Basic and Regulatory Mechanisms of In Vitro Release of Met-Enkephalin from the Dorsal Zone of the Rat Spinal Cord

Abstract: Under control conditions, superfused slices of the dorsal half of the lumbar enlargement from adult rats released Met-enkephalin-like material (MELM) that behaved as authentic Met-enkephalin under two different chromatographic procedures (Bio-gel filtration, HPLC). MELM release increased markedly on exposure of slices to batrachotoxin (0.5 μ M ) or to an excess of K⁺ (28 and 56 m M instead of 5.6 m M ). The K ⁺ -evoked release was totally dependent on the presence of Ca²⁺ in the super-fusing fluid whereas the spontaneous efflux of MELM was only partially Ca²⁺-dependent. Further experiments performed with tissues of polyarthritic rats indicated that the increase in their MELM levels was associated with a lower fractional rate constant of MELM release, therefore suggesting that spinal Met-enkephalin turnover might be reduced in chronically suffering animals. Examination of the possible modulation of MELM release by various neuroactive compounds present within the dorsal horn revealed that cholecystokinin (10 μ M ), but not its desulphated derivative, substance P-sulphoxide (10 μ M ), and to a lesser extent substance P, enhanced the K⁺-evoked MELM release. In contrast, γ-aminobutyric acid (10 μ M ) and (–)-baclofen (1 μ M ) partially prevented the stimulatory effect of K⁺ on MELM release. Other compounds such as serotonin, somatostatin, and neurotensin altered neither the spontaneous nor the K⁺-evoked release of MELM.     

Nitric Oxide Regulates Substance P Release from Rat Spinal Cord Synaptosomes

Abstract: In order to determine whether nitric oxide (NO) acts directly upon nerve terminals to regulate the synaptic transmission at the level of spinal cord, effects of NO-donors on release of substance P (SP) and glutamic acid (Glu) were investigated by superfusion of synaptosomes prepared from the rat spinal cord. Basal levels of endogenous SP and Glu release were 5.99 ± 2.50 fmol/min/mg of protein and 26.2 ± 4.8 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCI evoked 2.7- and 3.8-fold increases in SP and Glu release in a calcium-dependent manner, respectively. Sodium nitroprusside (NP) caused a reduction in the depolarization-evoked overflow of SP in a concentration-dependent manner without affecting its basal release, although it failed to affect either basal or evoked release of Glu. The reduction in SP overflow was also observed by the perfusion with S -nitroso- N -acetyl-penicillamine or membrane-permeable cyclic GMP, but not with cyclic AMP. NP caused the concentration-dependent increases in cyclic GMP levels in synaptosomes. Together with reports that excitatory amino acids stimulate NO synthase and release NO in the spinal cord, these data suggest that there may be an interaction between nerve terminals containing Glu and SP, and that NO may directly participate in the regulation of synaptic transmission in SP-containing nerve terminals, which may be mediated through the activation of guanylate cyclase and the increase in cyclic GMP levels.     

5-Hydroxytryptamine-Facilitated Release of Substance P from Rat Spinal Cord Slices Is Mediated by Nitric Oxide and Cyclic GMP

Abstract: The role of nitric oxide (NO) in the control of 5-hydroxytryptamine (5-HT)-induced release of substance P was investigated in rat spinal cord in vitro. 5-HT facilitated the 60 m M K⁺-evoked release of substance P-like immunoreactive materials (SPLI) from the superfused rat dorsal spinal cord slices without affecting spontaneous SPLI release. The facilitatory effect of 5-HT was significantly inhibited by ICS 205-930 or granisetron (potent and specific 5-HT₃ receptor antagonists), by N ^G-monomethyl- l -arginine (NMMA, a NO synthase inhibitor), and by methylene blue or 1 H -[1,2,4]oxadiazolo[4,3- a ]quinoxaline-1-one (MB or ODQ, respectively; both are inhibitors of soluble guanylyl cyclase) and was mimicked by 2-methylserotonin (2-m-5-HT, a selective 5-HT₃ receptor agonist), l -arginine (a precursor of NO), or 8-bromo-cyclic GMP. NMMA, MB, or ODQ inhibited the 2-m-5-HT-induced increase of cyclic GMP levels in the rat dorsal spinal cord slices. These data suggest that the facilitatory effect of 5-HT on the release of SPLI is mediated by the 5-HT₃ receptor and that the intracellular signaling is mediated via NO by an increase in cyclic GMP production.     

