Immunohistochemical identification of supportive cell types in the enteric nervous system of the rat colon and rectum

Summary The non-neuronal, supportive cells of the enteric nerve plexus were investigated in the colon and rectum of adult and developing rats by means of immunohistochemistry, utilizing antisera against GFA protein and S-100 protein. Immunoreactivity to GFA protein was almost exclusively found in cells associated with the myenteric plexus and a small number of cells within the submucous ganglia. On the other hand, the use of S-100 protein antiserum resulted in the visualization of all supportive elements in the enteric nervous system. However, two types of supportive cells could be tentatively differentiated in the enteric nerve plexus during the second week of postnatal development, using GFA protein and S-100 protein antisera; GFA protein-positive cells were clearly discernible from S-100 protein-positive cells in terms of both the morphological profiles and immunohistochemical properties. It was assumed that at least two different types of supportive cells are contained in the enteric nerve plexus. We suggest that in the enteric nervous system the terms glial cells and Schwann cells should be employed to designate the supportive cells containing GFA and S-100 proteins, and cells containing S-100 protein, respectively. We discuss the possibility that glial cells are associated with the parasympathetic preganglionic fibres directly derived from the central nervous system, while Schwann cells originate from the neural crest.     

Immunohistochemical localization of S-100 protein,glial fibrillary acidic protein,and neuron-specific enolase in the pars distalis of quail,rat, and human hypophyses

Summary In the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.     

Immunohistochemical localization of S-100 protein, glial fibrillary acidic protein, and neuron-specific enolase in the pars distalis of quail, rat, and human hypophyses

In the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.     

Neuronal and Glial Marker Proteins in the Evaluation of the Protective Action of MK 801

A quantitative dot immunobinding procedure was used to quantify glial [the S-100 protein and the glial fibrillary acidic (GFA) protein] and neuronal (the 68- and 200-kDa neurofilament polypeptides, neuron-specific enolase, and neuronal cell adhesion molecule) markers. A single intraperitoneal administration of 10 mg/kg of MK 801 blocked the increase of glial parameters and the decrease in content of neuronal marker proteins that occurred as the response to an N-methyl-D-aspartate (NMDA) lesion in the rat hippocampus. The degradation products of GFA protein and the 68-kDa neurofilament polypeptide that were induced by the NMDA lesion did not appear after MK 801 treatment. This study shows that brain-specific proteins are a set of precise tools for the evaluation of neuroprotective effects of antagonists to excitatory amino acids.     

Induction of S-100b (beta beta) protein in human teratocarcinoma cells

Human teratocarcinoma NT2/D1 cells undergo differentiation into a variety of cell types, including neurons, treated with retinoic acid. In the present study, the concentrations of alpha S-100 and beta S-100 proteins (alpha and beta subunits of S-100 proteins), and three subunits (alpha, beta and gamma) of enolase in NT2/D1 cells were measured using the sensitive enzyme immunoassay method. The concentration of beta S-100 was markedly increased in the cells after treatment with retinoic acid, whereas the concentration of alpha S-100 was undetectably low, indicating that the S-100b (beta beta) protein was induced by retinoic acid. On the other hand, the concentrations of the three forms of enolase isozymes did not change in the same culture. The induction of S-100b protein was not observed in the NT2/D1 cells after treatment with forskolin, dibutyryl cyclic AMP or cholera toxin. The indirect double-labeled immunofluorescence, using antibodies specific to beta S-100 and monoclonal antibodies specific to neurofilaments, revealed that both the S-100b protein and the neurofilaments were induced in the same subpopulation of cells which underwent neuronal differentiation.     

Effect of protein S-100 on phosphorylation of nuclear proteins of cells of rat brain and liver

The effect of acidic neurospecific protein S-100 on the phosphorylation of brain and liver nuclear proteins with 1 and 10 microM ATP was investigated. It was shown that protein S-100 increases the phosphorylation of brain nuclear proteins, while antigen D, another acidic neurospecific protein half-identical to 14-3-2 protein, inhibits this process. Ca2+ and cAMP at concentration of 10(-6) M do not affect the phosphorylation of brain nuclear proteins. In control assays the tracer 32P is presumably incorporated into high molecular weight nuclear protein fractions (Mr greater than 40000). After addition of protein S-100 the tracer is mainly incorporated into these proteins as well independently of ATP concentration (1 or 10 microM). The phosphorylation of nuclear proteins with molecular weights above 100000 is mostly increased in this case. At ATP concentration of 1 microM protein S-100 decreases histone phosphorylation 2.3 times but does not affect that of non-histone proteins. However, at 10 microM ATP the inhibitory action of this protein on histone phosphorylation is absent. The possible mechanisms of protein S-100 action on nuclear proteins phosphorylation are discussed.     

