Biomedical subjects

S D Skaper

Publications and source records attributed to S D Skaper.

At least 109 records · Page 6Linked to original sources

PubMed1987

Ionic responses and growth stimulation in rat astroglial cells: differential mechanisms of gangliosides and serum.

Rat astroglial cells respond to fetal calf serum (FCS) and gangliosides, including GM1, by undergoing proliferation. Here, we show that addition of FCS but not GM1 causes an increase in Na+, K+-pump activity, as measured by ouabain-sensitive 86Rb+ influx. The increase of Na+, K+-pump activity by FCS was due to increased Na+ influx (measured with 22Na+). This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+/H+ exchange. Amiloride also blocked the FCS-stimulated incorporation of [3H]thymidine into DNA. Two defined polypeptide growth factors, epidermal growth factor and fibroblast growth factor were also able to elicit an amiloride-sensitive Na+ influx and an ouabain-sensitive K+ uptake in these astroglial cells, in the presence of FCS or insulin. Thus, GM1 differs from serum and growth factors in the mechanisms by which these agents stimulate the proliferation of the astroglial cells used here.

Amiloride↗

PubMed1987

Catalase protection of neuronal survival in vitro is not directed to the accumulation of peroxides in the culture medium.

Walicke et al. (1986, J. Neurosci. 6, 1114-1121) have shown that catalase can replace the pyruvate requirement for survival of CNS neurons cultured in vitro. Since presently the only known function of catalase is the enzymatic degradation of hydrogen peroxide to water and oxygen, the simplest interpretation of the ability of catalase to support neuronal survival would be that catalase removes from the culture medium hydrogen peroxide. To test this hypothesis 8-day embryonic chick forebrain cells were cultured for 24 hr in a modified Eagle's Basal Medium with the serum-free supplement N1 (HEBM/N1) in the presence or absence of Phenol Red, 20 micrograms/ml catalase, 1 mM pyruvate, and/or 25 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES) on a polyornithine-laminin substratum. The various media were then assayed for peroxide content using the potassium iodide method described by Wang and Nixon (1978, In Vitro 14, 714-722). The present data reveal that (1) HEBM/N1 normally contains approximately 50 microM peroxides, little of which is hydrogen peroxide, (2) the organic peroxide levels accumulating in this medium are not reduced by either catalase or pyruvate, and (3) medium modifications can reduce to no longer detectable levels the peroxides accumulating in the medium, but catalase or pyruvate is still required for neuronal survival. We conclude that catalase must exert its survival-promoting action at levels other than peroxides accumulating in the culture medium.

Animals↗

PubMed1987

Dissociation of the stellate morphology from intracellular cyclic AMP levels in cultured rat brain astroglial cells: effects of ganglioside GM1 and lysophosphatidylserine.

Secondary microcultures of newborn rat cerebrum astroglial (AG) cells, maintained in a serum-free, chemically defined medium, were treated with various agents known to elevate intracellular cyclic AMP (cAMP) levels. Earlier studies had shown these drugs to induce a process-bearing (stellate) morphology in the AG cells, a response that was antagonized by the presence of gangliosides. One millimolar dibutyryl cyclic AMP (dBcAMP), 10 microM forskolin, 12 nM cholera toxin, and 30 microM isoproterenol all raised intracellular cAMP levels, from basal values of 3 pmol/10(6) cells to 30-30,000 pmol/10(6) cells, depending on the agent tested. dBcAMP caused the greatest elevation, and forskolin the least. The timing and/or the level of the AMP response did not precisely correlate with those of the stellation response. Values of ED50 with the four agents, as determined for the cAMP response, were always higher than stellation ED50 values in all treatments, and ED50 did not correlate with the maximal levels of cyclic AMP induced by the four agents. The capacity of ganglioside GM1 to block the stellation response to the four agents was not accompanied by a similar capacity to block the cAMP responses. Lysophosphatidylserine (lysoPS) had the capacity to induce AG cell stellation as well, without altering the basal level of cAMP. Both lysoPS and gangliosides, therefore, may act directly on the cellular machinery underlying the stellation response without involving changes in intracellular AMP.

Animals↗

PubMed1986

Control of Na+, K+-pump activity in dorsal root ganglionic neurons by different neuronotrophic agents.

