High-sensitivity phenylthiohydantoin amino acid analysis on-line to a gas phase protein sequencer.
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Amino Acids↗
Biomedical subjects
M Costa
Publications and source records attributed to M Costa.
Sustained nerve-mediated contractions of guinea-pig ileum during morphine withdrawal by washout.
The effects of morphine withdrawal by washout were examined in ilea from guinea-pigs pretreated by subcutaneous implantation of two pellets, each containing 155 mg morphine. One week later preparations of the longitudinal muscle-myenteric plexus were set up in vitro in a modified Krebs solution containing morphine (1 microM). Within 5-15 min after washing in morphine-free Krebs, the preparations exhibited marked spontaneous contractions which became maximal after 20-60 min. The contractions resulting from morphine washout were greatly reduced by TTX and by hyoscine but not by hexamethonium, suggesting that excitation involved predominantly the cholinergic myenteric motor neurons, and was thus similar to that underlying the contraction induced by naloxone. In contrast to the naloxone-induced contractions, however, those resulting from morphine washout were well sustained during periods of recording up to 5 h. Morphine washout therefore provides conditions suitable for investigating the mechanisms underlying the variety of withdrawal signs observed in the ileum in vitro.
Animals↗
Comparison of the localization of chromosome damage induced by calcium chromate and nickel compounds.
Chromosomal aberrations were studied in Chinese hamster ovary cells and in C3H10T1/2 cells following treatment with NiCl2, crystalline NiS, and CaCrO4. All three compounds caused an increase in chromosomal aberrations in a concentration- and time-dependent fashion. The chromosomal aberrations induced by NiCl2 and crystalline NiS occurred predominantly in heterochromatic regions of the chromosomes. Additionally, treatment of cells with crystalline NiS and to a smaller extent long-term treatment with NiCl2 caused a preferential effect on the condensation state of the heterochromatic long arm of the X-chromosome in hamster cells. In contrast, treatment of cells with CaCrO4 did not induce aberrations preferentially in heterochromatin. These results are interesting because nickel(II), which is thought to be the ultimate carcinogen of nickel compounds, binds poorly to DNA, is weakly mutagenic, but induces chromosome damage, probably because of its interaction with nuclear proteins in heterochromatin. Chromate binds to DNA, is mutagenic, and interacts with chromatin randomly.
Calcium Compounds↗
Effect of nickel(II) on DNA-protein binding, thymidine incorporation, and sedimentation pattern of chromatin fractions from intact mammalian cells.
Nuclear uptake and chromatin binding of nickel(II) was investigated in Chinese hamster ovary (CHO) cells. The cytoplasmic:nuclear ratio of nickel immediately following treatment was 5:1, but by 24 and 48 hours this ratio decreased to 4:1 and 2:1, respectively, indicating that nickel is retained longer in the nucleus than cytoplasmic nickel. Chromatin was fractionated by sonication and centrifugation into fast-sedimenting, magnesium-insoluble, or magnesium-soluble components. The magnesium-insoluble portion bound more nickel ions and retained the metal longer than either the magnesium-soluble or the fast-sedimenting fractions. Treatment of cells with nickel chloride (NiCl2) decreased the amount of DNA in the magnesium-insoluble fraction but increased the amount of DNA in the fast-sedimenting chromatin fraction. The magnesium-insoluble fraction isolated from nickel-treated cells contained approximately ten times more [35-S]-methionine-labeled protein per milligram DNA compared with untreated cells. The magnesium-soluble and the fast-sedimenting fractions isolated from the nickel-treated cells did not exhibit a similar increase in [35-S]-methionine-labeled protein per milligram of DNA. Nickel treatment suppressed [14-C]-thymidine incorporation into total DNA by 30% compared with untreated cells. However, the magnesium-insoluble chromatin fraction from nickel-treated cells had a tenfold to 20-fold increase in thymidine incorporation, while the other chromatin fractions did not exhibit an increase in thymidine incorporation. These findings indicate that nickel induced widespread alterations in chromatin conformation and preferentially interacted with an Mg-insoluble component of chromatin.
Animals↗
Inhibitory effects of opioids in a circular muscle-myenteric plexus preparation of guinea-pig ileum.