Differential immunocytochemical staining for glial fibrillary acidic (GFA) protein,S-100 protein and glutamine synthetase in the rat subcommissural organ,nonspecialized ventricular ependyma and adjacent neuropil

Summary Antibodies raised against glial fibrillary acidic protein (GFA), S-100 protein (S100) and glutamine synthetase (GS) are currently used as glial markers. The distribution of GFA, S100 and GS in the ependyma of the rat subcommissural organ (SCO), as well as in the adjacent nonspecialized ventricular ependyma and neuropil of the periaqueductal grey matter, was studied by use of the immunocytochemical peroxidase-antiperoxidase technique. In the neuropil, GFA, S100 and GS were found in glial elements, i.e., in fibrous (GFA, S100) and protoplasmic astrocytes (S100, GS). The presence of S100 in the majority of the ventricular ependymal cells and tanycytes, and the presence of GFA in a limited number of ventricular ependymal cells and tanycytes confirm the glial nature of these cells. The absence of S100, GFA and GS from the ependymocytes of the SCO, which are considered to be modified ependymal cells, suggests either a non-astrocytic lineage of these cells or an extreme specialization of the SCO-cells as glycoprotein-synthesizing and secreting elements, a process that may have led to the disappearance of the glial markers.     

The pH dependence of breakdown of various purified brain proteins by cathepsin D preparations

In a continuing study of control processes of cerebral protein catabolism we compared the activity of cathepsin D from three sources (rat brain, bovine brain, and bovine spleen) on purified CNS proteins (tubulin, actin, calmodulin, S-100 and glial fibrillary acidic protein). The pH optimum was 5 for hydrolysis with tubulin as substrate for all three enzyme preparations, and it was pH 4 with the other substrates. The pH dependence curve was somewhat variable, with S-100 breakdown relatively more active at an acidic pH range. The formation of initial breakdown products and the further catabolism of the breakdown products was dependent on pH; hence the pattern of peptides formed from glial fibrillary acidic protein was different in incubations at different pH's. The relative activity of the enzyme preparations differed, depending on the substrate: with tubulin and S-100 as substrates, rat brain cathepsin D was the most active and the bovine spleen enzyme was the least active. With calmodulin and glial fibrillary acidic protein as substrates, rat brain and spleen cathepsin D activities were similar, and bovine brain cathepsin D showed the lowest activity. Actin breakdown fell between these two patterns.The rates of breakdown of the substrates were different; expressed as μg of substrate split per unit enzyme per h, with rat brain cathepsin D activity was 8–9 with calmodulin and S-100, 4 with glial fibrillary acidic protein, 1.8 with actin, and 0.9 with tubulin. The results show that there are differences in the properties of a protease like cathepsin D, depending on its source; furthermore, the rate of breakdown and the characteristics of breakdown are also dependent on the substrate.We recently measured the breakdown of brain tubulin by cerebral cathepsin D in a continuing study of the mechanisms and controls of cerebral protein catabolism (Bracco et al., 1982a). We found that tubulin breakdown is heterogeneous, that membrane-bound tubulin is resistant to cathepsin D but susceptible to thrombin (Bracco et al., 1982b), and that cytoplasmic tubulin was in at least two pools, one with a higher, another with a lower, rate of breakdown. The pH optimum of tubulin breakdown by cerebral cathepsin D differed significantly from the pH optimum of hemoglobin breakdown by the same enzyme.These findings showed that the properties of breakdown by a cerebral protease depend on the substrate. To further examine this dependence of properties of breakdown on the substrate, we now report measurements of pH dependence of breakdown of several purified proteins (tubulin, actin, calmodulin, S-100, glial fibrillary acidic protein [GFA]) from brain by cathepsin D preparations from three sources, rat brain, bovine brain, and bovine spleen. We also compare the rate of breakdown of the various proteins with the rate of hemoglobin breakdown.     

