Neuropeptides modulate the transmitter-induced glycogenolysis and gluconeogenesis in leech segmental ganglia

The effects of four neuropeptides (AKH, FMRFamide, proctolin, VIP) on the alterations in glycogen levels induced by other transmitters were studied in isolated leech segmental ganglia. With the exception of FMRFamide, which produced a small increase (14%) in glycogen, the peptides were by themselves without effect on the glycogen levels. Proctolin abolished the glycogenolytic effects of 5-HT, dopamine and octopamine but not histamine. AKH in combination with ACh produced glycogenolysis although each by themselves were ineffective. AKH modified the effects of other transmitters in different ways i.e. by reduction or reversal of effect. VIP and noradrenaline produced an increase in glycogen (cf. noradrenaline alone which decreased glycogen), but did not modify the effects of the other transmitters. FMRFamide produced a complex variety of modulatory effects on the other transmitters. It is concluded that the glycogen stores in leech ganglia, which are localized principally in the glial cells, may be controlled in complex ways by the different combinations of monoamines, amino acids and neuropeptides.     

Transmitter mediated regulation of energy metabolism in nervous tissue at the cellular level

The Monoamines 5-hydroxytryptamine (5-HT), noradrenaline (NA) and histamine, and the peptide Vasoactive Intestinal Polypeptide (VIP), regulate energy metabolism in nervous tissue, in addition to producing excitation and/or inhibition. These transmitters induce glycogen hydrolysis in a concentration dependent manner. The glycogen breakdown is brought about by increased cyclic AMP formation, or translocation of calcium ions to activate phosphorylase, and is partially localized in glial cells. Data from a diversity of nervous systems, including leech and snail ganglia, and rodent cortex, point towards important roles for neurons containing these transmitters in the regulation of the glycogen turnover. It is proposed that energy metabolism may be controlled within domains defined by the geometric arrangements of the neurons releasing these transmitters. The different domains may overlap temporally and spatially to coordinate energy metabolism in relation to increases in neuronal activity. The non-myelin forming glial cells, which contain glycogen whose turnover rate is altered by the transmitters, appear to be important in the local supply of energy substrate to neurons.     

Modulation by Monoamines of Somatostatin-Sensitive Adenylate Cyclase on Neuronal and Glial Cells from the Mouse Brain in Primary Cultures

Primary cultures of mouse embryonic neuronal or glial cells from the cerebral cortex, striatum, and mesencephalon were used to identify and determine the cellular localization of somatostatin receptors coupled to an adenylate cyclase. Somatostatin inhibited basal adenylate cyclase activity on neuronal but not on glial crude membranes in the three structures examined. The somatostatin-inhibitory effect on neuronal crude membranes was still observed in the presence of (-)-isoproterenol, 3,4-dihydroxyphenylethylamine (dopamine, DA), or 5-hydroxytryptamine (5-HT, serotonin) used at a concentration (10(-5) M) inducing maximal adenylate cyclase activation. In addition, in most cases biogenic amines modified the pattern of the somatostatin-inhibitory effect, triggering either an increase in the peptide apparent affinity for its receptors or an increase in the maximal reduction of adenylate cyclase activity or both. However, 5-HT did not modify the somatostatin-inhibitory response on striatal and cortical neuronal crude membranes. The changes in somatostatin-inhibitory responses were interpreted as a colocalization of the amine and the peptide receptors on subtypes of neuronal cell populations. Finally, somatostatin was shown to inhibit adenylate cyclase activity following its activation by (-)-isoproterenol on glial crude membranes of the striatum and the mesencephalon but not on those of the cerebral cortex.     