The actions of opioids were examined in a strip preparation of the external muscle and myenteric plexus of the guinea-pig ileum cut parallel to the circular muscle. Contractions of the circular muscle induced by electrical stimulation of myenteric neurons were depressed in a concentration-dependent manner by the mu agonists, morphine and DAGO, and by the kappa agonist, U-50,488H. The concentrations of morphine, DAGO and U-50,488H which depressed nerve-mediated contractions by 50% (IC50) were 86 nM, 11 nM and 5.0 nM, respectively. The equilibrium dissociation constants (KD) for naloxone as an antagonist of the inhibitory effects of DAGO and of U-50,488H were 5.6 nM and 29.4 nM, respectively. In contrast to the potent inhibitory effects of mu and kappa agonists, the delta-selective agonist, D-Pen-L-Pen, produced only weak inhibition of nerve-mediated contractions. Even at a concentration of 3 microM, there was less than 50% inhibition, which was not antagonised by the delta receptor antagonist, ICI 174864. The experiments indicate that both mu and kappa opioid receptors are present on the myenteric neurons supplying the circular muscle and that delta receptors are either absent or ineffectively activated.
Animals↗
The source of the nerve fibres forming the deep muscular and circular muscle plexuses in the small intestine of the guinea-pig.
A quantitative ultrastructural study was made of the neurites forming the deep muscular and circular muscle plexuses of the guinea-pig small intestine following microsurgical lesions designed to interrupt intrinsic and extrinsic nerve pathways within the intestinal wall. Removal of a collar of longitudinal muscle with attached myenteric plexus from the circumference of a segment of small intestine resulted in the underlying circular muscle. The few surviving neurites in the deep muscular plexus and circular muscle disappeared completely from lesioned segments that were, in addition, extrinsically denervated surgically. These results indicate that the majority of nerve fibres in the deep muscular and circular muscle plexuses of the guinea-pig small intestine is intrinsic to the intestine and originates from nerve cell bodies located in the overlying myenteric plexus. At the light-microscopic level, nerve bundles were traced from the myenteric plexus to the circular muscle.
Animals↗
Distribution of peptide-containing neurons and endocrine cells in the rabbit gastrointestinal tract, with particular reference to the mucosa.
The distribution patterns of peptide-containing neurons and endocrine cells were mapped in sections of oesophagus, stomach, small intestine and large intestine of the rabbit, by use of standard immunohistochemical techniques. Whole mounts of separated layers of ileum were similarly examined. Antibodies raised against vasoactive intestinal peptide (VIP), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), enkephalins (ENK) and gastrin-releasing peptide (GRP) were used, and for each of these antisera distinct populations of immunoreactive (IR) nerve fibres were observed. Endocrine cells were labelled by the SP, SOM or NPY antisera in some regions. VIP-IR nerve fibres were common in each layer throughout the gastrointestinal tract. With the exception of the oesophagus, GRP-IR nerve fibres also occurred in each layer of the gastrointestinal tract; they formed a particularly rich network in the mucosa of the stomach and small intestine. Fewer nerve fibres containing NPY-IR or SOM-IR were seen in all areas. SOM-IR nerve fibres were very scarce in the circular and longitudinal muscle layers of each area and were absent from the gastric mucosa. The SP-IR innervation of the external musculature and ganglionated plexuses in most regions was rather extensive, whereas the mucosa was only very sparsely innervated. ENK-IR nerve fibres were extremely rare or absent from the mucosa of all areas, although immunoreactive nerve fibres were found in other layers. These studies illustrate the differences in distribution patterns of peptide-containing nerve fibres and endocrine cells along the gastrointestinal tract of the rabbit and also show that there are some marked differences in these patterns, in comparison with other mammalian species.
Animals↗
Pathway-specific patterns of the co-existence of substance P, calcitonin gene-related peptide, cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig.
