Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus

Summary The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and antineurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon, GLP1 and neurotensin are shown to be colocalized in the EG-cells.     

Brain injury: New insights into neurotransmitter and receptor mechanisms

The studies reviewed here represent a continuing search for mechanisms which play a role in neurological disturbances resulting from brain injury. Focal cortical freezing lesions in rats were shown to cause a widespread decrease in local cerebral glucose utilization (LCGU) in cortical areas of the lesioned hemisphere and this was interpreted as reflecting a depression of cortical activity. Such an interpretation was supported by the finding that in lesioned brain reduction of cerebral metabolism by pentobarbital and isoflurane was limited by the metabolic depression that has already occurred as a result of injury and by the demonstration that the energy status and substrate (glucose) supply in the cortical areas in the injured brain have not been compromised at the time when LCGU was decreased. Both the serotonergic and the noradrenergic neurotransmitter systems were implicated in functional alterations associated with injury. Cortical serotonin (5-HT) metabolism was increased throughout the lesioned hemisphere and complete inhibition of 5-HT synthesis withp-chlorophenylalanine ameliorated the decrease in cortical LCGU, interpreted as reflecting cortical functional depression. Cortical norepinephrine metabolism was bilaterally increased in focally injured brain, while prazosin, a selective ₁-noradrenergic receptor blocker, normalized cortical LCGU in the lesioned hemisphere. Low-affinity in vivo binding of [¹²⁵I]HEAT, another selective ₁-receptor ligand, was specifically increased in cortical areas of the lesioned hemisphere at the time of the greatest depression in LCGU, suggesting that ₁-adrenoreceptors may be of functional importance in injured brain. The general conclusion from this series of studies on mechanisms underlying functional disturbances in injured brain is that both the serotonergic and the noradrenergic neurotransmitter systems are involved in the widespread cortical depression which develops with time as a consequence of a focal lesion. The data are compatible with the inhibitory effects of NE and 5-HT in the cortex and with the hypothesis that these two transmitter systems affect cortical information processing.     

Developmental Pattern for Phosphatidylserine Decarboxylase in Rat Brain

In adult rats, a significant portion of brain ethanolamine glycerophospholipids are synthesized by a pathway involving phosphatidylserine decarboxylase, a mitochondrial enzyme. We have now examined whether this enzyme plays a particularly prominent role during development. Activities for both phosphatidylserine decarboxylase and succinate dehydrogenase (another mitochondrial enzyme) were determined in brain homogenates from rats 5 days of age to adulthood. Succinate dehydrogenase activity, expressed on a per unit brain protein basis, increased markedly during development. This pattern has been reported previously and is as expected from the postnatal increase in oxidative metabolism. In contrast, phosphatidylserine decarboxylase activity decreased 40% from 5 to 30 days of age. The apparent Km for brain phosphatidylserine decarboxylase was 85 microM in both young (8- and 20-day-old) and adult animals. Parallel studies in vivo were carried out to determine the contribution of the phosphatidylserine decarboxylase pathway, relative to pathways utilizing ethanolamine directly, to the synthesis of brain ethanolamine glycerophospholipids. Animals were injected intracranially with a mixture of L-[G-3H]serine and [2-14C]ethanolamine and incorporation into the base moieties of the phospholipids determined. The 3H/14C ratio of ethanolamine glycerophospholipids decreased about 50% during development. Our studies in vitro and in vivo both suggest that phosphatidylserine decarboxylase plays a significant role in the synthesis of brain ethanolamine glycerophospholipids at all ages, although it is relatively more prominent early in development.     

Isolation and Sequencing of a Genomic Clone for Mouse Brain Specific Small RNA

We isolated a mouse genomic clone that hybridized with small RNA present in the cytoplasm of the brain. The RNA was about 150 nucleotides long. This RNA seemed to be specific to the brain, since it was not found in the liver or kidney. The clone DNA contained a sequence homologous to 82-nucleotide "identifier" core sequence of cDNA clones of rat. The sequence contained a split promoter for RNA polymerase III and was flanked by a 12-nucleotide direct repeat (ATAAATAATTTA).     

Enhancement of ATPase Activity by a Lipid Peroxide of Arachidonic Acid in Rat Brain Microvessels

The effects of 15-hydroperoxyarachidonic acid (15-HPAA) on Na+, K+- and Mg+-ATPase activities in the blood-brain barrier (BBB) were examined using rat brain microvessels (MV). 15-HPAA markedly stimulated these ATPase activities in MV at low concentrations whereas the synaptosomal Na+, K+-ATPase activity was inhibited in a dose-dependent manner. Further neurochemical analysis revealed that this stimulatory effect of 15-HPAA in MV was not due to a simple detergent-like action of the compound on the membranes but rather to stimulation of the phospholipase A2 and lipoxygenase activity within MV. In addition, it was shown that free radical reactions were involved in the mechanism. Since such anti-edema drugs as 1,2-bis(nicotinamido)propane were proved to be potent suppressors of the enhanced ATPase activity, further speculations on the role of this effect for ischemic brain edema are offered.     

Development and Characterization of Pantothenic Acid Transport in Brain

In vitro, the transport of [3H]pantothenic acid into and from rabbit brain slices was studied. In newborn rabbits and throughout development, forebrain and cerebellar slices were able to accumulate and phosphorylate [3H]pantothenic acid comparably to slices from adults. The accumulation and phosphorylation of [3H]pantothenic acid by adult forebrain slices were not decreased by substitution of LiCl for NaCl in the artificial CSF or by addition of short-chain fuels (e.g., 5 mM pyruvate or acetoacetate) to the medium. However, probenecid and ouabain (both 1 mM) and medium-chain fatty acids (e.g., 0.1 mM octanoate, nonanoate, and decanoate) profoundly inhibited [3H]pantothenic acid accumulation by forebrain slices but not intracellular phosphorylation and conversion to [3H]CoA. There in vitro results suggest that brain slices accumulate pantothenic acid by a saturable system (probably facilitated diffusion) that is sensitive to inhibition by probenecid and medium-chain fatty acids.     