Energy utilization and gluconeogenesis in isolated leech segmental ganglia: Quantitative studies on the control and cellular localization of endogenous glycogen

The isolated segmental ganglia of the horse leech Haemopis sanguisuga were used as a model system to study the utilization and control of glycogen stores within nervous tissue. The glycogen in the ganglia was extracted and assayed fluorimentrically and its cellular localization and turnover studied by autoradiography in conjunction with [³H]glucose. We measured the glycogen after various periods of electrical stimulation and after incubation with K⁺, Ca²⁺, ouabain and glucose. The results for each experimental ganglion were compared to a paired control ganglion and the results analysed by paired t-tests. Electrical stimulation caused sequential changes in glycogen levels: a reduction of up to 67% (5–10 min); followed by an increase of up to 124% (between 15–50 min); followed by a reduction of up to 63% (60–90 min). Values were calculated for glucose utilization (e.g. 0.53 μmol glucose/gm wet weight/min after 90 min) and estimates derived for glucose consumption per action potential per neuron (e.g. 0.12 fmol at 90 min). Glucose (1.5–10 mM) increased the amount of glycogen (1.5 mM by 30% at 60 min) and attenuated the effects of electrical stimulation. Ouabain (1 mM) blocked the effect of 5 min electrical stimulation. Nine millimolar K⁺ increased glycogen by 27% after 10 min and decreased glycogen by 34% after 60 min; 3 mM Ca²⁺ had no effect after 10 or 20 min and decreased glycogen by 29% after 60 min. Other concentrations of K⁺ and Ca²⁺ reduced glycogen after 60 min. Autoradiographic analysis demonstrated that the effects of elevated K⁺ were principally within the glial cells. We conclude that (i) the glycogen stores in the glial cells of leech segmental ganglia provide an endogenous energy source which can support sustained neuronal activity, (ii) both electrical stimulation and elevated K⁺ can induce gluconeogenesis within the ganglia, (iii) that electrical activation of neurons produces changes in the glycogen in the glial cells which are controlled in part by changes in K⁺.     

Vasoactive Intestinal Polypeptide Receptors Linked to an Adenylate Cyclase, and Their Relationship with Biogenic Amine- and Somatostatin-Sensitive Adenylate Cyclases on Central Neuronal and Glial Cells in Primary Cultures

The presence of vasoactive intestinal polypeptide (VIP) receptors coupled to an adenylate cyclase was demonstrated on membranes of neurons or glial cells grown in primary cultures originating from the cerebral cortex, striatum, and mesencephalon of mouse embryos. A biphasic pattern of activation was observed in all these cell types, involving distinct high- and low-apparent-affinity mechanisms. The absence of additive effects of VIP and 3,4-dihydroxyphenylethylamine (DA, dopamine), isoproterenol (ISO), and 5-hydroxytryptamine (5-HT, serotonin) suggests that the peptide receptors are colocated with each of the corresponding amine receptors on neuronal membranes of the three structures studied. The nonadditivity between the VIP- and ISO-induced responses on cortical and striatal glial membranes reveals as well a colocation of VIP and beta-adrenergic-sensitive adenylate cyclases on the same cells. A subpopulation of mesencephalic glia could possess only one of the two types of receptors, as a partial additivity of the VIP and ISO responses was seen. In addition, VIP modified the characteristics of the somatostatin inhibitory effect on adenylate cyclase activity of neuronal membranes from the cerebral cortex and striatum but not from those of the mesencephalon. On striatal and mesencephalic glial membranes the somatostatin inhibitory effect was observed only in the presence of VIP. However, as previously seen with ISO, the presence of VIP did not allow the appearance of a somatostatin inhibitory response on cortical glial membranes. This suggests that cortical glia are devoid of somatostatin receptors.     

