Cyclic Nucleotide Phosphohydrolase Activity Research Articles

Maintenance of glial plasticity with aging in C-6 glial cells and normal astrocytes in culture: responsiveness to opioid peptides.

In this study we used as glial cell models, early and late passage C-6 glial cells, 2B clone, and advanced passages of glial cells derived from aged mouse cerebral hemispheres (MACH) to examine responsiveness to opioids. We have previously reported that early passage C-6 glial cells, 2B clone, are bipotential and can be geared toward oligodendrocyte or astrocytic expression, whereas late passage C-6 glial cells are astrocytic. In addition, MACH cultures have been previously characterized and consist of astrocytes type 1 and 2, some oligodendrocytes, and few glial precursors. In this study, early passage (17-20) and late passage (106-108) C-6 glial cells or MACH cells of passages 16-19 were grown from plating time until harvesting, day 7 or 8, in DMEM + 10% FBS in the presence or absence of opioid peptides, Leu-enkephalin (10(-8) to 10(-10) M) or its synthetic analog, dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg; 10(-8) to 10(-10) M). We examined for the activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), enzyme markers for astrocytes and oligodendrocytes, respectively. We found that CNP activity was markedly increased in the early passage following opioid treatment, indicative of a shift to oligodendrocytic expression. In the late passage cells, already committed to astrocytic expression, opioid treatment enhanced GS activity suggesting that astrocytes respond to opioids. GS activity was markedly increased in MACH cultures grown in the presence of opioids with no changes in CNP. Thus, type 1 astrocytes, the predominant glial type in MACH cultures, responded to opioids.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of culture substrata on the differentiation of advanced passage glial cells in cultures from aged mouse cerebral hemispheres

We have previously reported that glial cells derived from aged mouse cerebral hemispheres (MACH) in primary cultures and after several passages consist of protoplasmic astrocytes (Type 1), differentiated stellate astrocytes (Type 2), a few oligodendrocytes and also glial precursors.4,33,34 In this study, we examined the influence of culture substrata: plastic, poly-l-lysine, laminin or collagen on the differentiation of MACH glial cells of advanced passages (P18–19) using glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) activity as biochemical markers for astrocytes and oligodendrocytes, respectively. Cultures were also examined morphologically using light microscopy. In general, GS activity was increased in cultures grown on the three chemical substrata versus plastic alone with the most striking effect being the 2-fold increase observed in those cells grown in laminin. No differences were noted in CNP activity. Morphologically, proliferation of protoplasmic (Type 1) astrocytes was enhanced by culture day 2 on polylysine substratum and stellate differentiated (Type 2) astrocytes were noted on collagen. The striking feature in cultures grown on laminin was the presence of astrocytes with markedly long processes. Thus, morphological astrocyte differentiation appears to correspond to the increased GS activity. We propose that the extracellular matrix components such as collagen and laminin may play an important role in promoting glial precursors to differentiate into astrocytes.

Effect of ultrasound on the neonatal rat brain

Six-day-old rat neonates were exposed to 2.25 MHz continuous wave (CW) ultrasound for 5 min at an intensity of 2.5 W/cm 2 (SATA). The temperatures on the head surface and in the mouth were measured. There was a higher average temperature elevation in the mouth (9°C) than on the head surface (7°C). Survival differs between control and exposed groups at 30 days after exposure. Ninety percent of the control group lived to 30 days, versus 59.7% for the exposed group. At differing times following exposure, the brains of the pups were removed and tested for enzymatic activities. Changes in acetylcholine esterase and in 2′,3′-cyclic nucleotide phosphohydrolase activities were not statistically significantly different from controls. There were no significant differences in brain weight and total protein between control and exposed pups.

Comparative biochemical, morphological, and immunocytochemical studies between C‐6 glial cells of early and late passages and advanced passages of glial cells derived from aged mouse cerebral hemispheres

