Differences in monoamine oxidase activity between cultured noradrenergic and cholinergic sympathetic neurons

Two types of monoamine oxidase activity (MAO-A and MAO-B) help regulate the levels of biogenic amines such as catecholamines and serotonin. Although MAO-A has greater activity toward most catecholamines than MAO-B, no direct experiments have determined the types and levels of MAO activity that are normally expressed in noradrenergic neurons. Noradrenergic neurons from neonatal rat superior cervical ganglia were isolated and cultured under conditions that permit either continued expression of the noradrenergic phenotype or promote a transition to a predominantly cholinergic phenotype. After 14-21 days in vitro, neurons from both types of cultures were assayed for the type and amount of monoamine oxidase activity using tryptamine, a common substrate for both MAO-A and MAO-B. Neurons cultured under noradrenergic conditions expressed sevenfold greater MAO activity than neurons cultured under cholinergic conditions. Essentially all MAO activity in the noradrenergic cultures was inhibited by preincubation with 10(-8)-10(-9) M clorgyline, which indicated that this activity was primarily MAO-A. Cultures grown under cholinergic conditions exhibited 6- to 10-fold lower MAO-A activity and an 8- to 10-fold lower level of catecholamine synthesis from labeled precursors compared to neurons grown under noradrenergic conditions. These results directly demonstrate that high MAO-A activity is expressed in noradrenergic neurons in vitro. The corresponding decreases in both MAO-A specific activity and catecholamine synthesis as neurons become cholinergic in vitro suggest that the expression of the noradrenergic phenotype involves the coordinate regulation of degradative as well as synthetic enzymes involved in catecholamine metabolism.     

Induction of cholinergic function in cultured sympathetic neurons by periosteal cells: cellular mechanisms.

Periosteum, the connective tissue surrounding bone, alters the transmitter properties of its sympathetic innervation during development in vivo and after transplantation. Initial noradrenergic properties are downregulated and the innervation acquires cholinergic and peptidergic properties. To elucidate the cellular mechanisms responsible, sympathetic neurons were cultured with primary periosteal cells or osteoblast cell lines. Both primary cells and an immature osteoblast cell line, MC3T3-E1, induced choline acetyltransferase (ChAT) activity. In contrast, lines representing marrow stromal cells or mature osteoblasts did not increase ChAT. Growth of periosteal cells with sympathetic neurons in transwell cultures that prevent direct contact between the neurons and periosteal cells or addition of periosteal cell-conditioned medium to neuron cultures induced ChAT, indicating that periosteal cells release a soluble cholinergic inducing factor. Antibodies against LIFRbeta, a receptor subunit shared by neuropoietic cytokines, prevented ChAT induction in periosteal cell/neuron cocultures, suggesting that a member of this family is responsible. ChAT activity was increased in neurons grown with periosteal cells or conditioned medium from mice lacking either leukemia inhibitory factor (LIF) or LIF and ciliary neurotrophic factor (CNTF). These results provide evidence that periosteal cells influence sympathetic neuron phenotype by releasing a soluble cholinergic factor that is neither LIF nor CNTF but signals via LIFRbeta.     

Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture

Ciliary neurotrophic factor (CNTF) influences the levels of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in cultures of dissociated sympathetic neurons from newborn rats. In the presence of CNTF both the total and specific activity of ChAT was increased 7 d after culture by 15- and 18-fold, respectively, as compared to cultures kept in the absence of CNTF. Between 3 and 21 d in culture in the presence of CNTF the total ChAT activity increased by a factor of greater than 100. Immunotitration demonstrated that the elevated ChAT levels were due to an increased number of enzyme molecules. In contrast to the increase in ChAT levels, the total and specific activity levels of TH were decreased by 42 and 36%, respectively, after 7 d in culture. Half-maximal effects for both ChAT increase and TH decrease were obtained at CNTF concentrations of approximately 0.6 ng and maximal levels were reached at 1 ng of CNTF per milliliter of medium. The effect of CNTF on TH and ChAT levels were seen in serum-containing medium as well as in serum-free medium. CNTF was shown to have only a small effect on the long-term survival of rat sympathetic neurons. We therefore concluded that the effects of CNTF on ChAT and TH are not due to selective survival of cells that acquire cholinergic traits in vitro, but are rather due to the induction of cholinergic differentiation of noradrenergic sympathetic neurons.     