Opioid modulation of capsaicin-evoked release of substance P from rat spinal cord in vivo

Capsaicin has been shown to evoke the release of substance P (SP) from small diameter primary afferent fibers. Using an in vivo perfusion of the rat spinal cord, this study examined the pharmacology of opioid receptor systems which modulate the capsaicin-evoked release of SP. The addition of capsaicin (200 μM) to the perfusate raised SP-like immunoreactivity (SP-LI) from resting levels of 31±5 to 74±14 pg/ml or an increase of 139% above the baseline. Using high pressure liquid chromatography (HPLC) the identity of the released SP-LI was determined to coelute primarily with authentic SP or the oxidized form of SP. Opioid receptor agonists were added to the perfusate and their ability to inhibit capsaicin-evoked release of SP-LI was assessed. Morphine (10–100 μM), DAGO (1–100 μM), DPLPE (10–100 μM), but not U50488H (100 μM) produced a dose-dependent reduction in the capsaicin-evoked release of SP-LI. Pretreatment with the opioid receptor antagonist naloxone (1 mg/kg, IP) had no effect on the basal or capsaicin-evoked release of SP-LI. Naloxone pretreatment was able to antagonize completely the opioid-produced inhibition of capsaicin-evoked SP-LI release. These data indicate that the release of SP from primary afferent fibers can be modulated by the activation of mu or delta but not kappa opioid receptors. Further, these data support the hypothesis that spinally administered mu and delta opioid agonists may produce their antinociceptive effect through the presynaptic inhibition of neuropeptide release from small diameter primary afferent fibers.     

Ventral Horn Neuropeptides Modulate the Release of Noradrenaline from Tissue Slices of Rat Brainstem and Ventral Thoracic Spinal Cord

The release of endogenous noradrenaline (NA) from slices of adult rat brainstem and ventral thoracic spinal cord was investigated using a fixed-volume incubation technique and HPLC with electrochemical detection. Incubation with potassium (15-50 mM) produced a dose-related increase in basal NA release that was calcium dependent. The potassium-evoked release of NA from spinal cord or brainstem slices was potentiated according to dose by preincubation with either (a) the selective alpha 2-adrenoceptor antagonist idazoxan (10(-6)-10(-4) M) or (b) the thyrotrophin-releasing hormone (TRH) analogue RX 77368 (pGlu-His-3,3'-dimethyl ProNH2; 10(-5) and 10(-4) M). Incubation of spinal cord slices with the NA uptake inhibitor maprotiline (1 microM) enhanced the effect of idazoxan but inhibited that of RX 77368. The effects of RX 77368 and potassium alone (15 mM) on NA release from both spinal cord and brainstem slices were reduced to basal levels with tetrodotoxin (10(-7) M). Similarly, preincubation of spinal cord, but not brainstem, slices with the insect neuropeptide proctolin (10(-4) M) significantly attenuated the potassium- or RX 77368-induced release of NA, whereas substance P (3 X 10(-5) and 1 X 10(-4) M) had no effect on either tissue. These results suggest that changes in NA release in the spinal cord and brainstem may mediate some of the actions of neuropeptides in ventral spinal cord, although the peptides may not be acting directly on the noradrenergic nerve terminals in these tissues.     