We have used C6 glial cells (2B clone), early and late passage, as well as advanced passages (8-17) of glial cells derived from aged (18-month-old) mouse cerebral hemispheres (MACH), as model systems for studying glial properties. In this study passages 20-24 were considered "early" and passages 73-90 were considered "late." Activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) were used as biochemical markers for astrocytes and oligodendrocytes, respectively. Glial phenotypes were identified immunocytochemically using double staining for glial fibrillary acidic protein (GFAP) and A2B5 antigen (type 1 and type 2 astrocytes) or galactocerebroside (GalC) and A2B5 antigen (oligodendrocytes); cells positive for A2B5 and negative for both GFAP and GalC were considered to be precursor cells. Cultures were grown either in DMEM supplemented with 10% fetal bovine serum or in serum-free chemically defined medium (CDM) supplemented with insulin and transferrin. We report that early-passage C6 glial cells continue to be bipotential cells and when grown in the absence of serum express high GS and CNP activities correlating with the high number of GFAP- and GalC-positive cells, respectively. Late-passage cells continued to be committed to the type 2 astrocytic phenotype regardless of media composition (+/- serum). MACH cultures consist of protoplasmic type 1 astrocytes, differentiated type 2 astrocytes, and oligodendrocytes as well as glial progenitor cells. When these cultures were grown in CDM+transferrin, both GS and CNP activities increased, suggesting that transferrin has provided the signal for progenitor cells present in these cultures derived from aged brain to differentiate into type 2 astrocytes and oligodendrocytes.

Reversible biochemical changes in the developing rat central nervous system following ultrasound exposure.

Rat embryos in utero at 10 days of gestation (gd) were exposed to the intensity of 2.5 W/cm2 (SATA) of 1 MHz continuous-wave ultrasound for 5 min. The right uterine horn was exteriorized, exposed to the ultrasound beam, and then reinserted into the abdominal cavity. Embryos from the unexposed left uterine horn served as controls. The cerebral hemispheres of each fetus were removed and analyzed for enzymatic activity at four different stages of development. There was an increase in the temperature at the surface of the intact uterus during the ultrasound exposure. No gross anatomical malformations were observed in the exposed embryos. There were no differences in brain weight or protein content between exposed and control fetuses. However, the ultrasound exposure caused a transient decrease in acetylcholine esterase and 2',3'-cyclic nucleotide phosphohydrolase activities of the insonated fetal brains compared to the control brains. Because of the relatively high temporal-average intensity, the relatively large volume of tissue exposed, and the minimal sound path through tissue, the biochemical changes observed in this experiment would be unlikely to occur in clinical examinations.

2′,3′-Cyclic nucleotide phosphohydrolase activity determined using an image analyzer detection system

The activity of 2′,3′-cyclic nucleotide phosphohydrolase (CNPase) was assayed using high-performance thin-layer chromatography (HPTLC) and an image analyzer detection system. The assay system was used to study a possible inhibitory effect by aminoguanidine on CNPase specific activity. One advantage of using a fixed-time HPTLC system over a real-time spectrophotometric system for an enzyme activity study was that apparent inhibition of the enzyme due to interference of the assay system (chromophore inhibition, etc.) was avoided. In addition, due to the increased accuracy of the image analyzer over conventional methods of TLC plate analysis, a rapid and more accurate measurement of HPTLC plates was possible which required only nanomole amounts of substrate. Also, a digital image of each plate analyzed was stored indefinately in the computer's memory for future reference. The measurements of CNPase specific activity made using this system compared favorably to those found in recent literature.

Early and late passage C-6 glial cell growth: similarities with primary glial cells in culture.

Earlier studies in our laboratory have shown that C-6 glial cells in culture exhibit astrocytic properties with increasing cell passage. In this study, we tested the responsiveness of early and late passage C-6 glial cells to various cultures conditions: culture substrata (collagen, poly-L-lysine, plastic), or supplements for the culture medium, DMEM, [fetal calf, or heat inactivated (HI) serum, or media conditioned from mouse neuroblastoma cells (NBCM) or primary chick embryo cultured neurons (NCM)]. Glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), astrocytic and oligodendrocytic glial markers, were used. Cell number and protein content increased exponentially with days in culture regardless of the type of the substratum or cell passage. Differences in cell morphology among the three types of substratum were also reflected on GS activity, which rose by three-fold on culture day 3 for cells grown on collagen; thereafter, GS profiles were similar for all substrata. This early rise in GS is interpreted to reflect differential cell adhesion processes on the substrata; specifically, cell adhesion on the collagen stimulated differentiation into "astrocytic phenotype". Analogous to immature glia cells in primary cultures, early passage C-6 glial cells responded to neuronal factors supplied either from NCM or NBCM by expressing reduced GS activity, the astrocytic marker and enhanced CNP activity, the oligodendrocytic marker. Thus, early passage cells can be induced to express either astrocytic or oligodendrocytic phenotype. In accordance with our previous reports on primary glial cells, late passage C-6 cells exhibit their usual astrocytic behavior, responding to serum factors with GS activity. Moreover, whereas NCM or NBCM alone markedly lowered GS activity, a combination with serum restored activity. The present findings confirm our previous observations and further establish the C-6 glial cells as a reliable model to study immature glia.