The co-existence of immunoreactivities to substance P (SP), calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and dynorphin (DYN) in neurons of the dorsal root ganglion (DRG) of guinea-pigs has been investigated with a double-labeling immunofluorescence procedure. Four main populations of neurons could be identified that contained different combinations of these peptides and had distinctive peripheral projections: (Neurons that contained immunoreactivity to SP, CGRP, CCK and DYN were distributed mainly to the skin. Neurons with immunoreactivity to SP, CGRP and CCK, but not DYN, were distributed mainly to the small blood vessels of skeletal muscles. Neurons with immunoreactivity to SP, CGRP and DYN, but not CCK, were distributed mainly to pelvic viscera and airways. Neurons containing immunoreactivity to SP and CGRP, but not CCK and DYN, were distributed mainly to the heart, systemic blood vessels, blood vessels of the abdominal viscera, airways and sympathetic ganglia. Other small populations of DRG neurons containing SP, CGRP or CCK alone also were detected. Perikarya containing these combinations of neuropeptides were not found in autonomic ganglia. The peripheral axons of neurons containing immunoreactivity to at least SP and CGRP were damaged by chronic treatment with capsaicin. However, some sensory neurons containing CCK alone were not affected morphologically by capsaicin. These results clearly show that individual DRG neurons can contain many different neuropeptides. Furthermore, the combination of neuropeptides found in any particular neuron is related to its peripheral projection.
Animals↗
The projections of chemically identified nerve fibres in canine ileum.
The projections of nerve fibres with immunoreactivity for the peptides enkephalin (ENK), gastrin-releasing peptide (GRP), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP) were studied in canine small intestine by analysing the consequences of lesions of intrinsic and extrinsic nerves. Of peptides present in fibres supplying myenteric ganglia, GRP, SOM and VIP were in anally directed nerve pathways, whereas ENK and NPY were in orally directed pathways. Pathways ran for up to about 30 mm. SP fibres ran for short distances in both directions in the myenteric plexus. The circular muscle was supplied with ENK, NPY, SP and VIP fibres arising from the myenteric ganglia, whereas most mucosal SP and VIP fibres were deduced to arise from submucous ganglia. There were projections of fibres reactive for ENK, GRP, SOM, SP and VIP from myenteric ganglia to submucous ganglia. Antibodies to tyrosine hydroxylase were used to locate noradrenaline nerve fibres supplying the intestine; these fibres all disappeared when extrinsic nerves running through the mesentery to the small intestine were cut. It is deduced that there is an ordered pattern of projections of peptide-containing fibres in the canine intestine.
Animals↗
Galanin-immunoreactive neurons in the guinea-pig small intestine: their projections and relationships to other enteric neurons.
Galanin immunoreactivity was observed in nerve cell bodies and nerve fibres, but not in enteroendocrine cells, in the small intestine of the guinea-pig. Nerve terminals were found in the myenteric plexus, in the circular muscle, in submucous ganglia, around submucous arterioles, and in the mucosa. Lesion studies showed that all terminals were intrinsic to the intestine; those in myenteric ganglia arose from cell bodies in more orally placed ganglia. Myenteric nerve cells were also the source of terminals in the circular muscle. Galanin (GAL) was located in a population of submucous nerve cell bodies that also showed immunoreactivity for vasoactive intestinal peptide (VIP) and in a separate population that was immunoreactive for neuropeptide Y (NPY). Processes of the GAL/VIP neurons supplied submucous arterioles and the mucosal epithelium. Processes of GAL/NPY neurons ran to the mucosa. It is concluded that galanin immunoreactivity occurs in several functionally distinct classes of enteric neurons, amongst which are neurons controlling (i) motility, (ii) intestinal blood flow, and (iii) mucosal water and electrolyte transport.
Animals↗
Abnormalities of peptide-containing nerve fibers in infantile hypertrophic pyloric stenosis.
The distributions of nerve cells and fibers with immunoreactivity for the peptides enkephalin, gastrin-releasing peptide, neuropeptide Y, somatostatin, substance P, and vasoactive intestinal peptide were examined in specimens of myenteric plexus and external muscle from the pylorus of 20 infants with hypertrophic pyloric stenosis. These were compared with peptide distributions in pyloric samples from unaffected infants and adults. In the normal pylorus the circular muscle was richly supplied with fibers reactive for enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide. In pyloric stenosis, these immunoreactive fiber bundles were either missing or less than 5% of normal. In contrast, there were reactive cell bodies and nerve fibers in the myenteric plexuses of both normal and affected specimens. In the samples from cases of stenosis, swollen nerve fibers that appeared to be in the process of degeneration were frequently encountered. It is concluded that infantile hypertrophic pyloric stenosis is associated with a loss of peptide immunoreactivity in nerve fibers in the circular muscle, although the same peptides are still revealed in fibers and in nerve cell bodies in the myenteric plexus.
