Demonstration of enkephalin-, substance p- and glutamate decarboxylase-like immunoreactivity in cultured cells derived from newborn rat neostriatum

Summary The presence of cells exhibiting leucine-enkephalin-, substance P- and glutamate decarboxylase-like immunoreactivity was demonstrated in dissociated cultures from newborn rat neostriatum. The size and shape of the enkephalin-immunoreactive cells varied, but they were generally larger than substance P- and glutamate decarboxylase-immunoreactive cells, which formed relatively uniform cell populations. Cells of apparently non-neuronal origin did not show any immunoreactivity. It is unlikely that enkephalin is present in the same cells that contain substance P or glutamate decarboxylase because of norphological differences between these cells. The possible coexistence of substance P and glutamate decarboxylase in the same cells however, could not be excluded. The results of this study confirm that the cell bodies of neurons containing three possible neurotransmitters are located in the neostriatum.     

Neurokinin A-like immunoreactivity in rat primary sensory neurons; coexistence with substance P

Rat spinal cord, dorsal root ganglia and skin were investigated employing immunohistochemical technique with specific antisera to neurokinin A and substance P. Neurokinin A-like immunoreactivity was detected in the spinal dorsal horn and skin with a similar distribution pattern as that of substance P-like immunoreactivity. After dorsal root transection a parallel decrease of neurokinin A and substance P-like immunoreactivity was observed in the dorsal horn. Using colchicine pretreatment a population of neurokinin A positive cell bodies was seen in the dorsal root ganglia, and by comparison of consecutive sections of the same cells stained for substance P it was revealed that these neurons also display substance P-like immunoreactivity. However, substance P-, but not neurokinin A-, immunoreactive cells were also observed. It is concluded that neurokinin A- and substance P-like immunoreactivity coexist in a population of rat primary sensory neurons.     

Neurokinin A-like immunoreactivity in rat primary sensory neurons; coexistence with substance P

Summary Rat spinal cord, dorsal root ganglia and skin were investigated employing immunohistochemical technique with specific antisera to neurokinin A and substance P. Neurokinin A-like immunoreactivity was detected in the spinal dorsal horn and skin with a similar distribution pattern as that of substance P-like immunoreactivity. After dorsal root transection a parallell decrease of neurokinin A and substance P-like immunoreactivity was observed in the dorsal horn. Using colchicine pretreatment a population of neurokinin A positive cell bodies was seen in the dorsal root ganglia, and by comparison of consecutive sections of the same cells stained for substance P it was revealed that these neurons also display substance P-like immunoreactivity. However, substance P-, but not neurokinin A-, immunoreactive cells were also observed. It is concluded that neurokinin A- and substance P-like immunoreactivity coexist in a population of rat primary sensory neurons.     

Substance P-like immunoreactivity in the nervous system of hydra

Summary Using immunocytochemistry we find substance P-like material in nerve cells of hydra. These nerve cells are situated in the ectoderm of the basal disk and tentacles. Radioimmunoassay of hydra extracts gives dilution curves parallel to that of synthetic substance P, from which it can be calculated that one animal contains at least 0.6 fmol substance P-like immunoreactivity. After chromatography on Biogel P-100, the substance P-like immunoreactivity elutes as a peak in the void volume and a peak at the position of synthetic substance P.     

Substance P-like immunoreactivity in the nervous system of hydra

Using immunocytochemistry we find substance P-like material in nerve cells of hydra. These nerve cells are situated in the ectoderm of the basal disk and tentacles. Radioimmunoassay of hydra extracts gives dilution curves parallel to that of synthetic substance P, from which it can be calculated that one animal contains at least 0.6 fmol substance P-like immunoreactivity. After chromatography on Biogel P-100, the substance P-like immunoreactivity elutes as a peak in the void volume and a peak at the position of synthetic substance P.     

