pg Met-enkephalin equivalents per 5 min. During a 30-min period in which pinches were applied to the muzzle, the MELM content in the superfusates increased markedly (by 120.5 +/- 32.9%). This effect was totally suppressed following bilateral lesions of the dorsolateral funiculi (DLF), under both chronic and acute conditions. It is concluded that strong mechanical stimuli applied in the trigeminal region can induce the release of MELM within the lumbar spinal cord via mechanisms involving the DLF. This heterosegmental release of Met-enkephalin may participate in the management of pain by methods involving high intensity stimulation.
Animals↗
Noxious mechanical stimuli increase the release of Met-enkephalin-like material heterosegmentally in the rat spinal cord.
Although the physiological functions of the endogenous opioid systems are not yet clearly established, it is widely accepted that they exert an inhibitory control on pain transmission. However, the well-documented hypoalgesic effects of low doses of the opiate antagonist naloxone both in animals and humans do not fit in with this concept. The present investigations, at two different spinal/medullary levels (viz. cervicotrigeminal and lumbar) demonstrate that, in the rat, a noxious mechanical stimulus does not alter the release of Met-enkephalin-like material (MELM) from neural segments related to the stimulated area of the body, but does increase its release from other segments. Electrophysiological studies have already demonstrated the existence of such heterosegmental mechanisms, notably 'diffuse noxious inhibitory controls' (DNIC), which are naloxone-reversible and could play an important role in pain perception. The involvement of spinal enkephalins in DNIC would seem to mean that the heterosegmental spinal release of MELM triggered by noxious stimuli participates in pain processes.
Animals↗
Opioid receptors and neuropeptides in the CNS in rats treated chronically with amoxapine or amitriptyline.
The central mechanism responsible for the potentiation by antidepressant drugs of analgesia induced by morphine, was explored by measuring the levels of various neuropeptides (met-enkephalin, leu-enkephalin, dynorphin, substance P and cholecystokinin-like materials) and the density of delta and mu opioid binding sites in the spinal cord of rats treated for 14 days with amoxapine (10 mg/kg i.p., daily) or amitriptyline (10 mg/kg i.p., daily). Similar measurements were made in the hypothalamus and cerebral cortex for comparison. Chronic treatment with amoxapine or amitriptyline did not affect the levels of dynorphin, substance P and cholecystokinin, but markedly enhanced the levels of leu-enkephalin in the three structures examined. The levels of met-enkephalin were also increased after treatment with amitriptyline but only in the spinal cord and hypothalamus. No changes in opioid receptors were found in the cerebral cortex, but the densities of delta and mu opioid binding sites were increased in the spinal cord, and decreased in the hypothalamus of rats treated with amoxapine or amitriptyline. These changes induced by antidepressants in opioidergic markers at the spinal level might account for the potentiation of the action of morphine in amoxapine- or amitriptyline-treated rats. In addition, the observed alterations in the same markers in the hypothalamus could be associated with changes induced by antidepressants in neuroendocrine regulation.
Amitriptyline↗