Interleukin-6 expression and regulation in rat enteric glial cells

As yet, little is known about the function of the glia of the enteric nervous system (ENS), particularly in an immune-stimulated environment. This prompted us to study the potential of cultured enteroglial cells for cytokine synthesis and secretion. Jejunal myenteric plexus preparations from adult rats were enzymatically dissociated, and enteroglial cells were purified by complement-mediated cytolysis and grown in tissue culture. Cultured cells were stimulated with recombinant rat interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, and IL-6 mRNA expression and secretion were assessed using RT-PCR and a bioassay, respectively. Stimulation with TNF-alpha did not affect IL-6 mRNA expression, whereas IL-1beta stimulated IL-6 mRNA and protein synthesis in a time- and concentration-dependent fashion. In contrast, IL-6 significantly and dose-dependently suppressed IL-6 mRNA expression. In summary, we have presented evidence that enteric glial cells are a potential source of IL-6 in the myenteric plexus and that cytokine production by enteric glial cells can be regulated by cytokines. These findings strongly support the contention that enteric glial cells act as immunomodulatory cells in the enteric nervous system.     

Spatial relationship between telocytes,interstitial cells of Cajal and the enteric nervous system in the human ileum and colon

Telocytes (TCs) are recently described interstitial cells, present in almost all human organs. Among many other functions, TCs regulate gastrointestinal motility together with the interstitial cells of Cajal (ICCs). TCs and ICCs have close localization in the human myenteric plexus; however, the exact spatial relationship cannot be clearly examined by previously applied double immunofluorescence/confocal microscopy. Data on TCs and submucosal ganglia and their relationship to intestinal nerves are scarce. The aim of the study was to analyse the spatial relationship among these components in the normal human ileum and colon with double CD34/CD117 and CD34/S100 immunohistochemistry and high-resolution light microscopy. TCs were found to almost completely encompass both myenteric and submucosal ganglia in ileum and colon. An incomplete monolayer of ICCs was localized between the TCs and the longitudinal muscle cells in ileum, whereas only scattered ICCs were present on both surfaces of the colonic myenteric ganglia. TC-telopodes were observed within colonic myenteric ganglia. TCs, but no ICCs, were present within and around the interganglionic nerve fascicles, submucosal nerves and mesenterial nerves, but were only observed along small nerves intramuscularly. These anatomic differences probably reflect the various roles of TCs and ICCs in the bowel function.     

Circadian rhythms and glial cells of the central nervous system

Glial cells are the most abundant cells in the central nervous system and play crucial roles in neural development, homeostasis, immunity, and conductivity. Over the past few decades, glial cell activity in mammals has been linked to circadian rhythms, the 24-h chronobiological clocks that regulate many physiological processes. Indeed, glial cells rhythmically express clock genes that cell-autonomously regulate glial function. In addition, recent findings in rodents have revealed that disruption of the glial molecular clock could impact the entire organism. In this review, we discuss the impact of circadian rhythms on the function of the three major glial cell types – astrocytes, microglia, and oligodendrocytes – across different locations within the central nervous system. We also review recent evidence uncovering the impact of glial cells on the body's circadian rhythm. Together, this sheds new light on the involvement of glial clock machinery in various diseases.     

Development of the enteric nervous system

The mature enteric nervous system (ENS) is characterized by a degree of neuronal phenotypic diversity and independence of central nervous system control unequaled by any other region of the peripheral nervous system. Studies that have utilized the immunocytochemical demonstration of neurofilament protein and explanation of primordial gut with subsequent growth in culture have indicated that the neural crest precursors of enteric neurons are already committed to the neuronal lineage when they colonize the bowel; however, neuronal phenotypic expression occurs within the gut itself. It is likely that precursors able to give rise to each type of neuron found in the mature ENS are present among the earliest neural crest émigrés to reach the bowel. In mice a proximodistal wave of neuronal phenotypic expression occurs that does not appear to reflect the descent of neuronal precursors. This observation, the known plasticity of developing neural crest-derived neurons, and the demonstration of a persistent population of proliferating neuroblasts in the gut raise the possibility that enteric neuronal phenotypic expression is influenced by the enteric microenvironment.     

Characterization of immunoreactive somatostatin released from rat nervous system in vitro.

