Biochemical characterization of FMRF-NH2-like peptides in spinal cords of various mammalian species using specific radioimmunoassays

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2) and Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-NH2 (A-18-F-NH2), originally detected by FMRF-NH2 antiserum and subsequently isolated from bovine brain, were found to be highly localized in the bovine spinal cord. Using specific radioimmunoassays coupled with HPLC, F-8-F-NH2 and A-18-F-NH2 immunoreactivities in spinal cord of bovine, rat, mouse, guinea pig and human were studied. One major F-8-F-NH2 immunoreactivity was detected in the spinal cord of every species except in human, however, the retention time of F-8-F-NH2 immunoreactivity appears to vary from species to species. In the human spinal cord three major F-8-F-NH2 immunoreactivities are detected and one of them was eluted in the position of F-8-F-NH2. Two major A-18-F-NH2 immunoreactivities were detected in every species except guinea pig; one of these immunoreactivities can be identified as F-8-F-NH2 immunoreactivity due to the high affinity of the A-18-F-NH2 antiserum to F-8-F-NH2. F-8-F-NH2 and A-18-F-NH2 immunoreactivities can also be clearly detected by FMRF-NH2 antiserum, however, the quantities of these peptides can be grossly underestimated by the FMRF-NH2 RIA. These results confirm that there is a novel system of FMRF-NH2-like peptides in mammalian CNS and some of them are more closely related to the bovine peptides, F-8-F-NH2 and A-18-F-NH2 than to FMRF-NH2.     

IgG from antiserum against endogenous mammalian FMRF-NH2-related peptides augments morphine- and stress-induced analgesia in mice

Two mammalian FMRF-NH2-like peptides have been isolated from bovine brain; an octapeptide with the structure Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2) and an octadecapeptide, Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe- NH2 (A-18-F-NH2). In the present study determinations were made of the effects of intracerebroventricular administration of IgG prepared from antisera raised against these peptides on nociception and morphine- and immobilization-induced opioid analgesia in mice. Both F-8-F-NH2-IgG and A-18-F-NH2-IgG antisera increased nociception (thermal response latency) and significantly augmented morphine- and immobilization-induced analgesia in a naloxone reversible manner, with F-8-F-NH2-IgG antisera having a greater effect than A-18-F-NH2-IgG antisera. These results provide further evidence that mammalian FMRF-NH2-like peptides function as endogenous opiate antagonists and have a role in the mediation of antinociception.     

FMRF-NH2-like peptide is deficient in the pituitary gland of the Brattleboro rat

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2), isolated from bovine brain, is an FMRF-NH2-like peptide with morphine-modulating activity. In the rat, F-8-F-NH2 immunoreactivity (IR) is highly localized in the neurohypophysis. In this study, F-8-F-NH2-IR was studied in the hypothalamo-neurohypophyseal system of an Arg8-vasopressin (AVP)-deficient animal, the Brattleboro (DI) rat, and the normal control Long-Evans (LE) strain. F-8-F-NH2-IR in the DI pituitary is below the level of detection in contrast to that in the LE (0.50 +/- 0.04 pmol/gland). Neuropeptide Y (NPY) levels are increased two-fold in the DI pituitary while AVP levels are below detection. The content of F-8-F-NH2-IR in the hypothalami and spinal cords of DI and LE rats is not statistically different, suggesting that the absence of F-8-F-NH2-IR in the Brattleboro pituitary is not due to a genetic defect in F-8-F-NH2 biosynthesis. The results of this study raise the question whether AVP could be involved in the regulation of F-8-F-NH2 immunoreactivity in the neurohypophysis.     

FMRF-NH2-like mammalian octapeptide: possible role in opiate dependence and abstinence

Yang et al. have isolated from bovine brain an octapeptide, FLFQPQRF-NH2 (F-8-F-NH2), with certain antiopiate properties. Malin et al. previously found that ICV injection of this peptide could precipitate an opiate abstinence syndrome in dependent rats. RIA revealed significantly higher levels of F-8-F-NH2 immunoreactivity in CSF withdrawn from the cisterna magna of morphine-dependent rats as opposed to CSF withdrawn from sham-implanted controls. ICV infusion of IgG from antiserum against F-8-F-NH2 significantly reduced the number of abstinence signs subsequently precipitated by naloxone in morphine-dependent rats.     