Chemical phenotypes of P2X2 purinoreceptor immunoreactive cell bodies in the area postrema

Purines such as adenosine 5'-triphosphate (ATP) act as extracellular messengers through specific purinergic receptors. Three different classes of purinergic receptors have been identified and termed P1, P2X, and P2Y. The purinergic receptor subunit P2X2 is a ligand-gated ion channel that is widely expressed by neurons in the CNS. In the brainstem medulla oblongata, the ionotropic P2X2 receptor (P2X2R) is enriched in the area postrema (AP). Two different antisera to P2X2R were used to determine the chemical nature of P2X2R immunoreactive cell bodies in the rat AP, an area lacking a blood-brain barrier. Subcellularly, P2X2R immunoreactivity was located to the periphery of individual cell bodies. The majority of P2X2R-immunoreactive cells were shown to contain tyrosine hydroxylase (TH) (63.5 ± 7.7%) and dopamine β-hydroxylase (61.5 ± 5.1%). Phenylethanolamine N-methyltransferase (PNMT)-containing cells were not detected in the AP, supporting a noradrenergic nature of P2X2R cells in the AP. There were no P2X2R-immunoreactive cells in the AP that contained the GABA-synthesizing enzyme glutamic acid decarboxylase 65. Only single vesicular glutamate transporter 2-immunoreactive cell bodies that were not P2X2R-positive were demonstrated in the AP. Some P2X2R-positive cells in the AP were immunoreactive for the neuropeptides substance P and pituitary adenylate cyclase-activating polypeptide, whereas dynorphin-, enkephalin-, or cholecystokinin-positive cells were not P2X2R-immunoreactive. Presence of P2X2R in a majority of noradrenergic cells of the AP implies that ATP may have a regulatory action on neuronal noradrenaline release from the AP, a circumventricular organ with a strategic position enabling interactions between circulating substances and the central nervous system.     

Neurotransmitters and putative neuromodulators in the gut of Anguilla anguilla (L.). Localizations in the enteric nervous and endocrine systems

The gut of silver eels (Anguilla anguilla L.) was investigated in order to describe both the cholinergic and adrenergic intramural innervations, and the localization of possible accessory neuromediators. Histochemical reactions for the demonstration of nicotinamide adenine dinucleotide phosphate, reduced form-(NADPH-)diaphorase and acetylcholinesterase (AChEase) were performed, as well as the immunohistochemical testing of tyrosine hydroxylase, met-enkephalin, substance P, calcitonin gene-related peptide (CGRP), bombesin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), somatostatin, cholecystokinin-octapeptide (CCK-8), serotonin, cholineacetyl transferase. The results evidenced a different pattern in comparison with other vertebrates, namely mammals, and with other fish. Both NADPH-diaphorase and AChEase activities were histochemically detected all along the gut in the myenteric plexus, the inner musculature and the propria-submucosa. Tyrosine hydroxylase immunoreactivity was observed in the intestinal tract only, both in the myenteric plexus and in the inner musculature. Several neuropeptides (metenkephalin, CGRP, bombesin, substance P, VIP, NPY, somatostatin) were, in addition, detected in the intramural innervation; some of them also in epithelial cells of the diffuse endocrine system (met-enkephalin, substance P, NPY, somatostatin). Serotonin was only present in endocrine cells. Tyrosine hydroxylase immunoreactivity was present in localizations similar to those of NADPH-diaphorase-reactivity, and in the same nerve bundles in which substance P- and CGRP-like-immunoreactivities were detectable in the intestinal tract. In addition, NADPH-diaphorase-reactive neurons showed an anatomical relationship with AChEase-reactive nerve terminals, and a similar relationship existed between the latter and substance P-like immunoreactivity.     

GABA metabolism in venezuelan equine encephalomyelitis virus infection

Mice infected with the Venezuelan equine encephalomyelitis virus showed a significant decrease in the GABA content of cerebral hemispheres. Activity of the enzyme which synthetizes GABA, glutamate decarboxylase, is also reduced in whole cerebral hemispheres, neostriatum, and frontal cortex of infected animals, as compared to values obtained from the same regions of control mice. No significant difference was demonstrated in the activities of GABA transaminase, glutamate dehydrogenase, lactate dehydrogenase, succinate dehydrogenase and NAD-malate dehydrogenase in any of the regions studied. The results suggest that the viral infection produced an alteration in the mechanism of GABA synthesis.     