Cysteine: Depolarization-Induced Release from Rat Brain In Vitro

Compounds released on depolarization in a Ca2+-dependent manner from rat brain slices were screened to identify candidates for neuroactive substances. Lyophilized superfusates were analyzed by reversed-phase HPLC after derivatization with 9-fluorenyl N-succinimidyl carbonate. One of the compounds that showed an increase of concentration in superfusates in the presence of iodoacetamide was identified as the cysteine (Cys) derivative, S-carboxamidomethylcysteine, by fast atom bombardment mass spectrometry and other methods. This stable Cys derivative originates from endogenous, extracellular Cys. The finding led to a method for quantification of Cys in superfusates by immediate cooling of the superfusates to 0 degrees C and reaction of Cys with N-ethylmaleimide. Depolarization-induced Ca2+-dependent release of Cys was most prominent in the neocortex, followed by the mesodiencephalon, striatum, and cerebellum. This suggests that Cys is released from a neuronal compartment and might be involved in neurotransmission.     

Differences in Binding Properties of μ and δ Opioid Receptor Subtypes from Rat Brain: Kinetic Analysis and Effects of Ions and Nucleotides

Differences in binding properties of mu and delta opioid receptors were investigated using DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), which occur, respectively, as the most selective mu and delta radioligands available. At high concentration, each agonist is able to interact with its nonspecific sites. Competition experiments indicated that a two-site competitive model was adequate to explain the interactions of DAGO and DTLET with [3H]DTLET and [3H]DAGO binding sites, respectively. The weak cross-reactivity (congruent to 10%) of DTLET for mu sites was taken into account in these experiments. On the other hand, DAGO and DTLET exhibit differential binding kinetics. Thus, at 35 degrees C, the lifetime of DTLET within its receptor site is about 14 times longer than that of the mu agonist. Sodium and manganese ions decrease the maximal number of high affinity mu and delta sites, but the sensitivity of mu receptors is three times higher towards Na+ and 20-fold higher towards Mn2+ than that of delta receptors. GTP reduces similarly the mu and delta binding whereas only the DAGO binding was modified by the nonhydrolyzable analogue guanylylimidodiphosphate [GMP-P(NH)P]. However, in the presence of Na+ ions, GMP-P(NH)P inhibits the DTLET binding in a concentration-dependent manner. The effects of Na+ and GMP-P(NH)P could be explained by a sequential transformation of delta receptors to low-affinity states. This model predicts that Na+, by lowering the affinity of a fraction of sites, produces a decrease in the maximal number of high-affinity delta receptors and that GMP-P(NH)P enhances the Na+ effect. Moreover, the binding kinetic to this high-affinity state was also modified by Na+ and nucleotides. All of these data support the existence of two independent mu and delta binding sites, the properties of which are differentially regulated by these endogenous effectors.     

Capsaicin Does Not Change Tissue Levels of Glutamic Acid, Its Uptake, or Release in the Rat Spinal Cord

Capsaicin treatment (50 mg/kg, subcutaneous) of newborn rats resulted in 1 75% decrease of substance P immunoreactivity in the dorsal spinal cord of the adult animal, but failed to affect levels of the proposed sensory neurotransmitter glutamic acid or to alter high-affinity uptake of [3H]glutamic acid into synaptosomes of the same tissue. Furthermore, capsaicin (30 microM) in vitro had no influence on the release of [3H]glutamic acid from spinal cord P2 fractions of untreated adult rats, but induced a marked release of substance P. The results suggest that, in contrast to substance P fibers, neurons containing glutamic acid are not sensitive to capsaicin. Eleven other neurochemical parameters measured in the spinal cord did not appear to be changed by the treatment with capsaicin, suggesting a considerable neurochemical selectivity of the lesion.     

Evidence for Ascending and Descending Intraspinal as Well as Primary Sensory Somatostatin Projections in the Rat Spinal Cord