Medullary and mesencephalic neuronal groups reacting to antibodies against VIP, somatostatin and bombesin in adult Gallus gallus domesticus

The caudo-cranially intermediate one-third of medullary dorsal region, the periaqueductal grey and the rostro-ventral portion of the midbrain tegmentum of adult chickens were studied in detail by means of the PAP-DAB procedure, to define further the main morphological features of the neuronal populations that in previous studies had shown VIP (Vasoactive Intestinal Polypeptide),-Somatostatin (SRIF)-, and Bombesin-like immunoreactivities. In the medulla, VIP-like immunoreactivity was detected within neuronal bodies and processes and extended down to the cervical spinal cord. SRIF-like immunoreactivity was seen only within nerve cell processes, at least a part of which could be sensitive fibre terminals. Bombesin-like immunoreactivity was observed only within neuronal processes. In the periaqueductal grey, all 3 immunoreactivities were detected within perikarya and neuronal processes, with a higher density cranially. In the rostro-ventral portion of the midbrain tegmentum, VIP-like and Bombesin-like immunoreactivities were detected (the latter being located somewhat more cranially) both in neuronal bodies and in processes. SRIF-like immunoreactivity was found in this region only in long neuronal processes.     

A-18-famide and F-8-famide, endogenous mammalian equivalents of the molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2), inhibit colonic bead expulsion time in mice

Morphine and the two endogenous mammalian FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides known as morphine-modulating neuropeptides, F-8-Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) and A-18-Famide (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe -NH2), were administered intracerebroventricularly (ICV) to mice and the effect of each on colonic bead expulsion time was measured. Each of the three compounds delayed expulsion of a 3 mm glass bead placed in the distal colon. A-18-Famide was more potent than F-8-Famide [ED 50 = 2.3 micrograms (1.2 nmole) and 13.9 micrograms (13.0 nmole), respectively]. A-18-Famide: 1) did not block morphine-induced delay of bead expulsion time, and 2) was blocked by simultaneous administration (ICV) of 1.0 microgram of the competitive opiate antagonist naloxone. These data demonstrate apparent opioid modulatory or agonist-like, rather than antagonist-like, properties of A-18-Famide and F-8-Famide.     

Identification by radioimmunoassay and HPLC of morphine modulatory peptide-immunoreactivity in rat spinal cord and brain

Two so-called morphine modulatory peptides, an octapeptide and an octadecapeptide, have recently been isolated from bovine spinal cord. We have raised antibodies to the octapeptide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH₂: FF-8), which in radioimmunoassay react with peptides terminating in Arg-Phe-NH₂. This dipeptide is common to both the morphine modulatory peptides and the molluscan neuropeptide FMRF amide. The distribution and molecular forms of immunoreactive peptides were examined in the rat central nervous system and gastrointestinal tract. Highest concentrations of FF-8-like immunoreactivity were found in the dorsal spinal cord, brain stem and hypothalamus. The immunoreactive material in central nervous system extracts was resolved by reversed phase HPLC into three peaks of activity, the two largest peaks eluted in similar positions to the standard octapeptide and octadecapeptide. It appears that previously observed FMRF amide-like immunoreactivity in the rat central nervous system corresponds to peptides immunochemically and chromatographically similar to the two bovine spinal cord peptides.     

Unique distributions of natriuretic hormones in dog brain

We examined the regional distributions of atrial natriuretic polypeptide (ANP) and digoxin-like immunoreactivity (DLI) in dog brain, using specific radioimmunoassay. The molecular form of the dog brain ANP was similar to that of alpha-hANP, in gel filtration and reversed-phase HPLC. Distribution of ANP in dog brain differed from rat brain. A significant amount of ANP-like immunoreactivity (ANPLI) was observed in the periaqueductal grey, ventral thalamus and spinal cord, however, only a trace amount was seen in the hypothalamus. The DLI was widely distributed in the dog brain, especially with over 2 ng/g wet wt. of the immunoreactivity content in mammillary body, septum, striatal body, hypothalamus and periaqueductal grey. Different from the localizations of natriuretic hormones in rat brain, the periaqueductal grey matter in dog brain may be an important source of both natriuretic hormones.     