Enkephalin, Leucine↗
Distributions of neuropeptides in the human esophagus.
The distributions of nerve cells and fibers with immunoreactivity for the peptides substance P, somatostatin, enkephalin, vasoactive intestinal peptide, gastrin-releasing peptide, and neuropeptide Y and the enzyme tyrosine hydroxylase were examined in 25 samples of human esophagus. These were compared with samples of stomach and intestine. In the smooth muscle of the muscularis externa, the muscularis mucosae, and beneath the epithelium, the most abundant nerve fibers contained vasoactive intestinal peptide and neuropeptide Y, in contrast to the scarcity of substance P, enkephalin, somatostatin, and gastrin-releasing peptide. Gastric and intestinal samples contained dense populations of fibers containing vasoactive intestinal peptide, neuropeptide Y, substance P, and enkephalin in the equivalent layers, but somatostatin- and gastrin-releasing peptide-immunoreactive fibers were scarce. Complete coexistence of vasoactive intestinal peptide and neuropeptide Y in nerve fibers within the muscle layers was demonstrated in the esophagus, but not in gastric and intestinal samples. The myenteric plexus along the length of the esophagus contained cell bodies and fibers reactive for vasoactive intestinal peptide, neuropeptide Y, enkephalin, and substance P. Somatostatin-immunoreactive cell bodies were very rare in the myenteric plexus, no gastrin-releasing peptide-immunoreactive cell bodies were seen, and both somatostatin and gastrin-releasing peptide-immunoreactive fibers were rare. In the upper esophagus, striated muscle bundles did not contain nerve fibers reactive for these peptides but immunoreactive fibers were seen in the muscularis mucosae and subepithelium. It is concluded that the esophagus has a different pattern of innervation by peptide-containing neurons than the stomach and intestines. Esophageal neurons can be classified into separate classes on the basis of their peptide content.
Adult↗
The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, attenuates the Bezold-Jarisch reflex in the anaesthetized rat.
The effects of MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, on the Bezold-Jarisch reflex elicited by i.v. doses of 5-hydroxytryptamine (5-HT) was investigated in urethane-anaesthetized rats. Activation of the Bezold-Jarisch reflex with 5-HT (0.5-16 microgram/kg) produced pronounced dose-dependent hypotensive and bradycardic responses which were attenuated by MK-801 (1 mg/kg, i.v.) but not by saline. The data suggests that activation of the Bezold-Jarisch reflex by 5-HT involves a glutamatergic synapse presumably located within the brainstem vagal reflex arc.
Anesthesia↗
Sources of excitatory synaptic inputs to neurochemically identified submucous neurons of guinea-pig small intestine.
The locations of the cell bodies of axons responsible for synaptic potentials evoked in neurochemically identified submucous neurons of the guinea-pig small intestine were investigated using a combination of intracellular recording, immunohistochemical and lesioning techniques. The myenteric plexus was removed from an 8-15 mm wide ring of small intestine in 15 anaesthetized guinea-pigs. After the operations, the animals were allowed to recover for 3-7 days so that nerve terminals that were disconnected from their cell bodies would degenerate. Preparations of submucous plexus were then made from the region under the lesion. Submucous neurons were impaled with electrodes containing a mixture of KCl and the fluorescent dye, Lucifer yellow CH, and their electrophysiological properties determined. They were then filled with the dye for subsequent reidentification after processing for immunohistochemical localization of vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY). The synaptic inputs to 33 neurons were characterized: 19 of these were found to be VIP-reactive, 7 were NPY-reactive and 7 were negative for both VIP and NPY. These results were compared to those obtained from 43 neurons in control preparations: 25 VIP-reactive, 9 NPY-reactive and 9 negative for both VIP and NPY. Removal of the myenteric plexus caused a significant reduction in the number of inputs providing fast excitatory synaptic potentials to each of the neurochemically defined classes of neurons. The lesions also caused a significant reduction in the number of VIP-reactive neurons that exhibited slow excitatory synaptic potentials (other neurochemical types do not normally exhibit such responses).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Naloxone-induced depolarization and synaptic activation of myenteric neurons in morphine-dependent guinea pig ileum.