Na+, K+-pump activity is indispensable for neuronal survival in vitro and a specific role in its regulation has been demonstrated for the NGF action on its target neurons. We have extended these earlier studies to include two other neuronotrophic agents: the chick eye-derived ciliary neuronotrophic factor (CNTF); and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). CNTF and TPA individually supported the survival of an identical (and maximal) number of embryonic day 10 (E10) dorsal root ganglion (DRG) neurons as did NGF. E10 DRG neurons, seeded as monolayer cultures with 86Rb+ (as K+ tracer) but no trophic supplement in their medium, received NGF, CNTF, TPA, or no agent at 2, 4 or 6 h after seeding. The cultures were analyzed at 6 and 24 h for Na+, K+-pump performance and at 24 h for neuronal survival. Neurons receiving no agent lost their pump activity over the first 6 h and died over the 10-24 h incubation period. Both pump performance and survival were fully supported by any one of the 3 agents when provided at seeding time. Delayed presentation of NGF also led to full restoration of pump activity and survival support, as expected. In contrast, CNTF and TPA failed to correct the increasing pump deficits incurred with increasing times of trophic deprivation, and neuronal survival was proportionally reduced. Delayed addition of CNTF and TPA did, however, prevent further losses of both pump and viability. Close similarities were observed between pump failure and cell losses, demonstrating a linear correlation between pump performance and neuronal survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Transamination of glutamate to tricarboxylic acid-cycle intermediates in cultured neurons correlates with the ability of oxo acids to support neuronal survival in vitro.

Cultures of central-nervous-system neurons at low densities require for their survival exogenous pyruvate, alpha-oxoglutarate or oxaloacetate, even in the presence of high glucose concentrations. Most other alpha-oxo acids support cell survival only in the presence of alpha-amino acids which transaminate to alpha-oxoglutarate, oxaloacetate or pyruvate. The alpha-oxo acids therefore operate as acceptors of amino groups from appropriate donors to generate tricarboxylic acid-cycle-relevant substrates, and these alpha-oxo acids provide for neuronal support only insofar as they make it possible for exogenously supplied alpha-amino acid precursors to generate intracellularly one of the three critical metabolites. To examine more closely the relationship between transamination activity and neuronal survival, we measured 14CO2 production from [14C]glutamate in the presence of appropriate alpha-oxo acid partners by using 8-day-embryonic chick forebrain, dorsal-root-ganglion and ciliary-ganglion neurons. Neuronal survival was measured concurrently in monolayer neuronal cultures maintained with the corresponding amino acid/oxo acid pairs. Forebrain and ganglionic cell suspensions both produced 14CO2 from [14C]glutamate, which accurately correlated with 24 h neuronal survival. Concentrations of glutamate or alpha-oxo acid which provide for maximal neuronal survival also produced maximal amounts of 14CO2. The same ability to generate CO2 from glutamate (in the presence of the appropriate alpha-oxo acids) can ensure neuronal survival in 24 h cultures and therefore must meet energy or other metabolic needs of those neurons which glucose itself is unable to satisfy.

Animals↗

PubMed1986

Purification of adult rat sciatic nerve ciliary neuronotrophic factor.

The ciliary neuronotrophic factor (CNTF), a protein required for the survival of cultured avian embryonic parasympathetic ciliary ganglionic neurons, was recently purified from extracts of selected chick intraocular tissues. Here we report the purification of a mammalian CNTF activity from extracts of adult rat sciatic nerve using a fractionation procedure similar to that employed for isolating chick eye CNTF. About 2 micrograms of CNTF protein can be obtained from each 1.5 g batch of nerve tissue. Like the chick CNTF, the mammalian factor displays trophic activity for dorsal root and sympathetic as well as ciliary ganglionic neurons. The nerve CNTF activity differs from its chick counterpart in molecular weight and chromatographic behavior on ion-exchange columns. Unlike purified nerve growth factor (NGF), nerve CNTF activity is insensitive to anti-NGF antibodies and is unable to support the survival of 8-day chick embryo dorsal root ganglion neurons.

Animals↗

PubMed1986

Gangliosides stimulate astroglial cell proliferation in the absence of serum.

Previous studies with microcultures of astroglial (AG) cells from newborn rat cerebrum had shown an ability of gangliosides to interact with AG cells cultured under defined conditions. We have now investigated the capability of gangliosides to stimulate DNA synthesis and cell number increases in similar secondary microcultures of newborn rat cerebrum AG cells. At a concentration of 6 X 10(-5)M, GM1 ganglioside stimulated DNA synthesis and increased cell numbers, with DNA synthesis leading cell increases by 12-24 hr. The ganglioside-induced AG cell proliferative response occurred with GD1a, GD1b and GT1b, GT1b being the most potent at 10(-5)M--while asialo GM1 and sialic acid were without effect. In the standard test cultures, DNA synthesis declined very steeply after the first day, with cell numbers stabilizing at the level reached after 2 days. Ganglioside was not itself responsible for the restricted proliferative response, as serum produced the same behaviors.