Somatostatin distribution was measured quantitatively in the rat spinal cord by radioimmunoassay. Rostro-caudally, somatostatin content was about 50% higher in lumbar-sacral cord than in cervical or thoracic levels. The dorso-ventral distribution is more uneven: somatostatin is highest in the dorsal horn, where the peptide is 15 times as concentrated as it is in the ventral white matter, the region of lowest concentration. However, measurable amounts of the peptide were found in all regions studied. Dorsal root ganglionectomy decreased somatostatin levels in the dorsal cord, supporting the previously proposed role for this peptide as a primary sensory neurotransmitter or modulator; but somatostatin content also was decreased both rostral and caudal to spinal transection, indicating the presence of ascending and descending somatostatin pathways within the spinal cord. Brain levels did not change. Met-enkephalin and substance P were also measured after the above surgical manipulations. Met-enkephalin content was not altered and substance P content was lowered significantly only after ganglionectomy. Although this study confirms the primary sensory neuron as the origin of a part of spinal cord somatostatin, it further indicates the presence of ascending and descending somatostatin pathways within the rat spinal cord.     

Release of Neuropeptide FF, an Anti-Opioid Peptide, in Rat Spinal Cord Slices Is Voltage- and Ca2+-Sensitive: Possible Involvement of P-Type Ca2+ Channels

Abstract: Neuropeptide FF (NPFF), an FMRFamide-like peptide with antiopioid properties, inhibits morphine-induced analgesia but also produces hyperalgesia. In the present study, the mechanisms of NPFF release were investigated in an in vitro superfusion system with rat spinal cord slices. The opening of voltage-sensitive Na⁺ channels with veratridine (20 µ M ) induced calcium-dependent NPFF release, which was abolished by tetrodotoxin (1 µ M ), suggesting that NPFF release depends on nerve impulse activity. We also showed that NPFF release was a function of the extent of depolarization and was calcium dependent. The 30 m M K⁺-induced release was blocked by Co²⁺ or Ni²⁺ (2.5 m M ) but was unaffected by Ca²⁺ channel blockers of the L type—Cd²⁺ (100 µ M ), nifedipine or nimodipine (10 µ M ), diltiazem (20 µ M ), or verapamil (50 µ M )—or the N type—ω-conotoxin GVIA (1 µ M ). In contrast, ω-agatoxin IVA (1 µ M ) led to a 65% reduction in NPFF release, suggesting that P-type Ca²⁺ channels play a prominent role. The 35% remaining release resulted from activation of an unknown subtype. The NPFF-like material in superfusates recognized spinal NPFF receptors, suggesting that NPFF release in the spinal cord has a physiological role.     

Effect of Pressure on the Release of Radioactive Glycine and γ-Aminobutyric Acid from Spinal Cord Synaptosomes

Exposure to high hydrostatic pressure produces neurological changes referred to as the high-pressure nervous syndrome (HPNS). Manifestations of HPNS include tremor, EEG changes, and convulsions. These symptoms suggest an alteration in synaptic transmission, particularly with inhibitory neural pathways. Because spinal cord transmission has been implicated in HPNS, this study investigated inhibitory neurotransmitter function in the cord at high pressure. Guinea pig spinal cord synaptosome preparations were used to study the effect of compression to 67.7 atmospheres absolute on [3H]glycine and [3H]gamma-aminobutyric acid ([3H]GABA) release. Pressure was found to exert a significant suppressive effect on the depolarization-induced calcium-dependent release of glycine and GABA by these spinal cord presynaptic nerve terminals. This study suggests that decreased tonic inhibitory regulation at the level of the spinal cord contributes to the hyperexcitability observed in animals with compression to high pressure.     

Differential Release of Endogenous 5-Hydroxytryptamine, Substance P., and Neurokinin A from Rat Ventral Spinal Cord in Response to Electrical Stimulation