There is considerable evidence that somatostatin is released from nerve terminals throughout the central nervous system in response to presynaptic stimulation, thus suggesting a neuromodulator role for the peptide. We here report the partial characterization of immunoreactive somatostatin released from rat nervous system in vitro (hypothalamus, spinal cord and hypothalamic, cortical, thalamic and striatal synaptosomes). Serial dilutions of released somatostatin immunoreactivity showed parallelism with dilutions of synthetic somatostatin standard. Somatostatin immunoreactivity released from all tissue areas coeluted with synthetic tetradecapeptide on Sephadex G-25 (fine grade) gel chromatography; more than 85% of this immunoreactivity bound to Sepharose-anti-somatostatin-serum immunoaffinity columns. In addition, immunoreactive material released from hypothalamus, spinal cord and hypothalamic and cortical synaptosomes inhibited somatotropin (growth hormone, 'STH', 'GH') release from perifused anterior pituitary in a dose-related manner, indicating biological similarity to synthetic somatostatin.     

Peroxynitrite stimulates L-arginine transport system y(+) in glial cells. A potential mechanism for replenishing neuronal L-arginine

We have reported previously that peroxynitrite stimulates L-arginine release from astrocytes, but the mechanism responsible for such an effect remains elusive. To explore this issue, we studied the regulation of L-[(3)H]arginine transport by either exogenous or endogenous peroxynitrite in glial cells. A 2-fold peroxynitrite-mediated stimulation of l-arginine release in C6 cells was found to be Na(+)-independent, was prevented by 5 mm L-arginine and, although only in the presence of Na(+), was blocked by 5 mm L-alanine or L-leucine. Peroxynitrite-mediated stimulation of L-arginine uptake was trans-stimulated by 10 mm L-arginine and was inhibited in a dose-dependent fashion (k(i) of approximately 40 microm) by the system y(+) inhibitor N-ethylmaleimide in C6 cells. Endogenous production of peroxynitrite in lipopolysaccharide-treated astrocytes triggered an increased L-arginine transport activity without affecting Cat1 l-arginine transporter mRNA levels. However, Western blot analyses of peroxynitrite-treated astrocytes and C6 glial cells revealed a 3-nitrotyrosinated anti-Cat1-immunopositive band, strongly suggesting peroxynitrite-mediated Cat1 nitration. Furthermore, peroxynitrite stimulation of L-arginine release was abolished in fibroblast cells homozygous for a targeted inactivation of the Cat1 gene. Finally, peroxynitrite-triggered L-arginine released from astrocytes was efficiently taken up by neurons in an insert-based co-culture system. These results strongly suggest that peroxynitrite-mediated activation of the Cat1 transporter in glial cells may serve as a mechanism focused to replenish L-arginine in the neighboring neurons.     

Evidence for neural progenitor cells in the human adult enteric nervous system

Putative neural stem cells have been identified within the enteric nervous system (ENS) of adult rodents and cultured from human myenteric plexus. We conducted studies to identify neural stem cells or progenitor cells within the submucosa of adult human ENS. Jejunum tissue was removed from adult human subjects undergoing gastric bypass surgery. The tissue was immunostained, and confocal images of ganglia in the submucosal plexus were collected to identify protein gene product 9.5 (PGP 9.5) - immunoractive neurons and neuronal progenitor cells that coexpress PGP 9.5 and nestin. In addition to PGP-9.5-positive/nestin-negative neuronal cells within ganglia, we observed two other types of cells: (1) cells in which PGP 9.5 and nestin were co-localized, (2) cells negative for both PGP 9.5 and nestin. These observations suggest that the latter two types of cells are related to a progenitor cell population and are consistent with the concept that the submucosa of human adult ENS contains stem cells capable of maintenance and repair within the peripheral nervous system.     

Synthesis by a clonal line of rat glial cells of a protein unique to the nervous system

The synthesis of a protein unique to the nervous system, the “S100-protein,” has been studied in a clonal line of rat glial cells. It has been shown that these cells do not begin to accumulate “S100-protein” until the cultures enter a phase of density-dependent inhibition of cell proliferation. Further experiments indicate that the regulation of “S100-protein” accumulation resides at least in part in an interaction involving the cell surface.     