A biochemical study of optic nerve myelin isolated by a micromethod from different aged rats

A micromethod was used to study myelin from individual rat optic nerves as a function of age. Protein and galactolipid content as well as 2′,3′-cyclic nucleotide phosphohydrolase (CNPase) activity at ages 2, 4, 22, and 28 months were determined. Protein content increased significantly at 22 months and decreased at advanced age. Galactolipid content increased significantly at 28 months while CNPase activity decreased. The ratios of protein to galactolipid and CNPase to galactolipid changed with changing age. These data suggest that, unlike the central nervous system, specific biochemical parameters of the optic nerve change with age.

Insulin: its binding to specific receptors and its stimulation of DNA synthesis and 2',3'-cyclic nucleotide phosphohydrolase activity in cerebral cells cultured from embryonic mouse brain.

The presence and specificity of insulin receptors was investigated in culture cells obtained from 15-16 days old embryonic mouse cerebra. Developmental studies suggested that the maximum insulin binding occurred at about 11 days in vitro (DIV). Scatchard analysis of binding data revealed two types of binding sites. One type of receptor was the high affinity type (Kd = 7.77 x 10(-9) M; number of receptor sites, Bmax = 350 fmol/mg protein) while the other type was of low affinity type (Kd = 5.75 X 10(-8)M; Bmax = 1150 fmol/mg protein). The specificity of receptors for insulin was also confirmed by showing that [125I]insulin was displaced by non-radioactive insulin but not by glucagon or growth hormone. Insulin displayed a clear dose-dependent stimulation of thymidine incorporation. It also stimulated the activity of the enzyme 2', 3'-cyclic nucleotide phosphohydrolase (CNPase), which is specifically associated with myelin produced from oligodendroglia. Thus insulin has a positive influence on the proliferation and differentiation of brain cells.

Studies of the in vitro effect of methylmercury chloride on rat brain neurotransmitter enzymes.

The in vitro effect of methylmercury (MM) on the enzymatic activities of brain cell specific marker enzymes, choline acetyltransferase (CAT), glutamic acid decarboxylase (GAD), 2',3'-cyclic nucleotide phosphohydrolase (CNP), glutamine synthetase (GS) and enolase was examined. The results demonstrate that at 100 microM MM, GS activity was not affected whereas a small decrease in the activity of both GAD (20%) and enolase (10%) was observed. CNP and CAT activity appeared to be more sensitive toward MM with 100 microM MM producing inhibition of 50% and 30%, respectively. The addition of sulfhydryl protecting reagents such as DTT or sodium thioglycolate can restore the enzyme activities to normal control levels despite prior exposure of the enzymes to MM.

Effects of neuron-conditioned medium and fetal calf serum content on glial growth in dissociated cultures

The influence of the microenvironment as assessed by medium conditioned by 6-day-old chick embryo neurons in culture and of the nutrients derived from fetal bovine serum was evaluated in cultures of primary chick embryo glial cells. Glia-enriched cultures from 15-day-old chick embryo were incubated from culture days 3–9 with various concentrations of neuron-conditioned medium, with or without 10% fetal bovine serum in the final culture medium. Also, glial growth was studied in cultures with 5%, 10% or 20% fetal bovine serum in the medium. Glutamine synthetase and 2′,3′,-cyclic nucleotide 3′-phosphohydrolase were used as astrocytic and oligodendrocytic markers, respectively. Cultures were harvested at day 9. The presence of neuron-conditioned medium in the cultures was associated with persistence of immature glioblast-like cells. This persistence of glial immature cells was also reflected by the lower glutamine synthetase activity in the cultures with neuron-conditioned medium as compared to cultures with neuron-conditioned medium and fetal calf serum. In cultures with 5% neuron-conditioned medium without fetal bovine serum, cyclic nucleotide phosphohydrolase activity was increased. We are assuming that the input of neurons to the microenvironment is partially mediated through the neuron-conditioned medium. Thus, the present findings show that neurons influence the growth and differentiation of glial cells in culture.