To investigate the cellular basis of opiate dependence, intracellular microelectrodes were used to record from both electrophysiologically defined classes of neurons (S and AH) in myenteric plexus longitudinal muscle preparations from morphine pretreated guinea pigs. These preparations responded to naloxone with the characteristic contraction of the longitudinal smooth muscle, indicative of morphine dependence. Depolarization in response to naloxone was observed in 42% of S neurons, but there were no consistent changes in input resistance. In some cells the depolarization was reduced or abolished after blockade of synaptic transmission, suggesting that it was due in part to the release of an excitatory transmitter producing a slow depolarization in the impaled neuron. Synaptic activation of S neurons during withdrawal was further indicated by the observation that fast postsynaptic potentials appeared after abrupt displacement of morphine from its receptors by naloxone. Morphine withdrawal, therefore, involves both the final motor neurons and interneurons. During naloxone-induced withdrawal, 25% of S neurons discharged action potentials. In contrast, no action potentials were discharged in AH neurons. Furthermore, naloxone did not alter the resting membrane potential, input resistance, soma action potential configuration, or slow hyperpolarization following a soma spike in AH neurons. The specificity of the withdrawal response for S neurons and the relatively small proportion of neurons involved suggests that morphine withdrawal occurs in quite specific neuronal circuits in the myenteric plexus.
Animals↗
Physicochemical characteristics and biological effects of nickel oxides.
Ten nickel oxides and nickel-copper oxides, which all contained NiO (bunsenite) as the predominant crystalline phase, were assayed as follows: in vitro dissolution tests in water and body fluids; in vitro phagocytosis tests in Chinese hamster ovary and C3H-10T1/2 cells; morphological transformation and cytotoxicity tests in cultured Syrian hamster embryo (SHE) cells; erythropoiesis stimulation assay by intrarenal administration to Fischer-344 rats; and scoring the renal histopathologic responses in rats killed 3 months post-injection. The test compounds differed substantially in their biological effects when tested in the various experimental systems. Based upon highly significant concordance of ranked results in the assays (P less than 0.001), six colligative biological attributes of the compounds were identified: (i) dissolution half-times in rat serum and renal cytosol; (ii) phagocytosis by C3H-10T1/2 cells; (iii) morphological transformation of SHE cells; (iv) erythropoiesis stimulation in rats; (v) induction of tubular hyperplasia in rat kidneys; and (vi) induction of arteriosclerosis in rat kidneys. Strong rank correlation (P less than 0.01) between results of the cell transformation and erythropoiesis stimulation assays is especially notable, since the compounds were tested by blind protocols in independent laboratories. The presence of high surface area and demonstrable Ni(III) were two physicochemical characteristics that were associated with the greatest biological effects of nickel oxides.
Animals↗
Effect of magnesium on nickel-induced genotoxicity and cell transformation.
Raising the extracellular level of magnesium ions inhibited nickel-induced DNA strand breaks, DNA-protein crosslinks, sister chromatid exchanges, chromosomal aberrations and cell transformation. Carcinogenic nickel ions preferentially damaged centromeres and other heterochromatic regions of Chinese hamster ovary cell chromosomes. Elevation of extracellular magnesium levels prevented the effects of nickel on heterochromatin and inhibited cell transformation, but did not substantially reduce the DNA damage induced by nickel in euchromatic regions. This study suggests that heterochromatic DNA damage may be important to the nickel-induced neoplastic transformation process.
Animals↗
Principles of industrial toxicology.
This article offers a brief introduction to the general principles of toxicology, followed by a discussion of the toxicology of heavy metals, solvents, halogenated hydrocarbons, and alcohols. It concludes with an overflow of pesticide toxicology.
Herbicides↗