Depolarization elicits,while hyperpolarization blocks uptake of endogenous glutamate by retinal horizontal cells of the turtle

We have employed an immunoreaction against glutamate to qualitatively demonstrate varying levels of glutamate in retinal horizontal cells of the turtle. Glutamate-like immunoreactivity (GLI) in horizontal cells could be demonstrated after glutamate decarboxylase was inhibited by aminooxy acetic acid (AOAA) and its degradation to GABA was blocked. Depolarization of horizontal cells by kainic acid (KA) induces strong glutamate immunoreactivity in these cells, whereas hyperpolarization by 2,3-cis piperidine dicarboxylate (PDA) abolishes glutamate-like immunoreactivity in horizontal cells. When glutamate release from cones and bipolar cells is blocked in the absence of calcium, or when glutamate uptake is blocked by DL-threo -hydroxy aspartate, KA/AOAA treatment of the retina does not induce GLI in horizontal cells. Our data show that horizontal cells are capable of taking up glutamate from the endogenous retinal pool in an activity dependent way. Our interpretation of these findings is that retinal horizontal cells are capable of regulating glutamate levels in the extracellular space of the cone pedicle complex by an activity-dependent uptake system. We suggest that inhibition of glutamate uptake upon hyperpolarization rather than inhibition of GABA release may evoke the antagonistic surround response of retinal bipolar cells.     

Glutamate decarboxylase in developing rat neocortex: Does it correlate with the differentiation of GABAergic neurons and synapses?

Postnatal development of glutamate decarboxylase was studied in the rat cerebral cortex. Two methods were used: estimation of the enzymatic activity of glutamate decarboxylase in homogenates of developing cortical tissue and visualization of structures containing glutamate decarboxylase-like immunoreactivity. Glutamate decarboxylase-like immunoreactivity appeared first in perikarya and dendrites and only later in axons and axon varicosities. The most rapid increase in the glutamate decarboxylase activity took place during the second postnatal week and this coincided with a rapid increase in the density of axon varicosities containing glutamate decarboxylase-like immunoreactivity but preceded the most rapid phase in the formation of GABAergic synapses by several days. However, there was a change in the characteristics of glutamate decarboxylase which correlated with GABA synaptogenesis: two fractions of glutamate decarboxylase with different sensitivities to the activating effects of Triton X-100 could be distinguished as from about the time when most of the GABAergic synapses are formed.     

Ultrastructure and localization of substance P and met-enkephalin immunoreactivity in the human fetal gastric antrum

Summary The ultrastructural localization and relations of substance P- and met-enkephalin-labeled neuronal structures were examined in the wall of the human gastric antrum during early fetal life. By 14–16 weeks of gestation, clearly discernable neural plexuses and a well developed external muscle coat were present. In the submucous coat, neural plexuses varied from immature forms consisting of 1–4 neurites partially enveloped by Schwann cell processes to more mature plexuses where neurons were completely enclosed by Schwann cell processes. Neuronal profiles with substance P- and met-enkephalin-like immunoreactivities were observed in the submucous plexus. In the myenteric plexus met-enkephalin-like immunoreactivity was seen within cell bodies and neurites. By contrast, although substance P-like immunoreactivity was observed in neurites in the myenteric plexus, no substance P-labeled somata could be identified. Unlabeled terminals were seen in contact with both unlabeled dendrites and met-enkephalinergic neurons. An increase in electron density was observed at the sites of contact. These structures probably represent early stages in the development of synaptic specializations. In addition, met-enkephalin-labeled varicosities were seen in apposition to smooth muscle cells of the circular muscle coat. This suggests that antral smooth muscle cells are directly innervated by met-enkephalin neurons.     

A Study of the Alimentary Canal of the Brachyopterygian Fish Polypterus senegalus with Electron Microscopy and Immunohistochemistry

The gastro-intestinal tract of Polypterus senegalus was investigated by means of electron microscopy and immunohistochemistry. Cilia-bearing cells can be observed over the whole length of the intestine. All enterocytes along the intestinal tract are characterized by apical pinocytotic vesicles. However, a typical intestinal region, which in other fish is characterized by large supranuclear vacuoles, is lacking. By means of electron microscopy, four types of endocrine cells and three types of nerve cell processes can be identified. By means of immunohistochemistry, endocrine cells with immunoreactivity for bombesin-, enkephalin-, G/CCK-, 5-HT-, somatostatin- and substance P-antisera can be found. Nerve cell processes show immunoreactivity for bombesin-, enkephalin-, 5-HT-, substance P- and VIP-antisera. The number of immunoreactive endocrine cells, nerve cells and nerve cell processes is different for each part of the gut.     