Abstract: The release of endogenous 5-hydroxytryptamine (5-HT), substance P (SP), and neurokinin A (NKA) from superfused tissue slices of rat ventral lumbar spinal cord, where SP and NKA coexist with 5-HT in terminals of descending bulbospinal neurons, was investigated. Electrical field stimulation was performed using square-wave pulses of 2-ms duration and 30 mA stimulus intensity. The following four different patterns of stimulation were used: 2 Hz continuous, 20 Hz continuous, 20 Hz intermittent, and 50 Hz intermittent. 5-HT was measured in the slice superfusates by HPLC with electrochemical detection. SP and NKA were measured by radioimmunoassay. The release of 5-HT was significantly enhanced using all stimulation paradigms and the evoked release of 5-HT per pulse was independent of the stimulation frequency. The release was found to be calcium dependent and there was no increase in the efflux of 5-hydroxyindoleacetic acid in response to stimulation. At 2 Hz (continuous), no significant increase in the release of SP was observed. Stimulation at higher frequencies yielded a significant increase in the release of SP per pulse. At 20 Hz, the release was increased by 73% (continuous) and 74% (intermittent), and at 50 Hz (intermittent) by 175% of basal efflux. The evoked release of NKA was also frequency dependent. At 2 Hz (continuous), no significant increase in the release of NKA was observed. At 20 Hz (intermittent), the evoked release per pulse was increased by 33% and at 50 Hz (intermittent) by 53% compared with the basal efflux of NKA. The results suggest that coexisting neurotransmitters/neuromodulators in the spinal cord may be released in different proportions depending on the stimulation frequency and that only 5-HT is released when the nerve terminal is activated by low-frequency stimulation.     

Tachykinin Systems in the Spinal Cord and Basal Ganglia: Influence of Neonatal Capsaicin Treatment or Dopaminergic Intervention on Levels of Peptides, Substance P–Encoding mRNAs, and Substance P Receptor mRNA

The aim of the study was to test whether the synthesis of substance P (SP) and that of its receptor (also known as NK1 receptor) are coordinately regulated after chronic pharmacologic intervention in two neural systems, the spinal cord and basal ganglia. In one set of experiments, capsaicin was administered subcutaneously during the early postnatal period (day 3 after birth) to induce degeneration of afferent sensory neurons in the spinal cord. In the other set of experiments, interruption of dopaminergic transmission was achieved by two methods: (a) The neurotoxin 6-hydroxydopamine was used to denervate dopaminergic neurons during the early postnatal period, and (b) haloperidol was used in adult animals to block dopaminergic transmission by receptor blockade. The spinal cord, striatum, or both were used for the quantification of tachykinin [SP and neurokinin A (NKA)] and opioid peptides [[Met5]-enkephalin (ME) and dynorphin A (1-8) (DYN)] by radioimmunoassays. The abundance of total SP-encoding preprotachykinin (PPT) mRNA and SP receptor (SPR) mRNA in spinal cord (C5 to T1 segments), striatum, or microdissected substantia nigra was determined by northern blot or solution hybridization analysis. Amines and their acid metabolites were quantified by HPLC. Capsaicin administration (subcutaneously) during the early postnatal period increased latency in a hot-plate test, decreased SP and NKA levels, increased levels of PPT mRNAs, and did not affect SPR mRNA levels in the spinal cord. Intraspinal SP systems may attempt to compensate for the loss of afferent SP input, whereas spinal cord receptor mRNA levels do not appear to be altered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spinal Neurokinin NK1 Receptor Down-Regulation and Antinociception: Effects of Spinal NK1 Receptor Antisense Oligonucleotides and NK1 Receptor Occupancy

Abstract: To define the effects of antisense oligonucleotides on spinal neurokinin 1 (NK1) receptor function in nociceptive processing, several antisense oligonucleotides directed against the NK1 receptor mRNA were intrathecally injected into rats via an implanted catheter, and their effect on the behavioural response to formalin injected into the paw was assessed. We observed that there was no significant reduction of pain behaviour or immunostaining of spinal NK1 receptors after repeated daily intrathecal treatment with an antisense oligonucleotide. However, spinal application of substance P (SP) in the antisense oligonucleotide-treated animals resulted in a profound and long-lasting reduction in the behavioural response to formalin injection, and a parallel reduction in the NK1 receptor immunoreactivity normally observed in spinal dorsal horn. Intrathecal SP in the control groups, i.e., rats treated with an oligonucleotide containing four mismatched bases, the corresponding sense oligonucleotide, a mixture of the sense and the antisense oligonucleotides, in each case had no effect. The effects of SP were blocked by NK1 receptor antagonists and were not mimicked by NMDA. The mechanism underlying these effects is not clear. It may be due to partial degradation of the internalised receptors, which cannot be replaced by newly synthesised receptors because of the action of the NK1 antisense oligonucleotide.     