Enzyme Immunoassay for Cholecystokinin Octapeptide Sulfate and Its Application

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for cholecystokinin octapeptide sulfate (CCK-8S) has been developed using N-terminal specific antibody for CCK-8S. In this assay CCK-8S coupled with poly-L-Glu (CCK-poly-Glu), which is adsorbed on a solid phase, competes with CCK-8S for the binding sites of rabbit anti-CCK antibody, and the complex of the immobilized antibody and CCK-poly-Glu is measured using goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase. The total time for completion of the assay is less than 24 h. Near 50% bound levels, the intraassay coefficient of variation is 5.2-6.2% and the interassay coefficient of variation is 5.9-8.5%. This assay is sensitive enough to detect 9 pg of CCK-8S, and the data from rat brain regions using this ELISA are very similar to the data from those using radioimmunoassay (RIA). Therefore, this ELISA is simpler and more rapid in comparison with conventional RIA. In the preliminary experiments, we applied this method for determination of CCK content in the brain regions of adult rats treated with 6-hydroxy-dopamine or in newborn rats subjected to anoxia, and showed that this system is applicable to detection of changes of endogenous CCK content.     

Evolutionary Divergence in the Structure of Myelin Basic Protein: Comparison of Chondrichthye Basic Proteins with Those from Higher Vertebrates

Abstract: A basic protein has been purified from the CNS myelin of the gummy shark (Mustelus antarticus). Electroblotting was used to examine the capacity of rabbit antisera raised against this electrophoretically pure protein to recognize myelin basic protein from higher vertebrates. The antisera bound to two shark proteins including the original polypeptide antigen and to chicken, bovine, and human myelin basic proteins. Thus, the shark protein appeared to possess antigenic determinants that have been retained through evolutionary divergence of these proteins. Whereas bovine basic protein caused experimental allergic encephalomyelitis in guinea pigs, animals that received injections of the shark protein showed neither clinical nor histological signs of this disease. However, tests for delayed-type hypersensitivity and for Arthus reaction following injection with the shark protein revealed a T-cell-mediated response to this antigen and substantial cross-reactivity with higher vertebrate basic proteins. Analysis of the amino acid composition of the shark protein, and comparison of its tryptic peptide map with that of the bovine protein, revealed substantial changes in the amino acid sequence. Although the shark protein has some antigenic determinants in common with the proteins from higher vertebrates, it appears that much of the structure differs.     

Expression of Microtubule-Associated Proteins During the Early Stages of Neurite Extension by Brain Neurons Cultured in a Defined Medium

Immunoblotting analysis was used to identify the microtubule-associated proteins (MAPs) present in cultures of mouse brain neurons. Polyclonal antibodies were raised against the two main adult brain MAPs, i.e., MAP2 (300 kDa) and tau (60-70 kDa). Whatever the stage of the culture, which was performed in a defined medium (3 or 6 days), the anti-MAP2 serum detected several high-molecular-weight components (including MAP2) and an entity with 62-65 kDa. Anti-tau revealed essentially a major peak of 48 kDa (young tau) but also slightly cross-reacted with the 62-65 kDa entity. During the culture period (0-6 days) the cells developed progressively a dense neuritic network; the concentration of the different MAPs increased in parallel but at different rates depending on the different species. The increase in concentration of the high-molecular-weight components occurred before that of 48-kDa tau. This suggests that high-molecular-weight MAPs and 48-kDa tau might be involved respectively in the initiation and elongation of neurites. In contrast, and since the main developmental changes in tau composition seen in vivo did not occur during the time course of the culture, this transition might be related to later events of neuronal differentiation.     

Leukotriene C4 Transport and Metabolism in the Central Nervous System

The transport and metabolism of radiolabeled leukotriene (LT) C4 in the CNS were investigated after intraventricular injection. Under thiopental (Pentothal) anesthesia, New Zealand white rabbits were injected intracerebroventricularly with 0.2 ml of artificial CSF containing 2.5 microCi of [3H]LTC4 (36 Ci/mmol), 0.3 microCi of [14C]mannitol, and, in some cases, 0.9 mg of probenecid, 1.8 mg of cysteine, 1.4 micrograms of unlabeled LTC4, or 2 mg of tolazoline HCl. After 2 h, the conscious rabbits were killed, and the quantity and nature of the 3H and 14C were determined in CSF, choroid plexus, and brain. The [3H]LTC4 recovered in CSF and brain was not extensively metabolized, as greater than 70% of the 3H remained [3H]LTC4, although some spontaneous conversion to 11-trans-[3H]LTC4 occurred. Oxidized forms of [3H]LTC4, [3H]LTD4, and [3H]LTE4 did not exceed 18% in CSF and brain. After intraventricular injection of [3H]LTC4, 3H was transferred from the CSF to blood by a probenecid-sensitive, but tolazoline-insensitive, transport system in the CNS much more rapidly than mannitol. Cysteine decreased the retention of [3H]LTC4 in brain. These results are consistent with previous in vitro observations that [3H]LTC4 is transferred from CSF into blood by an efficient transport system for LTC4 in choroid plexus.