Regulation of enzymes responsible for neurotransmitter synthesis and degradation in cultured rat sympathetic neurons. I. Effects of muscle-conditioned medium

The enzymatic machinery for neurotransmitter synthesis and breakdown have been compared in sister cultures of newborn rat sympathetic neurons grown for 12-28 days either in the presence (CM+ cultures) or in the absence (CM- cultures) of a culture medium conditioned by rat skeletal muscle cells. Neuron numbers, total protein, and lactate dehydrogenase activities were identical in CM+ and CM- cultures. Choline acetyltransferase activity was 27- to 100-fold higher in homogenates of CM+ than CM- cultures, whereas acetylcholinesterase activity was 2.5-fold lower. The activities of tyrosine hydroxylase (TOH), DOPA decarboxylase, and dopamine beta-hydroxylase were all about twofold lower in homogenates from CM+ cultures. All these effects were also observed in homogenates of sympathetic neuron cultures grown with and without a macromolecular factor partially purified from CM (Weber, J. (1981). Biol. Chem. 256, 3447-3453.). Experiments of mixing homogenates from CM+ and CM- cultures suggested that the differences in each of the enzyme activities did not result from differences in the concentrations of hypothetical reversible enzyme activators and/or inhibitors. In addition, the deficit in TOH activity in CM+ cultures resulted from a decrease in the enzymatic Vmax with no significant variation in the apparent Km's for the substrate and the cofactor. An identical decrease in the Vmax was observed if TOH was assayed under phosphorylating or nonphosphorylating conditions, suggesting that this decrease did not result from differences in the state of enzyme phosphorylation. Immunoprecipitation curves of TOH activity by an anti-TOH antiserum were parallel when performed on homogenates from CM+ and CM- cultures, suggesting a difference in the number of enzyme molecules without detectable alteration of their kinetic properties.     

Leukocyte-deactivating factor from macrophages: Partial purification and biochemical characterization. A novel cytokine

A deactivating factor (MDF) is released from granuloma-like lesions of mice (giant and epithelioid macrophages) to the surrounding medium. Test cells incubated in the presence of MDF display dramatic inhibition of superoxide anion (O₂^?) release when stimulated. This failure to manifest O₂ release is observed whether PMA, all-transretinal, or fMet-Leu-Phe is the stimulating agent. MDF acts on different cell types from different species; mouse macrophages as well as guinea pig, human, and mouse neutrophils. Such results suggest that it is a universal regulatory cytokine with high affinity for phagocytic lineages. The factor was subjected to various purification methods: ultrafiltration, gel chromatography, and reversed phase HPLC. A crude preparation that resulted from conditioning of medium by old macrophages (MCM) shows two peaks of activity when subjected to gel filtration. These correspond to molecular weights for the active principle of 3 and 11 kD. When the factor was obtained by extraction of the same cells after washing and sonication. Only the former peak was seen. Fractions corresponding to a MW of 3 kD from several preparations were combined and subjected to HPLC. MDF activity then appeared in a single fraction. MDF is thus putatively a modulator of the cidal activity of phagocytic cells that utilize release of reactive oxygen species for cytocidal activity. © 1993 Wiley-Liss, Inc.     

Morphological and biochemical studies on the development of cholinergic properties in cultured sympathetic neurons. II. Dependence on postnatal age