Opioid Control of the In Vitro Release of Cholecystokinin-Like Material from the Rat Substantia Nigra

Possible interactions between Met-enkephalin and cholecystokinin (CCK)-containing neurons in the rat substantia nigra were investigated by looking for the effects of various opioid receptor ligands and inhibitors of enkephalin-degrading enzymes on the K(+)-evoked overflow of CCK-like material (CCKLM) from substantia nigra slices. The delta-opioid agonists D-Pen2, D-Pen5-enkephalin (50 microM) and Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET; 3 microM) enhanced, whereas the mu-opioid agonists Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO; 10 microM) and MePhe3, D-Pro4-morphiceptin (PL 017; 10 microM) decreased, the K(+)-evoked release of CCKLM. By contrast, the kappa-opioid agonist U-50488 H (5 microM) was inactive. The stimulatory effect of DTLET could be prevented by the delta antagonist ICI-154129 (50 microM), but not by the mu antagonist naloxone (1 microM). Conversely, the latter drug, but not ICI-154129, prevented the inhibitory effect of DAGO and PL 017. A significant increase in CCKLM overflow was observed upon tissue superfusion with the peptidase inhibitors kelatorphan or bestatin plus thiorphan. This effect probably resulted from the stimulation of delta-opioid receptors by endogenous enkephalins protected from degradation, because it could be prevented by ICI-154129 (50 microM). Furthermore the peptidase inhibitors did not enhance CCKLM release further when delta-opioid receptors were stimulated directly by DTLET (3 microM). These data indicate that opioids acting on delta and mu receptors may exert an opposite influence, i.e., excitatory and inhibitory, respectively, on CCK-containing neurons in the rat substantia nigra.(ABSTRACT TRUNCATED AT 250 WORDS)     

大鼠颈段脊髓灰质板层及其细胞构筑

取12只SD(Sprague-Dawley)大鼠颈段脊髓横断面冰冻切片,用焦油紫和Pal-Weigert染色法染色,光镜观察SD大鼠颈段脊髓的组织结构,对SD大鼠脊髓灰质板层的细胞构筑进行研究。结果显示,Ⅰ层边界呈长弧形,Ⅰ、Ⅱ、Ⅲ、Ⅳ层呈层叠状排列,细胞多为椭圆形。Ⅴ、Ⅵ层均可分为内侧部和外侧部,外侧部分布大型细胞,Ⅴ层内侧部以中型椭圆形、三角形细胞为主,Ⅵ层内侧部以中型梭形细胞为主。Ⅶ层位于中间带,C5-C7Ⅶ层向前角延伸和Ⅷ层一起占据前角大部,Ⅶ及Ⅷ层的中型和大型细胞呈集中分布。Ⅷ层在C1-C4及C8占前角大部,在C5-C7位于前角内侧部。Ⅸ层主要由含大型运动神经元的核团组成。Ⅹ层由中型梭形细胞和小型星状细胞组成。对比观察发现,SD大鼠脊髓颈段板层类似于猫的Rexed分层,但灰质轮廓、板层出现节段、板层形态及其变化等方面均有所不同。     

Effects of Ethanol In Vitro and In Vivo on the Release of Endogenous Catecholamines from Specific Regions of Rat Brain

Blocks of tissue from the hypothalamus, olfactory bulb, or striatum of rats were incubated in vitro to study the basal and potassium-stimulated release of endogenous catecholamines. When ethanol (100-250 mM) was added to these preparations in vitro no changes in release were observed. When ethanol (3.0 g X kg-1) was injected intraperitoneally in vivo, however, and 3,4-dihydroxyphenylethylamine (DA, dopamine) release was measured in vitro at various times after drug administration, significant increases in the basal release and decreases in the potassium-stimulated release were observed in striatum and olfactory bulb. In striatum, these changes showed a more rapid onset and a longer duration than in olfactory bulb. In both brain regions, DA release did not differ from controls at 4-6 h after the ethanol injection, although blood ethanol concentrations remained elevated. This may imply the tissue's acquisition of acute functional tolerance to the drug. Similar increases and decreases in the basal and the potassium-induced release of DA from striatal tissues were also found at 1 h after injection of a lower dose of ethanol (1.0 g X kg-1). In terms of behavior, this lower dose of ethanol produced only mild intoxication and ataxia, in contrast to the loss of righting reflex following the higher dose.     