Substance P- and cholecytokinin-like immunoreactivity during post-metamorphic development of the central nervous system in the ascidian Ciona intestinalis

Following metamorphosis, the neural ganglion of ascidians is thought to be formed via the proliferation of epithelial cells comprising the ciliated duct. In adults, neuronal cell bodies expressing substance P- and gastrin/cholecystokinin-like immunoreactivity exhibit clearly defined patterns of distribution. Previous work shows that these patterns are re-established during regeneration of the adult ganglion. We have used antisera against substance P and cholecystokinin to monitor the formation of these patterns during normal post metamorphic development in Ciona intestinalis. Substance P cells first appear in the ganglion in animals of 1 mm body length. Cholecystokinin antiserum was not used at this stage but revealed a clear adult-like pattern of cells in the anterior region at the 3 to 5-mm stage. Substance P cells do not exhibit an adult pattern until animals have a body length of more than 10 mm. Proliferation in the neural complex was studied using the bromodeoxyuridine/anti-bromodeoxyuridine technique. Results suggest a mechanism whereby cells are born in the ciliated duct and later migrate to the ganglion. Double-labelling experiments indicate that more than 11 days elapse between cell birthdates and the expression of either of the peptides. Data presented suggest that the distributional patterns for these peptides during normal development are similar to those seen during regeneration.     

The cohesive properties of variants of Neisseria gonorrhoeae strain P9: specific pilus-mediated and non-specific interactions

The cohesive properties of virulent pilated Neisseria gonorrhoeae strain P9 (P++) have been compared with those of a non-pilated isogenic variant (P-) possessing the same outer membrane components. The binding of P++ gonococci to buccal epithelial cells was dependent on pH, with an optimum at pH 6.5 to 7.0 . This adhesion was markedly inhibited by treatment of the buccal epithelial cells with a neuraminidase/exoglycosidase mixture. In contrast, the binding of P++ gonococci to erythrocytes was unaffected by pH. A possible explanation is that pili bind to a carbohydrate receptor present on buccal epithelial cells but lacking on erythrocytes. The adhesion of P- gonococci to erythrocytes and to buccal epithelial cells was unaffected by pH but enhanced by treatment of the cells with neuaminidase or periodate. Presumably, neuraminic acid residues on host cell surface carbohydrates inhibit adhesion. The finding that P- gonococci bind to amphipathic gels suggests hydrophobic interactions as a possible non-specific mechanism attaching P- gonococci to host cell surfaces.     

Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells

Summary By use of the indirect immunofluorescence technique the distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) has been analyzed in cervical and lumbar dorsal root ganglia of untreated and colchicine-treated rats. In addition, lumbar ganglia were examined 2 weeks after transection of the sciatic nerve. The occurrence of CGRP-positive cells in relation to ganglion cells containing substance P-, somatostatin-, galanin-, cholecystokinin (CCK)-, and vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucin (PHI)-LI has been evaluated on consecutive sections as well as using elution-restaining and double-staining techniques.CGRP-LI was observed in many ganglion cells of all sizes ranging in diameter from 15 m to 65 m. Thus, this peptide occurs also in the large primary sensory neurons. In contrast to the sensory peptides described to date, CGRP-positive cells constituted up to 50% of all and 70% of the medium-sized neurons, thus being the most frequently occurring peptide in sensory neurons so far encountered. Subpulations of CGRP-positive neurons were shown to contain substance P-, somatostatin-, or galanin-LI and some CGRP-positive neurons contained both substance P- and galanin-LI. In fact, most substance P-, somatostatin- and galanin-positive cell bodies were CGRP-immunoreactive. The coexistence analysis further revealed that galanin and substance P often coexisted and that some cells contained both substance P- and somatostatin-LI, whereas no coexistence between galanin and somatostatin has as yet been seen. VIP/PHI-LI was only shown in a few cells in untreated or colchicine-treated rats. However, after transcetion of the sciatic nerve numerous VIP/PHI-positive cells were observed, some of which also contained CGRP-LI.The present results indicate that a CGRP-like peptide is present in a wide range of primary sensory neurons probably not related to specific sensory modalities. Often this peptide coexists with other biologically active peptides. Taken together these findings suggest that CGRP may have a generalized function.     