Isolation and some physico-chemical properties of a new neurospecific protein 10-40-4 from human brain

A method for isolation of a neurospecific protein 10-40-4 from human brain has been elaborated. This procedure includes immunoaffinity chromatography of a Sepharose 4B-IgG fraction of rabbit antisera against the protein fraction containing the antigen. The isolated protein cannot be detected in protein extracts of various organs and human blood serum by immunochemical methods. This indicates that the protein is specific for nervous tissue. The values of molecular weight (74 000) and pI (4.7) of the isolated protein suggest that the protein does not contain the carbohydrate component and reveals limited tissue specificity. The properties of protein 10-40-4 differ from those of the well-known neurospecific proteins, such as S-100, enolase 14-3-2 and glial fibrillar acid protein GFA.     

Isolation of S-100 binding proteins from brain by affinity chromatography

S-100-binding proteins, and calmodulin-binding proteins were isolated from S-100- and calmodulin-depleted bovine brain extract by Ca2+-dependent affinity chromatography using S-100- and calmodulin-coupled Sepharose columns respectively. The majority of the protein (80 to 90%) including calcineurin that bound to S-100 also bound to calmodulin and vice versa, suggesting both proteins may regulate common targets. However these two regulatory proteins also bind few other proteins specific for each. These include cyclic nucleotide phosphodiesterase, 55k, and 220k proteins for calmodulin and 24k, 42k, and 90k proteins for S-100. Certain proteins also specifically bound to S-100 both in Ca2+-dependent and independent ways. In glial cells S-100 protein may replace calmodulin in regulating Ca2+-influenced functions.     

Perspectives in S-100 protein biology: Review article

The S-100 protein family constitutes a subgroup of Ca²⁺-binding proteins of the EF-hand type comprising three dimeric isoforms, S-100a₀, S-100a and S-100b, plus a number of structurally related proteins displaying 28–55% homology with S-100 subunits. S-100 protein was discovered in 1965; yet, its biological functions have not been fully elucidated. The present report will review the putative biological roles of S-100 protein. Both intracellular and extracellular roles have been proposed for S-100 protein. Within cells, S-100 protein has been reported to regulate protein phosphorylation, ATPase, adenylate cyclase, and aldolase activities and Ca²⁺-induced Ca²⁺ release. Also, cytoskeletal systems, namely microtubules and microfilaments have been reported to be regulated by the protein in the presence of Ca²⁺. Some molecular targets of S-100 protein within cells, have been identified. This is the case with microtubule proteins, caldesmon, and a brain aldolase. S-100 protein has been reported to be secreted; extracellular S-100 protein can stimulate neuronal differentiation, glial proliferation, and prolactin secretion. However, the mechanisms by which S-100 is secreted and stimulates the above processes are largely unknown. Future research should characterize these latter aspects of S-100 biology and find out the linkage between its intracellular effects and its extracellular activities.     

Molecular Interaction of S-100 Proteins with Microtubule Proteins In Vitro

Several procedures were employed to examine the in vitro interaction between S-100 proteins and microtubule proteins. Binding of S-100 to tau factors was observed under all experimental conditions. S-100 binding to microtubule-associated protein 2 (MAP2) was best detected by exposing nitrocellulose-immobilized MAP2 or MAPs to either 125I-labeled S-100 or biotinylated S-100. S-100 binding to tubulin was detected when the two protein fractions were first incubated with each other followed by exposure to the bifunctional cross-linker disuccinimidylsuberate, and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfered onto nitrocellulose paper. By this procedure, complex formation between S-100 and tubulin, as well as between S-100 and a relatively low-molecular-weight MAP, was evidenced by immunoblotting using an anti-S-100 antiserum. Alternatively, complex formation between biotinylated S-100 and either tubulin or MAPs was visualized by means of avidin-peroxidase, after SDS-PAGE of the complex mixtures and transfer of the separated proteins onto nitrocellulose. The interaction between S-100 and tubulin was strictly Ca2+ dependent, and resistant to high concentrations of KCl, colchicine, or vinblastine.     