Superior cervical ganglion (SCG) neurons taken from perinatal rats and dissociated in culture develop cholinergic properties. This report examines this "plasticity" of neurotransmitter function with regard to its dependence on the stage of neuronal development. Explants of SCG from rats ranging in age from 2 d to adult were cultured, and the number of neurons surviving after 6 wk in culture was evaluated. The activities of choline acetyltransferase (ChAc) and DOPA decarboxylase (DDC) were assayed for each age group over time in culture, and the cytochemistry of the synaptic vesicle population was studied after norepinephrine loading and KMnO4 fixation. The specific activity of ChAc in all explants fell during the first 3--4 d in culture (secondary to degeneration of presynaptic terminals), with an increase during the next 30 d in explants from all age groups except in those from the 22-d- old and adult rats. The highest activity found after 1 mo in culture was in explants from 2-d-old rats (62.5 mmol per kg dry wt per h); the lowest was in explants from adults (1.3 nmol per kg dry wt per h). After 1 mo in vitro, there were no significant differences in DDC activity among explants from animals of any age (similar to approximately 220 mmol per kg dry wt per h). Co-culture of the SCG explants with heart muscle increased even further the ChAc activity in explants from 2-d-old rats but not in explants from 16-d-old and 6.5-wk- old animals. The cytochemistry of the synaptic vesicle population in 1- mo-old cultures correlated well with the ChAc activity; when the ChAc activity was high, the proportion of synaptic vesicles with clear centers was 71--88%. In explants from adult animals, only 12% of the vesicles contained clear centers. From these data we conclude that the maturity of the SCG neuron influences the degree to which it is able to adjust its neurotransmitter mechanisms. That the axons of this neuron are interacting with target tissues during the time that neurotransmitter plasticity is retained suggests that interaction with the target may play a role in the determination of transmitter type.     

Membrane properties of cultured rat sympathetic neurons: Morphological studies of adrenergic and cholinergic differentiation

Dissociated neurons from the newborn rat superior cervical ganglion were grown under conditions which lead to either adrenergic or cholinergic differentiation. Lectins and toxins were used to detect differences in the cell membrane associated with transmitter status, age of the neurons, or location on the neurons. These ligands were made visible in the light or electron microscope by coupling to rhodamine or colloidal gold. The density of binding sites for concanavalin A (Con A), ricin (RCA₆₀), and wheat germ agglutinin (WGA) increased with age in culture on both adrenergic and cholinergic cells. Soybean agglutinin (SBA) binding increased about threefold on adrenergic axons, but failed to increase on neurons induced to become cholinergic by medium conditioned by rat heart cells (CM). The effect of CM on SBA binding paralleled previously described effects of CM on transmitter production; the CM binding pattern developed slowly and was not readily reversible. Mature adrenergic neurons also appeared to bind more WGA than neurons in CM cultures. Tetanus toxin gold binding was uniform, but low, on axons of adrenergic and cholinergic neurons at all ages. In contrast, cholera toxin binding decreased with age on adrenergic axons. Binding sites for SBA and tetanus toxin were found to be less numerous on the cell body surface than on the axonal surface. Thus growth in CM induces fundamental changes in the phenotype of developing sympathetic neurons involving the cell membrane as well as transmitter choice. Differences also appear with maturation and between axonal and somatic cell surface membranes.     

Calmodulin N-methyltransferase. Partial purification and characterization

The distribution, properties, and substrate specificity of S-adenosylmethionine:calmodulin (lysine) N-methyltransferse (EC 2.1.1.60, calmodulin N-methyltransferase) of the rat have been studied. This enzyme is cytosolic and is found at high levels in tissues with high levels of calmodulin and at low levels in tissues with little calmodulin. In liver, heart, and skeletal muscle, which have low levels of calmodulin and very low calmodulin N-methyltransferase activity (a low ratio of calmodulin N-methyltransferase to calmodulin), calmodulin was found to be incompletely methylated, as judged by its ability to act as a substrate for purified calmodulin N-methyltransferase. Calmodulin N-methyltransferase was purified 470-fold with a 33% yield from rat testis cytosol, using ammonium sulfate precipitation and chromatography on DEAE-cellulose, CM-Sepharose, and Sephadex G-100. At pH 7.4, calmodulin N-methyltransferase did not bind to DEAE-cellulose, but bound strongly to CM-Sepharose. The enzyme eluted from Sephadex G-100 with an apparent molecular weight of 55,000. Purified calmodulin N-methyltransferase was incubated with extracts of rat tissues, and [methyl-3H]AdoMet and methylated proteins were resolved by electrophoresis in an attempt to discover substances other than calmodulin, but this enzyme only catalyzed the methylation of calmodulin, indicating a high degree of substrate specificity. Conditions were established for the in vitro preparative methylation of des(methyl)-calmodulin from Dictyostelium discoideum. Three moles of methyl/mol of calmodulin were incorporated into lysine 115 of des(methyl)calmodulin, resulting in the formation of 1 mol of trimethyllysine at the site normally methylated in calmodulins from most species. Activation of cyclic nucleotide phosphodiesterase by des(methyl)calmodulin was indistinguishable from activation by in vitro methylated or sham methylated Dictyostelium calmodulin, indicating that methylation does not affect the ability of calmodulin to activate this enzyme.     