GABA synthesis by cultured fibroblasts obtained from persons with Huntington's disease.

Abstract— A consistent observation in particular regions of brains of persons having died with Huntington's disease (HD) is a reduction in the concentration of γ-aminobutyric acid (GABA) and a decrease in the activity of its synthetic enzyme, glutamate decarboxylase (EC 4.1.4.15). GABA levels are also reduced in HD cerebrospinal fluids. This study suggests that skin fibroblasts obtained from persons with HD can be used to study their GABA system. A rapid and specific assay for [¹⁴C]glutamate– [¹⁴C]GABA based on Aminex A-7 chromatography has been developed. Cell monolayers and homogenates of HD cells convert [¹⁴C]glutamate to [¹⁴C]GABA. GABA synthesis by HD cell homogenates is pyridoxal dependent and is inhibited by 1 mm -aminooxyacetic acid. GABA synthesis by HD and control cell homogenates also show the same thermal sensitivity as rat brain GAD. When compared to non-HD human cells the HD cells reveal disturbances in the non-neuronal GABA metabolic pathway. Concentrated HD cell homogenates synthesize approx 3 times the amount of GABA as control cells. When diluted both extracts made similar amounts of GABA. Synthesis of GABA by HD cell homogenates is not inhibited by cysteine sulfinate. Decarboxylation of glutamate in these cells is therefore most likely due to glutamate decarboxylase and not cysteine sulfinate decarboxylase. HD cells in monolayer also synthesize 3 times the amount of GABA as compared to control cells. In addition, glutamate upake is altered in HD cells. This report indicates there may be a different pattern of enzyme regulation between HD and control cells.     

Inhibition of uroporphyrinogen decarboxylase by halogenated biphenyls in chick hepatocyte cultures. Essential role for induction of cytochrome P-448.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was assayed in cultures of chick-embryo hepatocytes by the changes in composition of porphyrins accumulated after addition of excess 5-aminolaevulinate. Control cells accumulated mainly protoporphyrin, whereas cells treated with 3,4,3',4'-tetrachlorobiphenyl or 2,4,5,3',4'-pentabromobiphenyl accumulated mainly uroporphyrin, indicating decreased activity of the decarboxylase. 3-Methylcholanthrene and other polycyclic-hydrocarbon inducers of the P-448 isoenzyme of cytochrome P-450, did not affect the decarboxylase in the absence of the biphenyls. Induction of P-448 was detected as an increase in ethoxyresorufin de-ethylase activity. Pretreatment of cells with methylcholanthrene decreased the time required for the halogenated biphenyls to inhibit the decarboxylase. The dose response of methylcholanthrene showed that less than 40% of the maximal induction of cytochrome P-448 was needed to produce the maximum biphenyl-mediated inhibition of the decarboxylase. In contrast, induction of the cytochrome P-450 isoenzyme by propylisopropylacetamide had no effect on the biphenyl-mediated decrease in decarboxylase activity. Use of inhibitors of the P-450 and P-448 isoenzymes (SKF-525A, piperonyl butoxide and ellipticine) supported the concept that only the P-448 isoenzyme is involved in the inhibition of the decarboxylase by the halogenated biphenyls. The effect of preinduction with methylcholanthrene to enhance inhibition of the decarboxylase was also shown by the increased rate at which porphyrin accumulated from endogenously synthesized 5-aminolaevulinate after treatment of cells with the combination of propylisopropylacetamide and the biphenyls. Antioxidants, chelators of iron, and chromate affected the decrease in decarboxylase activity only if they prevented the induced increase in cytochrome P-448. We conclude that the P-448 and not the P-450 isoenzyme of cytochrome P-450 plays an obligatory role in the inhibition of uroporphyrinogen decarboxylase caused by halogenated biphenyls.     