Biochemical correlates to cortical dysplasia,gliosis, and astrocytoma infiltration in human epileptogenic cortex

The study provides detailed biochemical correlates to the common histopathological diagnoses in epilepsy. A dot immunobinding procedure was used for quantification of NSE, GFA, S-100, NCAM, NF 68 and NF 200. The material consisted of samples from 48 patients either selected for surgical treatment of partial epilepsy or for disorders not related to epilepsy. The histopthological diagnosis of the epileptic cases was: MCD (mild cortical dysplasia, microdysgenesis), gliosis, astrocytoma, ganglioglioma, oligodendroglioma and single cases. The concentration in non-epileptic white matter, in per cent of that in grey matter was: NSE, 85; GFA, 175; S-100, 117; NCAM, 43; NF 68,227 and NF 200, 173. The concentration of NSE as well as of GFA was close to normal in the specimens of the MCD and gliosis groups and of one subgroup of the astrocytomas. There was a striking inverse relationship of the GFA vs the NSE concentrations in the whole material. The concentrations of S-100 showed no such inverse relationship to NSE levels. In all the epileptic groups, total NCAM was lower than 50% of that of the non-epileptic group. The mean NF 68 and NF 200 concentration in the gliosis and astrocytoma groups was 75% of the of the non-epileptic group while the corresponding value for the MCD group was 50%. There was a positive correlation of immunochemically determined GFA and the histopathological gliosis score in the samples of epileptogenic cortex. There was no correlation between the concentration of GFA in the samples and the duration of epilepsy. The concentration of neuronal markers was relatively unaffected in the cortex of most patients with epilepsy related to MCD, gliosis and even to astrocytoma infiltration, even after years of seizures.Special issue dedicated to Dr. Claude Baxter.     

Interaction Between S-100 Proteins and Steady-State and Taxol-Stabilized Microtubules In Vitro

S-100 proteins are a group of three 21-kilodalton, acidic, Ca2+-binding proteins of the "E-F hand" type shown to regulate several cell activities, including microtubule (MT) assembly-disassembly. We show here that S-100 proteins interact with MTs assembled from either whole microtubule protein or purified tubulin, both in the absence and in the presence of the MT-stabilizing drug taxol. Evidence for the binding of S-100 to MTs comes from both kinetic (turbidimetric) and binding studies. Kinetically, S-100 enhances the disassembly of steady-state MTs in the presence of high concentrations of colchicine or vinblastine at 10 microM free Ca2+ and disassembles taxol-stabilized MTs at high Ca2+ concentrations. Experiments performed using 125I-labeled S-100 show that S-100 binds Ca2+ independently to a single set of sites on taxol-stabilized MTs assembled from pure tubulin with an affinity of 6 x 10(-5) M and a stoichiometry of 0.15 mol of S-100/mol of polymerized tubulin. Under certain conditions, S-100 proteins also cosediment with MTs prepared by coassembly of S-100 with MTs, probably in the form of an S-100-tubulin complex. Because S-100 binds to MTs under conditions where this protein fraction does not produce observable effects on the kinetics of assembly-disassembly, e.g., in the absence of Ca2+ at pH 6.7, we conclude that the S-100 binding to MTs does not affect the stability of MTs per se, but rather creates conditions for increased sensitivity of MTs to Ca2+.     

Selective Increase in S-100β Protein by Aging in Rat Cerebral Cortex

Changes in the concentrations of nervous tissue-related proteins and their isoproteins, such as S-100 proteins (S-100 alpha and S-100 beta), enolase isozymes (alpha-enolase and gamma-enolase), and GTP-binding proteins (Go alpha, Gi2 alpha, and beta-subunits), were determined in the CNS of male rats of various ages (from 2 to 30 months old) by means of enzyme immunoassay. The weights of brains and the concentrations of soluble proteins in the cerebral cortex, cerebellum, and brainstem were constant during the observation period. The concentration of S-100 beta protein, which is predominantly localized in glial cells, increased gradually in the cerebral cortex with age; levels in the 25-month-old rats increased to approximately 150% of the levels in the young (2-month-old) rats. However, the S-100 beta concentrations in the cerebellum and brainstem were relatively constant, showing similar values in rats 2-30 months old. Levels of other proteins, including both neuronal (gamma-enolase and Go alpha) and glial (alpha-enolase and S-100 alpha) marker proteins, did not change significantly with age in the cerebral cortex, cerebellum, and brainstem. These results suggest that there is a close relation between the age-dependent changes of the CNS function and S-100 beta protein levels in the cerebral cortex.     