Animals↗

PubMed1986

Morphological modulation of cultured rat brain astroglial cells: antagonism by ganglioside GM1.

Secondary cultures of neonatal rat astroglial cells, maintained in a serum-free, chemically defined medium were treated with several agents thought to activate cyclic AMP-synthesizing systems. Dibutyryl cyclic AMP (dBcAMP), forskolin and cholera toxin promoted, within 2 h, the near-complete conversion of 1-day-old (D1) astroglial cells from a flat, epithelioid morphology to a stellate (star-shaped) morphology. With all 3 agents, cell susceptibility to morphological change declined with culture age, 5-day-old cultures failing to respond altogether. D1 cultures, after 48 h of treatment, had reverted to the flat morphology. Gangliosides reported to stimulate adenylate cyclase were also tested, using purified GM1 X GM1 failed to stimulate the conversion to stellate morphologies. GM1, however, did affect these astroglial cells by causing a block or reversal of their morphological response to dBcAMP, forskolin or cholera toxin. The GM1 response was specific for the intact ganglioside molecule, asialo GM1 and sialic acid having no effect. Gangliosides GD1a, GD1b and GT1b were also active, being effective at ca. 4-fold lower concentrations. The response to GM1 appeared to involve a direct interaction with the astroglial cell, rather than influencing either substratum or medium components.

Animals↗

PubMed1986

Age-dependent control of dorsal root ganglion neuron survival by macromolecular and low-molecular-weight trophic agents and substratum-bound laminins.

Chick embryo dorsal root ganglion (DRG) neurons can be supported in vitro by nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF). Pyruvate is also required for survival of neurons from embryonic day 8 (E8) chick ciliary ganglia and from several chick and rat embryonic central nervous system sources. Here we have examined the survival requirements of chick DRG neurons between E6.5 and E15. These DRG neurons, initially dependent only on NGF, become dependent also on CNTF and later on increasingly independent from both factors. Pyruvate nearly doubles neuronal survival at all ages under all conditions. The pyruvate concentration permitting this additional survival was reduced two-fold with serine present. In the presence of polyornithine-bound laminins, nearly all seeded neurons were rescued by pyruvate plus NGF (E8 on), or pyruvate plus CNTF (E10 on), or pyruvate without trophic factors (E15). The same maximal survival was achieved without pyruvate by supplying E10 or older neurons with both NGF and CNTF. Unmodified polyornithine substrata yielded about one-half this number of surviving neurons.

Animals↗

PubMed1986

An automated colorimetric microassay for neuronotrophic factors.

A microassay is described for determining the number of neurons surviving after 24 h in response to added neuronotrophic factors. Neuronal cultures in 96-well microtiter plates are supplied with a yellow tetrazolium derivative, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), which is taken up selectively by viable neurons and converted to a blue formazan product. The amount of blue color development can be rapidly quantified using an automatic microplate spectrophotometer. The resulting optical density is directly proportional to the number of viable neurons. The spectrophotometer has been interfaced with a computer allowing a print out of individual absorbance values and calculation of half-maximal (one trophic unit) neuronal survival. The assay has been used for the quantification of the trophic activities of nerve growth factor and ciliary neuronotrophic factor using, respectively, dorsal root and ciliary ganglionic neurons from 8-day chick embryos. Assay parameters were optimized so that about 2000 individual cultures of ganglionic neurons can be set up and analyzed each day, thus allowing the serial titration in duplicate of 80-120 separate samples. The determination of neuronal number and titer calculation steps now requires about 2 min per microplate (96 cultures), a 50-fold reduction in time over existing methods.

Animals↗

PubMed1986

GM1 ganglioside treatment of PC12 cells stimulates ganglioside, glycolipid, and lipid, but not glycoprotein synthesis independently from the effects of nerve growth factor.

The incorporation of radioactive precursors into gangliosides and other glycolipids, glycoproteins, and total lipids has been studied in rat pheochromocytoma PC12 cells. Starting with the same PC12 cell pool, cultures displaying different degrees of neuritic expression in response to nerve growth factor (NGF) and combinations of serum ganglioside GM1 were produced. Attempts were then made to correlate neuritic regulation with biochemical performances of these cells. NGF stimulates the incorporation of [3H]galactose into gangliosides and other glycolipids and glycoproteins and [14C]acetate into total lipids, regardless of the serum concentration. NGF both increased their initial labeling rates and promoted additional and more extensive labeling from culture day 4 onward. Unexpectedly, exogenous GM1 also elicited an increase in ganglioside labeling as well as that of the other lipid classes, but not of glycoproteins. The GM1-induced increase was evident at higher serum concentrations (1%) regardless of the presence or absence of NGF, but not apparent in low (0.15%) serum. Serum levels themselves did not affect labeling patterns in the absence of NGF and GM1. GM1-induced stimulation of labeling reflects an increase in the synthetic activities of the cells, and not increased precursor uptake or reduced product degradation. For all constituents stimulated by GM1, concurrent treatment with NGF produces cumulative effects, suggesting independent mechanisms of action by the two molecules.