Modulation of glial glycogen metabolism by 5-hydroxytryptamine in leech segmental ganglia

Leech segmental ganglia (16 out of 23 per animal) were divided into experimental and control groups (4 ganglia per group). The amounts of glycogen in the ganglia were assayed by a specific extraction procedure and fluorimetry, or by liquid scintillation counting following labelling of the glycogen by [³H]glucose. Within any individual animal the amounts of glycogen in the ganglia were relatively constant (max. variation 16%). 5-HT (10⁻⁶–10⁻⁴ M) reduced in a dose-dependent manner the endogenous glycogen (max. 20% reduction), and the [³H]glycogen (max. 60% reduction). The glycogenolytic effect was studied by light-microscope autoradiography in serial sections of segmental ganglia previously exposed to [³H]glucose. The 5-HT-mediated glycogenolysis was localized principally in the glial cells surrounding the neuron perikarya. 5-HT, in addition to its conventional transmitter role, may regulate the supply of energy substrate from glial cells to neurons within domains defined by the projections of the neurons from which it is released.     

Distribution of NADPH-diaphorase activity in the central nervous system of the young and adult land snail Megalobulimus abbreviatus

Nitric oxide (NO) is a gas produced through the action of nitric oxide synthase that acts as a neurotransmitter in the central nervous system (CNS) of adult gastropod mollusks. There are no known reports of the presence of NOS-containing neurons and glial cells in young and adult Megalobulimus abbreviatus. Therefore, NADPH-d histochemistry was employed to map the nitrergic distribution in the CNS of young and adult snails in an attempt to identify any transient enzymatic activity in the developing CNS. Reaction was observed in neurons and fibers in all CNS ganglia of both age groups, but in the pedal and cerebral ganglia, positive neurons were more intense than in other ganglia, forming clusters symmetrically located in both paired ganglia. However, neuronal NADPH-d activity in the mesocerebrum and pleural ganglia decreased from young to adult animals. In both age groups, positive glial cells were located beneath the ganglionic capsule, forming a network and surrounding the neuronal somata. The trophospongium of large and giant neurons was only visualized in young animals. Our results indicate the presence of a nitrergic signaling system in young and adult M. abbreviatus, and the probable involvement of glial cells in NO production.     

Fluorescence histochemical studies of the uptake of exogenous noradrenaline and dopamine by in vitro cultured cerebrocortex explants of the rat

Cerebrocortex of the neonatal rats were cultivated (--14 days). The cultures were studied living and with histological and fluorescence histochemical methods. A differentiation of neuronal cell- and fiber elements, oligodendro glial cells and astrocytes was found. The glyoxylic acid technique to estimate biogenic monoamines (Lindvall et al. 1974) was adapted up the cultivated explants. The normal cultures have only 24 h post cultivationem a specific fluorescence granularly in small concentration of the surface of the explant and in the explant self. Incubations with noradrenaline and dopamine demonstrated a various accumulation of the exogenous transmitters in the various parts of the cultivated explants. Uptake and releasing mechanisms in the cultivated material of the cerebrocortex were discussed with respect to the results of the sympathetic ganglia in vitro.     

Neurotransmitter metabolism in murine neuroblastoma cells.

Murine neuroblastoma cells in culture are able to synthesize the putative neurotransmitters--acetylcholine, dopamine, norepinephrine, tyramine, octopamine, histamine, serotonin and γ-aminobutyric acid (GABA). They possess not only synthetic, but also degradative enzymes involved in metabolism of these transmitters, and many of these enzymes increase in activity as the cells “differentiate”. Catecholamines, and perhaps other transmitters, appears to be stored within membrane-limited vesicles which accumulate within the process endings of these cells. Uptake of some transmitters, GABA, glycine, dopamine and norepinephrine, shows characteristics of the high affinity transport systems observed in other neuronal populations; uptake of choline and other amino acids is similar to that in non-neuronal populations. Cells show receptor sensitivities to acetyl-choline, dopamine, norepinephrine, prostaglandins E₁ and morphine, as demonstrated by electrophysiologic, toxin binding and cyclic nucleotide studies.     