Effect of S-100 protein on assembly of brain microtubule proteins in vitro

S-100 protein inhibits the assembly of brain microtubule proteins in vitro in the presence of 10 microM free Ca2+. The S-100 effect is generally greater on the rate than on the extent of assembly, and even greater as the microtubule protein concentration decreases and the time of preincubation between S-100 and microtubule proteins before GTP addition increases, at a given S-100/tubulin dimer molar ratio. The S-100 effect is greatly enhanced in the presence of physiological concentrations of K+ and is completely reversed by EGTA.     

Calcium-independent, pH-regulated effects of S-100 proteins on assembly-disassembly of brain microtubule protein in vitro

At alkaline pH, Ca2+ is no longer required for S-100 proteins to inhibit the assembly and to promote the disassembly of brain microtubules in vitro, though the presence of Ca2+ significantly favors the S-100 effects. These effects are inversely related to the microtubule protein concentration and directly related to the S-100 concentration and the pH. Ca2+-independent, pH-regulated inhibition of assembly of phosphocellulose-purified tubulin by S-100 is also described. The microtubule disassembling effect of S-100 is additive to that of alkali (used to raise the pH), and S-100 further disassembles microtubules after alkalinization. Thus the larger inhibitory effect of S-100 on microtubule assembly at alkaline versus acid pH depends on both a decrease in the assembly rate and an increase in the disassembly rate. Together with previous data on this topic, the present findings indicate that S-100 proteins act on microtubule protein in vitro primarily by binding to tubulin, this event being Ca2+-regulated at a given pH, and pH-regulated at a given free Ca2+ concentration.     

Interaction of smooth muscle caldesmon with S-100 protein

The interaction of caldesmon with certain Ca-binding proteins was investigated by means of electrophoresis under non-denaturating conditions. In the presence of Ca2+ calmodulin, troponin C and S-100 protein form a complex with caldesmon. No complex formation takes place in the absence of Ca2+. Lactalbumin and pike parvalbumin (pI4.2) do not interact with caldesmon independently of Ca-concentration. Both S-100 protein and calmodulin effectively inhibit phosphorylation of caldesmon by Ca-phospholipid-dependent protein kinase. At low ionic strength S-100 protein reverses the inhibitory action of caldesmon on the skeletal muscle acto-heavy meromyosin ATPase more effectively than calmodulin. It is supposed that in certain tissues and cell compartments the proteins belonging to the S-100 family are able to substitute for calmodulin in the caldesmon-dependent regulation of actin and myosin interaction.     

Nervous System-Specific Proteins in Developing Rat Cerebral Cells in Culture

Abstract: The nervous system-specific proteins: synaptin, D1, D2, D3, glial fibrillary acidic protein (GFA) and 14-3-2, were quantified in dissociated cerebral cells from the foetal rat brain at various times of growth in culture. By approximately 1 week in culture, the neuronal membrane markers synaptin, D1, D2, and D3 could all be demonstrated. A maximum concentration of 10–20% for synaptin, D1, and D3 and 160% for D2, in comparison with the levels in adult forebrain, was attained during the 2nd week in vitro. The astroglial gliofilament marker GFA increased continuously, reaching by 38 days of cultivation an 18-fold higher level than the concentration in adult forebrain. The neuronal cytoplasm marker 14-3-2 could be demonstrated in trace amounts, and only after more than 1 week in vitro. Neuronal cell bodies and processes stained by indirect immunofluorescence using an anti-D2 serum were strongly fluorescent after 1 week in vitro. Immunofluorescence staining for GFA revealed a cytoplasmatic filamentous network in perinuclear areas and processes of, presumably, astroblasts.     

Neurospecific protein S-100 in the synaptosomes of rat cerebral cortex]

Neurospecific S-100 protein was revealed by the methods of microelectrophoresis in the 15% polyacrylamide gel with a 0.1% sodium dodecylsulfate and by a highly purified S-100 protein "marker" in the composition of low molecular acidic proteins of the rat brain synaptosomes. The S-100 protein conten constitutes about 15-2o% of the low molecular acidic synaptosomal porteins in the rat brain.