FMRF-NH2-like mammalian peptide precipitates opiate-withdrawal syndrome in the rat

Yang et al. (14) have isolated from mammalian brain an octapeptide FLFQPQRF-NH2 (F-8-F-NH2) with certain antiopiate properties. Third ventricular injection of 2 micrograms of this peptide together with the aminopeptidase inhibitor bestatin precipitated an opiate-withdrawal syndrome in morphine-dependent but not in nondependent rats. Third ventricular injection in nondependent rats of 15 micrograms of the peptide together with bestatin induced a morphine-withdrawal-like behavioral syndrome. This syndrome was not produced by injection of bestatin or saline vehicle alone and was preventable by injection of 3.5 mg/kg morphine sulphate SC.     

Effects of mammalian and avian neurotensins and neurotensin fragments on wet-dog shaking and body temperature in the rat

The ability of mammalian and avian neurotensins and some neurotensin fragments to reduce wet-dog shaking (WDS) induced by thyrotrophin-releasing hormone (TRH) and to influence rectal temperature was tested after their injection into the periaqueductal grey region of male rats. Both neurotensins inhibited TRH-induced WDS and reduced rectal temperature by 2 degrees C; this latter effect was prevented by prior TRH administration. Of the four neurotensin fragments tested, both (1-8)- and (8-13)-neurotensin reduced WDS but only (8-13)-neurotensin reduced rectal temperature significantly. (1-6)- and (1-11)-neurotensin were without effect in either test system. From the activity of the various peptides, further examples of the mutual antagonism between TRH and neurotensin have been demonstrated. It is suggested that there is a possible role for neurotensin in controlling body temperature via the periaqueductal grey and that this may be one function of neurotensin in avian species; there may also be more than one receptor system binding neurotensin in the brain.     

Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination

We investigated the molecular forms of endothelin (ET) related peptides in porcine spinal cord by high performance liquid chromatography coupled with radioimmunoassays using three antisera raised against ET-1 and C-terminal fragments of ET-1 and big ET-1. ET-1 and its oxidized form were isolated as major immunoreactive peptides and sequenced. Furthermore, immunoreactivities like ET-3 and big ET-1(22-39) (contents: less than 8% and less than 1% of ET-1, respectively) were detected based on their chromatographic retention times and characteristics of immunoreactivity to the antisera. Big ET-1 was only scarcely detected. Immunohistochemical study showed the presence of ET-1-like immunoreactivity in motoneurons, dorsal horn neurons and dot- and fiber-like structures in the dorsal horn of lumbar spinal cord. These results indicate that ET-1 is present not only in endothelial cells but also in spinal cord, and that big ET-1 is converted into ET-1 in spinal cord by specific processing between Trp21-Val22. The data also indicate that ET-1 may act as a neuropeptide in the central nervous system.     

Distribution of five peptides, three general neuroendocrine markers, and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: an immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I-II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I-III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I-II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

Distribution Pattern of Metorphamide Compared with Other Opioid Peptides from Proenkephalin and Prodynorphin in the Bovine Brain

Metorphamide is a [Met]-enkephalin-containing opioid octapeptide with a C-terminal alpha-amide group. It is derived from proenkephalin and is, so far, the only endogenous opioid peptide with a particularly high affinity for mu opioid (morphine) receptors, a somewhat lesser affinity for kappa opioid receptors, and a relatively low affinity for delta opioid receptors. The concentrations of metorphamide in the bovine caudate nucleus, the hypothalamus, the spinal cord, and the neurointermediate pituitary were determined by radioimmunoassay and chromatography separation procedures. Metorphamide concentrations were compared with the concentrations of eight other opioid peptides from proenkephalin and prodynorphin in identical extracts. The other opioid peptides were [Met]-enkephalyl-Arg6-Phe7 and [Met]-enkephalyl-Arg6-Gly7-Leu8 from proenkephalin; alpha-neoendorphin, beta-neoendorphin, dynorphin A(1-8), dynorphin A(1-17), and dynorphin B from prodynorphin; and [Leu]-enkephalin, which can be derived from either precursor. All opioid peptides were present in all four bovine neural tissues investigated. Metorphamide concentrations were lower than the concentrations of the other proenkephalin-derived opioid peptides. They were, however, similar to the concentrations of the prodynorphin-derived opioid peptides in the same tissues. Marked differences in the relative ratios of the opioids derived from prodynorphin across brain regions were observed, a finding suggesting differential posttranslational processing. Differences in the ratios of the proenkephalin-derived opioids across brain regions were less pronounced. The results from this study together with previous findings on metorphamide's mu opioid receptor binding and bioactivities suggest that the amounts of metorphamide in the bovine brain are sufficient to make this peptide a candidate for a physiologically significant endogenous mu opioid receptor ligand.     