Paralemmin interacts with D3 dopamine receptors: implications for membrane localization and cAMP signaling

Paralemmin is a novel lipid-anchored protein, which is highly expressed in neuronal plasma membranes. In this study, we demonstrate that paralemmin specifically interacts with the third intracellular loop of the D3 dopamine receptor. Utilizing co-immunoprecipitation and glutathione-S-transferase (GST) pulldown strategies, we demonstrate that paralemmin interacts exclusively with D3, but not D2 or D4 dopamine receptors or beta-adrenergic receptors. Immunocytochemistry demonstrated co-localization of paralemmin and D3 receptor in vivo in hippocampus and cerebellum and in vitro in glial and neuronal cultures. Deletion mutational analysis indicates that amino acids 154-230 of paralemmin strongly interacted with amino acids 211-227 and 281-330 of the third intracellular loop of D3 receptor. The consequences of these interactions were investigated by co-expression in HEK293 cells. Cell surface biotinylation experiments demonstrate that paralemmin decreased D3 receptor concentration at the plasma membrane. Consistent with this observation, paralemmin expression decreased dopamine-stimulated adenylate cyclase activity. However, paralemmin also decreased basal, isoproterenol and forskolin-stimulated adenylate cyclase activity, suggesting a more general cellular function for paralemmin. Taken together, paralemmin has been implicated as a potent modulator of cellular cAMP signaling within the brain.     

Nitric Oxide as an Orthograde Cotransmitter at Central Synapses of Aplysia: Responses of Isolated Neurons in Culture

Nitric oxide serves as an orthograde synaptic cotransmitterbetween identified neurons in the cerebral ganglion of Aplysia.Nitric oxide synthase, the enzyme that produces nitric oxide,is localized in a few specific neurons in the ganglia, includingneuron C2. Guanylyl cyclase the target enzyme of nitric oxide,is found in neurons C4 and MCC, which are synaptic followersof C2. Stimulation of C2 causes a vsEPSP in these neurons thatis reduced to 50% of its amplitude by nitric oxide synthaseinhibitors and guanylyl cyclase inhibitors. The remaining portionof the vsEPSP is mediated by histamine. Thus, nitric oxide andhistamine act as orthograde cotransmitters in producing thevsEPSP. Both cotransmitters cause closure of a background potassiumchannel, which depolarizes the neuron and enhances its responseto synaptic inputs. Exogenous nitric oxide (released by nitricoxide donor molecules) and histamine mimic the vsEPSP's depolarizationand decreased membrane conductance. When neurons C4 or MCC areisolated in cell culture they respond just as they do in theganglion, i.e., the nitric oxide response but not the histamineresponse is blocked by guanylyl cyclase inhibitors, and themembrane conductance is decreased by both histamine and nitricoxide. Aplysia hemolymph partially suppresses the response tonitric oxide, due to nitric oxide scavenging by hemocyanin,which contains copper and is the equivalent of hemoglobin. NeuronC2 followers that are hyperpolarized by histamine are insensitiveto nitric oxide. Thus, only select follower neurons respondto both transmitters.     

Glial cells in the central nervous system of earthworm, Eisenia fetida

Glial elements in the central nervous system of Eisenia fetida were studied at light- and electron microscopic level. Cells were characterized with the aid of toluidine blue, Glial Fibrillary Acidic Protein (GFAP), S100 staining. We identified neurilemmal-, subneurilemmal-, supporting-nutrifying- and myelinsheath forming glial cells. Both neuronal and non-neuronal elements are S100-immunoreactive in the CNS. Among glial cells neurilemmal and subneurilemmal cells are S100-immunopositive. With the antibody against the S100 protein one band is visible at 15 kDa. GFA P-immunopositive supporting-nutrifying glial cells are localized around neurons and they often appear as cells with many vacuoles. GFA P-positive cell bodies of elongated neurilemmal glial cells are also visible. Western blot analysis shows a single 57 kDa GFA P immunoreactive band in the Eisenia sample. At ultrastructural level contacts between neuronal and glial cells are recognizable. Glial cell bodies and their filopodia contain a granular and vesicular system. Close contacts between neuronal cell membranes and glial filopodia create a special environment for material transport. Vesicles budding off glial cell granules move towards the cell membranes, probably emptying their content with kiss and run exocytosis. The secreted compounds in return may help neuronal survival, provide nutrition, and filopodia may also support neuronal terminals.     