The absence of differentiation-promoting response of astroglioma cells to glia maturation factor

The effects of glia maturation factor (GMF) on cell proliferation and differentiation were investigated with 3 astroglioma cells (GE-12, C6, and GA-1), Schwannoma-like cells (354A), and mixed glioma cells (LRM-55). In the exponentially growing phase the growth rates of all glioma cells were enhanced by GMF regardless of the presence or absence of serum, but the factor failed to make the saturation density surpass the control level observed in the medium without GMF even in the chemically defined medium (N2 medium). GMF markedly lowered the saturation density of Schwannoma-like cells in N2 medium. Although GMF increased the intracellular content of S-100 protein 10-fold and 2′,3′-cyclic nucleotide phosphohydrolase activity 1.5-fold in Schwannoma-like cells, GMF conversely decreased the S-100 contents and glycerol phosphate dehydrogenase activity in astroglioma cells. All the astroglioma cells secreted into the culture medium large quantities of a growth-promoting factor(s) which had similar chemical properties to those of GMF and stimulated the proliferation of normal glioblasts; but Schwannoma-like cells did not, although they produced a small amount of such a factor(s). These findings imply that astroglioma cells are deprived of the differentiation-promoting response to GMF while Schwannoma-like cells still preserve the response in addition to the proliferative response to GMF.

Two independent mechanisms of induction of 2′:3′-cyclic-nucleotide 3′-phosphohydrolase in glioma cells by cyclic AMP and high cell density

Specific activity of the myelin enzyme, 2′:3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37), increases 2- to 10-fold when sparsely inoculated cultures of C 6 rat glioma cells are allowed to grow to high cell density. Cyclic-nucleotide phosphohydrolase specific activity is also induced in C 6 cells and in oligodendrocytes by dibutyryl cyclic AMP or by agents that elevate intracellular cyclic AMP. In this report, we have compared the density-dependent induction of cyclic-nucleotide phosphohydrolase activity with the cyclic AMP-dependent induction. Dibutyryl cyclic AMP induced cyclic-nucleotide phosphohydrolase specific activity in both sparse and dense cultures which had very different density-dependent cyclic-nucleotide phosphohydrolase activities. Induction of both cyclic-nucleotide phosphohydrolase specific activity and intracellular cyclic AMP content by norepinephrine also occurred to a similar degree in sparse and dense cultures. Similar results were obtained for several clones of C 6 cells, and for a clone of oligodendrocyte x C 6 cell hybrids. Induction of cyclic-nucleotide phosphohydrolase by norepinephrine or dibutyryl cyclic AMP was not due to a change in cell density or rate of cell proliferation, nor did cell density have any appreciable effect on cyclic AMP content of the cells. These results show that regulation of cyclic-nucleotide phosphohydrolase activity in C 6 cells involves two distinct mechanisms.

Biochemical changes of rat brain membranes with aging.

Modification of membrane composition and enzymatic activities both in total brain homogenate and purified synaptic plasma membrane of 3 and 24 month old rats has been investigated. Protein, cholesterol and phospholipid content and (Na+, K+)ATPase and 2',3' cyclic nucleotide phosphohydrolase activities were determined. The major changes occurred in the whole homogenate where a general increase in total protein and cholesterol content with age and a significant increase of the cholesterol/phospholipids molar ratio has been detected. In S.P.M. aging process induced a decrease of protein, cholesterol and phospholipids content associated with an increased membrane viscosity and a decrease of delta E. These data are consistent with a change in the structural organization and in the distribution pattern of different cell population in the aging brain. A possible artifactual effect of freezing on the reported parameter is also discussed.

Regional effects of hypothyroidism on 5′-nucleotidase and cyclic nucleotide phosphohydrolase activities in developing rat brain