家兔若干与下行抑制有关的中枢部位中去甲肾上腺素、5-羟色胺和脑啡肽在针刺镇痛中的变化

应用推挽灌流技术、去甲肾上腺素(NA)放射酶学法和亮-脑啡肽放射免疫法观察不同脑区 NA 和脊髓背角亮-脑啡肽的释放。应用分子筛柱层析分离家兔不同脑区的5-羟色胺(5-HT)和5-羟吲哚乙酸(5-HIAA),并对它们进行荧光微量测定。以此来阐明针刺镇痛时 NA、5-HT 和亮-脑啡肽在下行抑制中的作用。1.家兔电针20 min,痛阈显著提高,此时中脑导水管周围灰质(PAG)和中缝大核(NRM)的 NA 释放显著减少,而 Al 核团和脊髓背角的 NA释放显著增加。2.电针镇痛时,PAG、延脑中缝核区和脊髓的5-HT 和5-HIAA 含量均有显著增加,除 PAG 外,这种增加的出现较 NA 为晚。提示可能在针刺镇痛的下行抑制中,NA 的参予较5-HT 为早。3.针刺镇痛时脊髓背角亮-脑啡肽的释放也明显增加。     

Electrophysiological Study of Supraspinal Input and Spinal Output of Cat's Subnucleus Reticularis Dorsalis (SRD) Neurons

This work addressed the study of subnucleus reticularis dorsalis (SRD) neurons in relation to their supraspinal input and the spinal terminating sites of their descending axons. SRD extracellular unitary recordings from anesthetized cats aimed to specifically test, 1) the rostrocaudal segmental level reached by axons of spinally projecting (SPr) neurons collateralizing or not to or through the ipsilateral nucleus reticularis gigantocellularis (NRGc), 2) whether SPr fibers bifurcate to the thalamus, and 3) the effects exerted on SRD cells by electrically stimulating the locus coeruleus, the periaqueductal grey, the nucleus raphe magnus, and the mesencephalic locomotor region. From a total of 191 SPr fibers tested to cervical 2 (Ce2), thoracic 5 (Th5) and lumbar5 (Lu5) stimulation, 81 ended between Ce2 and Th5 with 39 of them branching to or through the NRGc; 21/49 terminating between Th5 and Lu5 collateralized to or through the same nucleus, as did 34/61 reaching Lu5. The mean antidromic conduction velocity of SPr fibers slowed in the more proximal segments and increased with terminating distance along the cord. None of the 110 axons tested sent collaterals to the thalamus; instead thalamic stimulation induced long-latency polysynaptic responses in most cells but also short-latency, presumed monosynaptic, in 7.9% of the tested neurons (18/227). Antidromic and orthodromic spikes were elicited from the locus coeruleus and nucleus raphe magnus, but exclusively orthodromic responses were observed following stimulation of the periaqueductal gray or mesencephalic locomotor region. The results suggest that information from pain-and-motor-related supraspinal structures converge on SRD cells that through SPr axons having conduction velocities tuned to their length may affect rostral and caudal spinal cord neurons at fixed delays, both directly and in parallel through different descending systems. The SRD will thus play a dual functional role by simultaneously regulating dorsal horn ascending noxious information and pain-related motor responses.     

Neuromedin B is a major bombesin-like peptide in rat brain: regional distribution of neuromedin B and neuromedin C in rat brain, pituitary and spinal cord

Neuromedin B and neuromedin C are the novel mammalian bombesin-like peptides isolated from porcine spinal cord. We have developed highly specific and sensitive radioimmunoassays for neuromedin B and neuromedin C, and determined their regional distribution in rat central nervous system. Prior to measurements of the tissue contents, immunoreactive neuromedin B and C were characterized by gel-filtration and high performance liquid chromatography. Neuromedin B and C immunoreactivities have similar regional distribution in rat brain, but the content of immunoreactive neuromedin B is 2-6 times higher than that of immunoreactive neuromedin C in every region. These results indicate that neuromedin B is a major endogenous bombesin-like peptide in rat brain and has specific functions of physiological importance.     