Basic fibroblast growth factor, neurofilament, and glial fibrillary acidic protein immunoreactivities in the myenteric plexus of the rat esophagus and colon

The enteric nervous system consists of a number of interconnected networks of neuronal cell bodies and fibers as well as satellite cells, the enteric glia. Basic fibroblast growth factor (bFGF) is a mitogen for a variety of mesodermal and neuroectodermal-derived cells and its presence has been described in many tissues. The present work employs immunohistochemistry to analyze neurons and glial cells in the esophageal and colic enteric plexus of the Wistar rat for neurofilament (NF) and glial fibrillary acidic proteins (GFAP) immunoreactivity as well as bFGF immunoreactivity in these cells. Rats were processed for immunohistochemistry; the distal esophagus and colon were opened and their myenteric plexuses were processed as whole-mount preparations. The membranes were immunostained for visualization of NF, GFAP, and bFGF. NF immunoreactivity was seen in neuronal cell bodies of esophageal and colic enteric ganglia. GFAP-immunoreactive enteric glial cells and processes were present in the esophageal and colic enteric plexuses surrounding neuronal cell bodies and axons. A dense net of GFAP-immunoreactive processes was seen in the ganglia and connecting strands of the myenteric plexus. bFGF immunoreactivity was observed in the cytoplasm of the majority of the neurons in the enteric ganglia of esophagus and colon. The two-color immunoperoxidase and immunofluorescence methods revealed bFGF immunoreactivity also in the nucleus of GFAP-positive enteric glial cells. The results suggest that immunohistochemical localization of NF and GFAP may be an important tool in the study of the plasticity in the enteric nervous system. The presence of bFGF in neurons and glia of the myenteric plexus of the esophagus and the colon indicates that this neurotrophic factor may exert autocrine and paracrine actions in the enteric nervous system.     

5'guanylyl-imidodiphosphate actions on adenylate cyclase in homogenates of rat cerebral cortex plus neuronal and capillary fractions

A variety of neurohumoral agents activate adenylate cyclase in homogenates of rat frontal cortex (norepinephrine, isoproterenol, dopamine, apomorphine, histamine, 4-Me-histamine and prostaglandins E₁, E₂ and A₂). The enzyme in homogenates of isolated cortical neurons is likewise sensitive to norepinephrine, isoproterenol, dopamine, apomorphine, histamine, 2-Me- and 4-Me-histamine, and prostaglandin F_2α. Capillary-enriched fractions from the cortex possess an enzyme that is activated by norepinephrine, isoproterenol and dopamine. Addition of 5′-guanylyl-imidodiphosphate (Gpp(NH)p) to the cortical homogenates and neuronal fractions resulted in enhanced enzyme responses to norepinephrine, isoproterenol, dopamine, 2-Me- and 4-Me-histamine and the prostaglandins E₁ and E₂. The actions of histamine and apomorphine were not increased by the GTP analog. The sensitivity of the catecholamine-induced adenylate cyclase activation in cortical capillaries was augmented by Gpp(NH)p. Thus various cellular types within the cerebral cortex may possess different receptor characteristics with respect to stimulation of adenylate cyclase by neurohormones.     

Desensitization by histamine of H2 receptor-mediated adenylate cyclase activation in the human gastric cancer cell line HGT-1

Short-term treatment of cultured HGT-1 cells with histamine produced a time-dependent (half-life: 20 min) and homologous desensitization of histamine H2 receptor activity mediating cAMP generation in HGT-1 cells and gastric acid secretion in normal gastric mucosa. Histamine treatment resulted in loss of response of the adenylate cyclase to histamine in purified plasma membranes, but had no effect on basal, vasoactive intestinal peptide (VIP)- or NaF-stimulated enzyme activities. We propose that the desensitization of gastric histamine H2 receptor by histamine evidenced in cellular or subcellular preparations from HGT-1 cells could be involved in the physiological regulation and pharmacological control of gastric cell function in man.     