Acetates↗

PubMed1985

Phenotypical changes of embryonic chick adrenal medullary cells in vitro induced by nerve growth factor and ciliary neuronotrophic factor.

This study investigates the survival properties and changes in the morphological phenotype of adrenal medullary (chromaffin and neuronal) cells cultured from embryonic chicks at different developmental ages (embryonic days E8 to E16) in response to nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF). The 4-day survival of medullary cells from all embryonic ages except E8 was about 80% of the seeded cells and was only slightly enhanced by the addition of saturating doses of CNTF (10 ng/ml). With no factors, after 4 days 10-30% of the surviving medullary cells extended neurites. NGF (100 ng/ml) and, even more, CNTF (10 ng/ml) and their combination substantially increased the proportions of neurite-bearing cells (up to 70%). The effect of the factors were maximal at E10 and E12 and declined at older developmental ages. Neurite growth was virtually unaffected by NGF and CNTF at E8. These results show that in vitro survival and neurite growth of chick adrenal medullary cells in response to trophic factors is developmentally regulated.

Adrenal Medulla↗

PubMed1985

Central nervous system-directed neuronotrophic activity present in red blood cells.

A new neuronotrophic factor has been identified in extracts of vertebrate red blood cells. The factor supports the survival in culture of neurons from vertebrate central nervous systems, and does not support the survival of several peripheral ganglionic neurons. The active molecule appears to be a slightly acidic protein of 30,000-100,000 daltons.

Animals↗

PubMed1985

Selected nutrients reduce the pyruvate requirement for survival in vitro of chick central nervous system neurons.

Central nervous system neurons, cultured as monolayers at low density, need exogenous pyruvate for their survival. The pyruvate concentrations required by embryonic day 8 (E8) chick forebrain neurons are reduced substantially by other low molecular weight agents present in Dulbecco's modified Eagle's medium (DMEM) and in astroglia-conditioned Eagle's basal medium (EBM). To examine the nature of these pyruvate-sparing molecules, E8 chick forebrain neurons were cultured in EBM containing a concentration of pyruvate too low to support neuronal survival, unless sparing agents were supplied. This strategy permitted the identification of the pyruvate-sparing DMEM constituents, omission of which had led to neuronal loss. Pyruvate sparing required a mixture of (i) serine, (ii) pyridoxal (or riboflavin and biotin), and (iii) six essential amino acids: arginine, cystine, leucine, isoleucine, tyrosine, and tryptophan (each replaceable by corresponding amino acids, except tryptophan). All pyruvate-sparing contributors were optimally needed at or near their DMEM concentrations.

Amino Acids↗

PubMed1985

Developmental changes in the responses of rat chromaffin cells to neuronotrophic and neurite-promoting factors.

This study describes the survival and neurite outgrowth behaviors of cultured adrenal medullary (chromaffin) cells obtained from postnatal rats 1 day (D1) to 100 days (D100) old in response to nerve growth factor (NGF), chick eye ciliary neuronotrophic factor (CNTF), and laminin. In the absence of trophic factors the 4-day survival of cultured chromaffin cells (relative to the number of cells attached at 2 hr) increased from one-third of the cells at D1 to 40% at D8 and 90-100% at D16 and older stages. At saturating concentrations NGF increased cell survival at D8 by 90%, but failed to support all chromaffin cells present at 2 hr. In contrast, CNTF supported the survival of all cells at D8. At D1 NGF and CNTF had only a very small effect on survival during the 4-day culture period, although both factors clearly enhanced the numbers of surviving cells after 8 days. Either NGF or CNTF also elicited neurite outgrowth from rat chromaffin cells, which amounted to approximately 15-20% at D1 and D8 and subsequently decreased to about 5-8% at D30 and virtually zero at D100. At this last age both factors applied together clearly elicited neurites. Such a potentiating effect of NGF and CNTF was also seen at earlier postnatal ages. Laminin did not affect neurite growth at D30 in the absence of trophic factors, as already described for D8 rat chromaffin cells. In the presence of NGF, however, it increased neurite length and branching during a 4-day culture period and even enhanced neurite recruitment at later culture times. These data suggest that rat chromaffin cells undergo age-related changes in their responses to NGF and CNTF and that laminin modulates their neurite outgrowth behaviors in the presence of trophic factors.