Clypeodytes geiseri sp. n., an enigmatic Bidessini from Laos (Coleoptera: Dytiscidae,Bidessini)

Here, we describe Clypeodytes geiseri sp. n. from Champasak Province in southwestern Laos. This very distinct species belongs to the subgenus Hypoclypeus Guignot, 1950 and can be distinguished from all other Clypeodytes species by its larger size and almost completely blackish or dark ferrugineous dorsal surface, except for some weak testaceous markings on the elytra. The habitus and the median lobe of the new species are illustrated, and a map is provided. Altogether, six species of subgenus Hypoclypeus are now known from Asia.

urn:lsid:zoobank.org:pub:FC01425F-9D3A-482E-8FE4-D2BF3D4943DF     

Interaction of opiate peptide and noradrenalin systems: Light microscopic studies

In this light microscopic immunocyto-chemical study β-Endorphin (β-END), leu-enkephalin and dopamine-βhydroxylase (DBH) antisera are used to obtain an overview of the interaction of the noradrenergic and opiate peptide systems in brain. Serial brain areas were analyzed for DBH and then for β-END or leu-enkephalin. Several areas were evaluated for cell and fiber interactions between these systems. The areas of richest possible contact between β-END and DBH positive systems include the rostral locus coeruleus region, the periaqueductal grey, possibly the dorsal thalamus, the paraventricular hypothalamus and the arcuate nucleus. Enkephalin cells and fibers were seen surrounding the locus coeruleus throughout its length with a few fibers in the nucleus itself.     

Dynorphin immunocytochemistry in the rat central nervous system

The distribution of dynorphin in the central nervous system was investigated in rats pretreated with relatively high doses (300–400 μg) of colchicine administered intracerebroventricularly. To circumvent the problems of antibody cross-reactivity, antisera were generated against different portions as well as the full dynorphin molecule (i.e., residues 1–13, 7–17, or 1–17). For comparison, antisera to [Leu]enkephalin (residues 1–5) were also utilized. Dynorphin was found to be widely distributed throughout the neuraxis. Immunoreactive neuronal perikarya exist in hypothalamic magnocellular nuclei, periaqueductal gray, scattered reticular formation sites, and other brain stem nuclei, as well as in spinal cord. Additionally, dynorphin-positive fibers or terminals occur in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate-putamen, globus pallidus, hypothalamus, substantia nigra, periaqueductal gray, many brain stem sties, and the spinal cord. In many areas studied, dynorphin and enkephalin appeared to form parallel but probably separate anatomical systems. The results suggest that dynorphin occurs in neuronal systems that are immunocytochemically distinct from those containing other opioid peptides.     

Spinal and supraspinal contributions to central sensitization in peripheral neuropathy

We will focus on spinal cord dorsal horn lamina I projection neurones, their supraspinal targets and involvement in pain processing. These spinal cord neurons respond to tonic peripheral inputs by wind-up and other intrinsic mechanisms that cause central hyper-excitability, which in turn can further enhance afferent inputs. We describe here another hierarchy of excitation - as inputs arrive in lamina I, neurones rapidly inform the parabrachial area (PBA) and periaqueductal grey (PAG), areas associated with the affective and autonomic responses to pain. In addition, PBA can connect to areas of the brainstem that send descending projections down to the spinal cord - establishing a loop. The serotonin receptor, 5HT3, in the spinal cord mediates excitatory descending inputs from the brainstem. These descending excitatory inputs are needed for the full coding of polymodal peripheral inputs from spinal neurons and are enhanced after nerve injury. Furthermore, activity in this serotonergic system can determine the actions of gabapentin (GBP) that is widely used in the treatment of neuropathic pain. Thus, a hierarchy of separate, but interacting excitatory systems exist at peripheral, spinal and supraspinal sites that all converge on spinal neurones. The reciprocal relations between pain, fear, anxiety and autonomic responses are likely to be subserved by these spinal-brainstem-spinal pathways we describe here. Understanding these pain pathways is a first step toward elucidating the complex links between pain and emotions.