Quantitative comparison of growth-associated protein-43 and substance P in ulcerative colitis.

The aim of this study was to compare immunoreactivities for substance P with other enteric neuropeptides and GAP-43, a general marker for enteric nerves, in normal human colon and in different stages of ulcerative colitis. Tissue samples from normal colon and regions of ulcerative colitis colon were obtained at surgery and immunostained for substance P, vasoactive intestinal polypeptide (VIP), somatostatin, calcitonin gene-related peptide (CGRP), enkephalin, galanin, GAP-43, and neuron-specific enolase (NSE). Visual examination and semiquantitative analysis revealed a clear increase in the immunoreactivity for substance P in ulcerative colitis, whereas no differences were observed in the distribution of the other peptides. Therefore, quantitative analysis was performed only for substance P immunoreactivity in the lamina propria, circular muscle layer, and myenteric ganglia. In the lamina propria, the score of total intensity of substance P immunoreactivity was 0.55 +/- 0.15 (mean +/- SEM) in normal colon, 1.30 +/- 0.35 (p = 0.087) in least affected colon, and 2.22 +/- 0.28 (p < 0.001) in moderately affected colon, whereas no significant differences were observed in immunoreactivities for GAP-43. Similar results were obtained for the mean substance P- or GAP-43-immunoreactive area. In the circular muscle layer, the number, density, total intensity, and perimeter of substance P- and GAP-43-immunoreactive fibers were essentially similar in normal colon, and in mild or moderately affected colon. We conclude that ulcerative colitis does not change the density of gut innervation as a whole. However, the density of substance P-containing nerves is specifically increased, probably due to increased peptide synthesis leading to better visibility of the fibers.     

Oxic and anoxic growth of a new Citrobacter species on amino acids

A facultatively anaerobic bacterium, strain P-88, was enriched selectively under dual limitation by glutamate and oxygen in a chemostat. The new strain is a gram-negative motile rod. The mol% guanine plus cytosine of the DNA is 51.4±0.6 mol%. The organism grows on citrate as a sole source of carbon and energy, does not form acetoin, does not induce lysine decarboxylase and was thus classified as a species of the genus Citrobacter. A remarkable characteristic of the new isolate is its ability to grow on several amino acids with either a respiratory or a fermentative type of metabolism. Under strictly anoxic conditions glutamate was fermented to acetate, H₂, CO₂ and ammonia. Asparagine, aspartate and serine could also be fermented. Furthermore, all type strains of the genus Citrobacter were shown to have the same fermentative abilities. Based on enzyme activities determined in cell-free extracts a combination of the methylaspartate pathway and the mixed acid fermentation of Enterobacteriaceae is proposed to explain the glutamate fermentation pattern observed in cultures of strain P-88. Analysis of the growth of strain P-88 in continuous culture with various degrees of oxygen supply, demonstrated that the bacterium can rapidly switch between oxic and anoxic metabolism. Cultures of strain P-88 grown under oxygen limitation simultaneously respire and ferment glutamate, suggesting that the organism is particularly well adapted to growth in microoxic environments.     

Chemical and immunochemical characterisation of substance P-like immunoreactivity and physalaemin-like immunoreactivity in a carcinoid tumour

Extracts of a carcinoid tumour, resected from the mid-portion of the ileum of a patient with no symptoms of endocrine disorder, were associated with a high concentration of substance P-like immunoreactivity. Using reverse-phase high performance liquid chromatography and antisera specifically directed against the C-terminal and N-terminal regions of substance P and against the N-terminal region of physalaemin, the following components were isolated and identified: substance P and its oxidised form, [pGlu⁵]substance P-(5–11) peptide and its oxidised form, and the oxidised form of physalaemin. The identity of tumour substance P with the undecapeptide was confirmed by amino acid analysis and high performance ion-exchange chromatography. In vitro incubation of tumour tissue from a lymph node metastasis from the same patient with [³H]leucine resulted in incorporation of radioactivity into newly synthesised substance P.