Adrenal Glands↗

PubMed1985

GM1 ganglioside accelerates neurite outgrowth from primary peripheral and central neurons under selected culture conditions.

Neurons from chick embryonic day 8 (E8) ciliary ganglia, E8 and E15 dorsal root ganglia, E8 forebrain, and from rat E18 hippocampus and striatum were cultured as monolayers in the presence or absence of GM1 ganglioside. All of the primary neurons tested were susceptible to an effect of GM1 on their neuritic outgrowth, resulting in a 2- to 3-fold stimulation over control, the recognition of which depended on selecting culture conditions appropriate to each case. The response of E8 ciliary ganglionic neurons required a serum-free medium containing ciliary neuronotrophic factor, and was most pronounced by 8 h at 3 X 10(-8) M GM1. The neuritic response by either E8 or E15 dorsal root ganglionic neurons required serum (greater than or equal to 0.3%), their appropriate neuronotrophic factor, and 100-fold higher GM1 concentrations (presumably reflecting the serum presence), with optimal response times of 12-24 h. For E8 chick forebrain and E18 rat central neurons, GM1 substantially increased the proportion of neurite-bearing neurons in a serum-free pyruvate-containing medium between 7 and 24 h, with an optimal GM1 concentration of 10(-7) M. In all cases, the response to GM1 was a time-related gain, i.e. an earlier onset of neuritic regeneration rather than permanent increase in the number of neurite-bearing neurons.

Animals↗

PubMed1985

Tumor-promoting phorbol diester mimics two distinct neuronotrophic factors.

The two purified neuronotrophic factor proteins, nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF), differ in molecular properties and in their action on certain neuronal cell types. The survival in culture of dissociated chick embryonic day 8 dorsal root ganglionic neurons is supported by NGF and day 8 ciliary ganglionic neurons by CNTF, but neither factor can be a substitute for the other. Here we report that a known tumor-promoting phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), can support the survival of both neuronal types in the absence of either NGF or CNTF and does so with the same efficacy as the corresponding trophic factor. The inactive phorbol derivative, 4-alpha-phorbol-didecanoate, does not support either neuronal type. The combined provision of suboptimal doses of TPA and NGF or CNTF does not reveal synergistic effects. These results suggest that TPA, NGF and CNTF may exert their neuronal survival promoting influences through a common molecular mechanism.

Animals↗

PubMed1985

Neuronotrophic and neurite-promoting factors: effects on early postnatal chromaffin cells from rat adrenal medulla.

Adrenal chromaffin cells from early postnatal rats maintained in culture have previously been shown to grow neuritic processes and survive better in the presence of nerve growth factor (NGF). In the present study we have quantitated the effects on chromaffin cell (postnatal day (D) 8) survival and neurite outgrowth of: NGF, ciliary neuronotrophic factor (CNTF), activities contained in various types of conditioned media (CM), and various substrata (laminin, fibronectin and polyornithine-binding neurite-promoting factor from RN 22 Schwannoma cells - PNPF). At saturating concentrations CNTF (50 ng/ml) and C6 glioma cell CM, (50-fold concentrated) supported survival over the 4-day culture period of all the chromaffin cells present in culture 2 h after seeding. NGF (50 ng/ml) and the non-concentrated CMs from primary Schwann cell and astrocytes as well as Schwannoma and C6 glioma cell cultures, achieved the maintenance of only about half the number of cells above the baseline survival as compared to CNTF and the concentrated C6-CM. These results are compatible with two subsets of D8 chromaffin cells, one only supported by CNTF and the concentrated CM and the other supported by either NGF or CNTF. Either NGF or CNTF elicited neurite outgrowth from 15-20% of the surviving cells. Combination of maximal doses of NGF and CNTF caused a small increase in neurite recruitment beyond that elicited by either factor alone. Low doses of CNTF added to the effect of NGF, shifting the NGF titration curve by about 4-fold. Neurite outgrowth was also induced by the concentrated, but not the unconcentrated C6-CM. Laminin, fibronectin and PNPF did not affect the fibronectin and PNPF did not affect the recruitment of neurites as compared to a polyornithine substratum unless the cultures were supplemented with a neuronotrophic factor and carried for 7 days. However, even before showing effects on neurite recruitment these substrata affected various neuritic performances, such as length, neurite numbers and endings per cell.

Adrenal Medulla↗