Identification of glycolipid binding sites for soybean agglutinin and differences in the surface glycolipids of cultured adrenergic and cholinergic sympathetic neurons

Cultured adrenergic neurons from newborn rat superior cervical ganglia bind the lectin soybean agglutinin (SBA) at a fivefold higher density than the same neurons which have been induced to become cholinergic (M. Schwab and S. L. Landis, 1981, Dev. Biol.84, 67–78). In the present experiments, the binding sites for this lectin on the surfaces of living neurons were identified by labeling the surfaces with the galactose oxidase-[³H]sodium borohydride reduction technique, with and without prior incubation with the lectin. SBA binds to and inhibits the labeling of two neutral glycolipids, a glycolipid comigrating with globoside on thin-layer chromatograms and an unidentified glycolipid. When neuronal proteins are extracted and separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, SBA shows only very faint labeling of this fraction. Thus the SBA binding sites on these neurons appear to be two neutral glycolipids. Further support for this conclusion comes from the finding that the two neutral glycolipids detected by SBA are present in smaller amounts or are less accessible on the cholinergic than on the adrenergic neurons as measured by surface labeling. In addition to the difference in neutral glycolipids, external labeling revealed quantitative differences in the major gangliosides of the two types of cultured neurons. Thus, by using pure cultures of sympathetic neurons which can be induced to become either adrenergic or cholinergic, specific glycolipid profiles were correlated with the two neurotransmitter phenotypes.     

A platelet factor stimulating the proliferation of vascular endothelial cells: Partial purification and characterization

Platelets have been shown to contain a novel growth factor that stimulates the proliferation of vascular endothelial cells in vitro. The factor potently stimulated both DNA synthesis and proliferation rate in serum-deprived endothelial cells. Gel exclusion chromatography showed at least two peaks of activity on endothelial cells, the major peak being at an apparent molecular weight of 20 000. Isoelectric focusing revealed that the pI of the factor was 4.0-4.8. It was adsorbed to a column of DEAE ion exchange chromatography and eluted with a salt gradient. The factor was heat-labile and trypsin-sensitive. The activity was not destroyed by a reducing agent including dithiothreitol. This factor stimulated the proliferation of vascular endothelial cells but was found to be inactive against normal rat kidney fibroblasts.     

A diffusible second messenger mediates one of the pathways coupling receptors to calcium channels in rat sympathetic neurons.

Muscarinic and alpha-adrenergic suppression of current through Ca2+ channels was studied in adult rat superior cervical ganglion neurons using whole-cell and cell-attached configurations of the patch-clamp technique. Oxotremorine methiodide suppressed ICa by both a rapid (much less than 1 s) and a slow (greater than 4 s) process, whereas norepinephrine suppressed ICa only by a rapid process. The slow muscarinic suppression could be prevented by adding 20 mM BAPTA, a Ca2+ chelator, to the recording pipette, whereas the adrenergic suppression was not affected. Muscarinic, but not alpha-adrenergic, receptors can couple to Ca2+ channels by a second messenger capable of diffusing into an on-cell patch. This signal seems not to be carried by intracellular Ca2+, cGMP, cAMP, or protein kinase C.     

Partial purification and characterization of epidermal growth factor in human breast milk

Human epidermal growth factor (hEGF), a potent growth stimulator of many tissues in culture, has been isolated from human urine and subsequently identified in many human biological fluids including breast milk. In this study, partial purification and characterization of hEGF-like substance(s) in human milk were performed using homologous hEGF radioimmunoassay (RIA) and radioreceptor assay (RRA). hEGF-like material(s) was extracted from pooled human milk by ethanol precipitation, followed by adsorption to cation- and anion-exchange resin. DEAE-Sephadex G-25 ion-exchange chromatography of human milk extracts revealed three major components with hEGF activity (peak I, II, III) eluted with a linear gradient by ammonium acetate. The competitive binding curves for these components were parallel to those for standard hEGF in both RIA and RRA. The apparent molecular weight of peak I was approximately 6,500 and that of peak II and III was approximately 7,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The pI value for peak I was approximately 4.5 and that for peaks II and III was approximately 5.0 by isoelectric focusing. These data are comparable to the size and charge heterogeneity of hEGF in human urine extracts. In conclusion, the major components of hEGF in human milk appear to be physicochemically, immunologically and biologically (receptor binding activity) indistinguishable from hEGF of urinary origin.     