Active Uptake of Substance P Carboxy-Terminal Heptapeptide (5–11) into Rat Brain and Rabbit Spinal Cord Slices

We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy-terminal heptapeptide, (5-11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5-11)SP, the uptake of (5-11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5-11)SP was accumulated by means of a high-affinity and a low-affinity uptake system. The Km and the Vmax values for the high-affinity system were 4.20 x 10(-8) M and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low-affinity system were 1.00 x 10(-6) M and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 x 10(-7) M and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.     

Heterogeneity of γ-Aminobutyric Acid/Benzodiazepine/β-Carboline Receptor Complex in Rat Spinal Cord

The properties of muscimol, beta-carboline (BC), and benzodiazepine (BZD) binding to crude synaptic membranes were studied in the spinal cord and cerebellum of rats. In cerebellar membranes, the density of high-affinity [3H]muscimol and [3H]6,7-dimethoxy-4-ethyl-beta-carboline ([3H]BCCM) binding sites is almost identical to that of [3H]flunitrazepam ([3H]FLU) or [3H]flumazenil (Ro 15-1788; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1-4]benzodiazepine-3-carboxylate). In contrast to the cerebellum, the number of muscimol and BC binding sites in rat spinal cord is approximately 20-25% of the number of FLU or flumazenil binding sites. Moreover, in spinal cord membranes, BC recognition site ligands displace [3H]-flumazenil bound to those sites, with low affinity and a Hill slope significantly less than 1; the potency of the different BCs in displacing [3H]flumazenil is 20-50-fold lower in the spinal cord than in the cerebellum. [3H]Flumazenil is not displaced from spinal cord membranes by the peripheral BZD ligand Ro 5-4864 (4'-chlorodiazepam), whereas it is displaced with low affinity and a Hill slope of less than 1 (nH = 0.4) by CL 218,872 (3-methyl-6-(3-trifluoromethylphenyl)-1,2,4-triazolol[4,3-b] pyridazine). These data suggest that a large number of BZD binding sites in spinal cord (approximately 80%) are of the central-type, BZD2 subclass, whereas the BZD binding sites in cerebellum are predominantly of the central-type, BZD1 subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic α2 Adrenoceptors Inhibit Glutamate Release from Rat Spinal Cord Synaptosomes

Abstract: The presynaptic regulation of amino acid release from nerve terminals was investigated using synaptosomes prepared from the rat spinal cord. The basal releases of endogenous glutamate (Glu), aspartate (Asp), and γ-amino-butyric acid (GABA) were 34.6, 21.5, and 10.0 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCl (30 m M ) evoked 2.7-, 1.5-, and 2.9-fold increases in Glu, Asp, and GABA release, respectively. Clonidine reduced the K⁺-evoked overflow of Glu to 56% of the control overflow with a potency (IC₅₀) of 17 n M , but it did not affect K⁺-evoked overflow of Asp, GABA, and their basal releases. Similarly, noradrenaline inhibited the K⁺-evoked overflow of Glu, although phenylephrine and isoproterenol showed no effect. The inhibitory effect of clonidine was counteracted by α₂-adrenoceptor antagonists, rauwolscine, yohimbine, and idazoxan, regardless of the imidazoline structures. Because Glu is considered a neurotransmitter of primary afferents that transmit both nociceptive and nonnociceptive stimuli in the spinal cord, these data suggest that part of Glu release may be regulated by the noradrenergic system through α₂ adrenoceptors localized on the primary afferent terminals.