The activities of 5′-nucleotidase (EC 3.1.3.5) and of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) (EC 3.1.4.37) were measured in crude membrane preparations from 6 brain regions of hypothyroid rats 31 days and 42 days after conception (8–9 days and 19–20 days after birth respectively) and from normal rats 31, 36 and 42 days after conception. At 42 days the activities of both enzymes were markedly and significantly lower than in controls in all hypothyroid brain regions. The pattern of increase in CNP activity in the brain regions of normal animals reflected the caudo-rostral progress of myelination. At 42 days postconception in the more caudal regions of hypothyroid brain, myelination, as assessed by CNP activity, was delayed by 5–7 days. In the more rostral regions where normal activity had not yet developed, the delay due to hypothyroidism was not clearly defined. The lowered activity of 5′-nucleotidase observed using crude membrane preparations from hypothyroid animals was shown not to be due to a redistribution of enzyme between cytosol and membranes or to losses into the supernatant fraction during preparation of the particulate fraction used for assays. In addition the lowered activity of 5′-nucleotidase could not be accounted for in terms of the delayed myelination and consequent absence of myelin-associated enzyme. In contrast to the results with CNP, the effects of hypothyroidism on 5′-nucleotidase in the 6 regions showed no caudo-rostral pattern. Although it was clear that the lowered activity of 5′-nucleotidase in hypothyroid brain at 20 postnatal days was not directly attributable to the state of delayed myelination, no identification of the structures or cell types affected could be made.

Effects of methadone on ornithine decarboxylase and cyclic nucleotide phosphohydrolase in neuronal and glial cell cultures.

Mixed neuronal and nonneuronal cell cultures were obtained from 8-day-old chick embryos cerebral hemispheres and glial-enriched cultures were obtained from fifteen-day-old chick embryo cerebral hemispheres. Cultures were exposed to methadone, a narcotic drug, from days four to six. The activity of ornithine decarboxylase (ODC) was determined at day eight and the activity of cyclic nucleotide phosphohydrolase (CNP) was determined at day fifteen. Both ODC and CNP activity were higher in mixed neuronal-nonneuronal cell cultures treated with methadone as compared to control. No effect was observed in the neuronal-enriched or glial-enriched cultures. These findings are interpreted to reflect that neuronal-glial interaction is important in the response of primary neural cells to methadone.

Sulfatide Synthesis by Neural Cell Lines

Several neural cell lines were examined for their ability to synthesize sulfatide and 2',3'-cyclic nucleotide phosphohydrolase, biochemical components characteristic of myelin. The mouse glioma G26 and the rat schwannoma TRM6B actively produced sulfatide, while the rat glioma C6 was inactive, supporting the probable oligodendroglial origin of the G26. In contrast, the C6 cell line had a high level of 2'-3'-cyclic nucleotide phosphohydrolase activity, while TRM6B showed 30% and the G26 75% lower activities. Thus, these two activities appear to be independently regulated.

Isolation of glial cell-enriched and -depleted populations from mouse cerebellum by density gradient centrifugation and electronic cell sorting

Preparative amounts of populations enriched and depleted in glial cells have been isolated from 10-day-old mouse cerebella. Discontinuous density bovine serum albumin (BSA) gradients provide two distinct populations: the one derived from the 10-15% BSA interface is enriched in cyclic nucleotide phosphohydrolase (CNPase) activity, and a percentage of S-100, GFA protein, and NS-1 antigen-positive cells; the other, located as the 15-31% interface, contains a cell population depleted in these compounds. This report also describes the first use of flow microfluorimetry and electronic cell sorting techniques for the analysis and isolation of cell populations derived from the mammalian central nervous system. Glial cell-enriched and -depleted populations obtained by BSA density gradient centrifugation were analyzed for their forward angle light scattering properties and their capacity to bind anti-corpus anti-serum to the cell surface. The glial cell-enriched fraction shows a different frequency distribution of light scattering than the glial-depleted fraction. Anti-corpus callosum antiserum binds preferentially to the glial cell-rich fraction. Cells can be sorted into corpus callosum antigen-positive and -negative cell fractions and recovered with a viability of more than 95% as judged by trypan blue exclusion. Anti-corpus callosum-positive sorted cells are enriched in CNPase activity, S-100, and glial fibrillary acidic proteins.

In vitro protein synthesis by oligodendroglial cells

Oligodendroglial cells were isolated from spinal cord and brain stem tissue from 30-day-old rats according to the method of Poduslo and Norton except that acetylated trypsin at a concentration of 0.05% was used instead of unmodified trypsin, and the density gradient was slightly changed. Isolated oligodendroglial cells were found to incorporate [U- 14C]leucine into TCA-insoluble protein up to 120 min. This incorporation was inhibited by cycloheximide and chloramphenicol. Purity of the cell preparation was assessed by phase contrast microscopy and by assay of 2′,3′-cyclic nucleotide phosphohydrolase activity.