Immunocytochemical studies of the Gi Protein mediated muscarinic receptor-adenylyl cyclase system

The localization of three key signal transduction components was indicated in rat heart tissue by immunocytochemical and histochemical experiment. It was shown that:

1. The M2 muscarinic receptors are localized along outer cell membranes and T-tubule membranes of cardiomyocytes but additionally at membranes of endothelial cells and fibroblasts.
2. G_iα was found along outer cell membranes of cardiomyocytes and other cells of the heart and also inside the cells of the perinuclear space in close contact to the nuclei envelope and the endoplasmic reticulum membranes. G_oα were found to be associated mainly in atrial tissue, especially at the nerval (neuronal) endings located among the cardiac muscle cells. This was shown in parallel incubation with specific neuronal antibody as a marker for these structures.
3. Adenylyl cyclase was localized along the sarcolemma and the T-tubule membranes in normal cardiomyocytes of rat and guinea pig hearts. Under ischemic conditions, the adenylyl cyclase was also seen in junctional sarcoplasmic reticulum membranes. The reasons for this changed localization need further elucidation. Binding of the adenylyl cyclase within the molecular structure of the membrane or variation of the marker penetration remain to be clarified.

     

Progenitor cells from embryonic chick dorsal root ganglia differentiate in vitro to neurons: biochemical and electrophysiological evidence.

We have analyzed the appearance of neurons and glial cells in chick dorsal root ganglia during development. Neurons were identified by the presence of polysialogangliosides recognized by tetanus toxin (GD1b, GT1) or by the monoclonal antibody Q211 directed against polysialogangliosides containing four, five and six sialic acid residues. Glial cells were identified by the presence of 04 antigen. A population of undifferentiated cells, i.e., cells which express neither neuronal nor glial cell surface antigens, present in dorsal root ganglia until embryonic day 7, was separated from the neuronal and glial population. This cell population contains neuronal progenitor cells which differentiate to neurons within 1 day in culture. This differentiation process is characterized by the appearance of neuronal morphology, of neuron-specific gangliosides and by the appearance of voltage-dependent sodium and calcium channels.     

L-arginine immunoreactive enteric glial cells in the enteric nervous system of rat ileum

L-arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.     

Analysis of glia cell differentiation in the developing chick peripheral nervous system: sensory and sympathetic satellite cells express different cell surface antigens.

To identify and analyse precursor cells of neuronal and glial cell lineages during the early development of the chick peripheral nervous system, monoclonal antibodies were raised against a population of undifferentiated cells of E6 dorsal root ganglia (DRG). Non-neuronal cells of E6 DRG express surface antigens that are recognized by four monoclonal antibodies, G1, G2, GLI 1 and GLI 2. The proportion of non-neuronal cells in DRG that express the GLI 1 antigen is very high during ganglion formation (80% at E4) and decreases during later development (15% at E14). GLI 2 antigen is expressed only on a minority of the cells at E6 and increases with development. The G1 and G2 antigens are expressed on about 60-80% of the cells between E6 and E14. All cells that express the established glia marker O4 are also positive for the new antigens. In addition, it was demonstrated that GLI 1-positive cells from early DRG, which are devoid of O4 antigen, could be induced in vitro to express the O4 antigen. Thus, the antigen-positive cells are considered as glial cells or glial precursor cells. Surprisingly, the antigen expression by satellite cells of peripheral ganglia is dependent on the type of ganglion: antigens G1, G2 and GLI 1 were not detectable on glial cells of lumbosacral sympathetic ganglia and GLI 2 was expressed only by a small subpopulation. These results demonstrate an early immunological difference between satellite cells of sensory DRG and sympathetic ganglia.(ABSTRACT TRUNCATED AT 250 WORDS)