Alternative processing pathways for preprovasoactive intestinal peptide in the enteric nervous system of the rat

In order to study biosynthetic processing of preprovasoactive intestinal peptide (prepro VIP) we have raised antisera to sequences that flank the biologically active peptides VIP and PHI (peptide with N-terminal His and C-terminal Ile). We have used these antisera in radioimmunoassays to identify the N-terminal flanking peptide (NFP) and C-terminal flanking peptide (CFP)-like immunoreactivities in rat brain and gastrointestinal tract. Concentrations of NFP-LI were similar to those of VIP in brain and throughout the gut. Concentrations of CFP-LI were 10-20% those of VIP-LI but could be increased 5-fold by digestion with carboxypeptidase B, suggesting that the C-terminal lysine residue of prepro VIP is not normally removed during processing. In rat stomach the NFP-LI was of higher molecular weight and greater hydrophobicity than the intestinal component. The data are consistent with alternative processing pathways for prepro VIP in enteric nerves of rat stomach and intestine.     

Structure of the glial cells in the nervous system of parasitic and free-living flatworms

This study is devoted to ultrastructural and immunosytochemical investigation of the nervous system in parasitic and free-living platyhelminthes to learn if glial cells exist in the nervous system of flatworms. We described the ultrastructure of different types of glial cells and the peculiarities of myelinization of gigantic axons; immunoreactivity to the S100b protein is revealed. Comparative analysis of the glia structure of annelids and platods is given; structural, functional, and evolutionary aspects of myelinization of gigantic axons, which are revealed in cestodes, are discussed.     

Development of the mammalian enteric nervous system.

The mammalian enteric nervous system is derived from neural crest cells which invade the foregut and hindgut mesenchyme. It has been established that signalling molecules produced by the mesenchyme of the gut wall play a critical role in the development of the mammalian enteric nervous system. Recent studies have characterised further the role of such molecules and have identified novel extracellular and intracellular signals that are critical for enteric ganglia formation.     

Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.     

Electrogenic sodium-dependent bicarbonate secretion by glial cells of the leech central nervous system

The ability to move acid/base equivalents across the membrane of identified glial cells was investigated in isolated segmental ganglia of the leech Hirudo medicinalis. The intracellular pH (pHi) of the glial cells was measured with double-barreled, neutral-ligand, ion-sensitive microelectrodes during step changes of the external pH (pHo 7.4-7.0). The rate of intracellular acidification after the decrease in extracellular pH (pHo) was taken as a measure of the rate of acid/base transport across the glial membrane. Taking into account the total intracellular buffering power, the maximum rate of acid/base flux was 0.4 mM/min in CO2/HCO3-free saline, and 3.92 mM/min in the presence of 5% CO2/10 mM HCO-3, suggesting that the acid/base flux was dependent upon HCO3-. The rate of acid influx/base efflux increased both with the external HCO3- concentration and with increasing pHi (and hence HCO3-i). This suggested that the decrease in pHi was due to HCO3- efflux. The rapid decrease of pHi was accompanied by a HCO3--dependent depolarization of the glial membrane from -74 +/- 5 mV (n = 20) to -54 +/- 7 mV (n = 13). Both this depolarization and the rate of intracellular acidification were greatly reduced by the anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.3-0.5 mM), but were not affected by the removal of external Cl-. Reduction of the external Na+ concentration to one-tenth normal affected the rate of intracellular acidification only in the presence of CO2/HCO3-: the rate increased within the first 3-5 min after lowering external Na+; after longer exposures in low external Na+ the rate decreased, presumably due to depletion of intracellular Na+. Amiloride (1 mM), which inhibits the Na+-H+ exchange in these cells, had no effect on the rate of intracellular acidification. The intracellular Na activity (aNai) of the glial cells was measured to be 5.2 +/- 1.0 mM (n = 8) in CO2/HCO3-free saline; aNai increased to 7.3 +/- 2.2 mM (n = 8) after the addition of 5% CO2/24 mM HCO3-. Upon a change in pHo to 7.0 in the presence of CO2/HCO3-, aNai decreased by an average of 2 +/- 1.1 mM (n = 5); in CO2/HCO3--free saline external acidification produced a transient increase in aNai. It is concluded that, in the presence of CO2/HCO3-, the rate of intracellular acidification in glial cells is dominated by an outwardly directed, electrogenic Na+-HCO3-cotransport. Neurons, which do not possess this cotransporter, acidify at much lower rates under similar conditions.(ABSTRACT TRUNCATED AT 400 WORDS)