Development of the cholinergic neurotransmitter phenotype in postganglionic sympathetic neurons

Sympathetic ganglia are composed of noradrenergic neurons and cholinergic neurons that differ in the expression of neurotransmitter-synthesizing enzymes, neurotransmitter transporters and neuropeptides. The analysis of the cholinergic differentiation during development revealed important principles involved in the generation of neuronal diversity, in particular the importance of signals from the innervated target. Some peripheral targets, such as the sweat glands in the mammalian footpads, are purely cholinergically innervated in the adult, whereas skeletal muscle arteries receive both noradrenergic and cholinergic innervation. For sympathetic neurons innervating sweat glands there is convincing evidence that these neurons are initially noradrenergic and that the interaction of innervating fibers and target tissue induces a shift in the neurotransmitter phenotype from noradrenergic to cholinergic. In addition to this target-dependent differentiation, an earlier expression of cholinergic characters was observed in sympathetic ganglia that occurs before target contact. These data raise the possibility that different subpopulations of cholinergic sympathetic neurons, innervating distinct peripheral targets, may develop along distinct schedules. In vitro studies suggest that growth factors of the family of neuropoietic cytokines are involved in the specification of the cholinergic sympathetic phenotype. Recent in vivo studies that interfered with cytokine receptor expression in developing avian sympathetic ganglia indicate that only the late, target-dependent differentiation depends on cytokine signaling. The signals involved in the early, target-independent expression of cholinergic properties remain to be determined, as well as the identity of the target-derived cytokine. Thus, cholinergic sympathetic differentiation seems to be more complex than expected, involving either both target-independent and target-dependent control or only target-induced differentiation, according to the specific neuronal subpopulation and target.     

Partial purification and characterization of a growth factor present in goat's colostrum. Similarities with platelet-derived growth factor.

A factor in goat's colostrum which stimulates DNA synthesis and cell proliferation in Swiss 3T3 fibroblasts has been purified approx. 350-fold by a sequence of acid precipitation, cation-exchange chromatography and gel filtration. The growth factor is a highly basic, heat stable (100 degrees C for 5 min) polypeptide with Mr approx. 35000. The polypeptide resists denaturation by guanidinium chloride or urea but is totally inactivated by treatment with reducing agents. The factor, which we have termed colostric basic growth factor ( CBGF ), inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 fibroblasts but does not inhibit 125I-EGF binding to epidermoid A431 cells. CBGF interacts synergistically with plasma in stimulating DNA synthesis in quiescent Swiss 3T3 cells. The chemical and biological properties of CBGF are thus very similar to the properties reported for the human platelet-derived growth factor. Although high concentrations of CBGF are present in the colostrum of goats, cows, and sheep, the milk of these species contains little or no factor. The origin and possible functions of CBGF are unknown.     

Partial purification and characterization of the messenger RNA for cell fibronectin.

Fibronectin mRNA has been partially purified by guanidine extraction, oligo-(dT)-cellulose chromatography and sucrose density gradient centrifugation. We obtain a fraction which programs a wheat germ in vitro translation system to synthesize a polypeptide species which co-electrophoreses with fibronectin in SDS-polyacrylamide gels and which is immunoprecipitated with affinity purified fibronectin-specific IgG. Analysis of this RNA fraction by methyl mercury hydroxide-agarose gel electrophoresis reveals the presence of a band accounting for 30 percent to 50 percent of the ethidium bromide-staining material in the fraction. The RNA of this band has an estimated molecular weight of about 3 million daltons and is greatly reduced in the corresponding RNA fraction from RSV transformed CEF. This RNA has been tentatively identified as fibronectin mRNA.