Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells

Freshly isolated neonatal porcine aortic tissue (smooth muscle with or without endothelium present) produced approximately 30 ng/mg wet tissue of 6-oxo-prostaglandin F1 alpha (the stable hydrolysis product from prostacyclin) and approximately 15 ng/mg of prostaglandin E2, as measured by radioimmunoassay after 24 h incubation in culture medium. Primary cultures of porcine endothelial and smooth muscle cells (isolated by enzymic digestion of aortic tissue) exhibited the same pattern of prostaglandin production, but absolute values were greater than for fresh tissue, particularly in the case of endothelium. Subcultures of endothelium produced smaller amounts of prostaglandins, although the pattern remained similar. In contrast, subcultures of smooth muscle cells produced a greater total amount of prostaglandins than did primary cultures, and the main product was prostaglandin E2. Experiments with [14C] prostaglandin H2 or [14C]arachidonic acid confirmed that aortic tissue, cultured endothelium, and primary cultures or aortic smooth muscle cells synthesized prostacyclin, and demonstrated that subcultured smooth muscle cells enzymically isomerised prostaglandin H2 to prostaglandin E2. Kinetic studies showed that prostaglandin production by cultured vascular cells was transiently increased by subculture or changing the growth medium, and that production per cell declined with increasing cell density. The change in pattern of prostaglandin production during culture was shown to be due to a rapid decline in the rate of prostacyclin production (which apparently began immediately after tissue isolation), together with a more gradual rise in prostaglandin E2 production. These results indicate that the amounts and ratios of prostaglandins produced by vascular endothelial and smooth muscle cells are greatly affected by the conditions used to isolate and culture the cells; vascular cells in vivo may similarly alter their pattern of prostaglandin production in response to local changes in their environment.     

Continuous cell propagation using low-charge microcarriers

Microcarrier cell culture technology has been extended by the finding that two mammalian epithelial cell lines can be continuously subcultured by simple bead-to-bead transfer in normal medium in which calcium concentrations have been reduced. Data are reported which show that the hamster ovary line CHO-Kl and the monkey kidney line LLC-MK₂ can be subcultured simply by adding fresh microcarriers to the stirred suspension culture. Thirteen generations of continuous exponential growth are demonstrated with two such subcultures for the CHO-Kl cells and with four such subcultures for the LLC-MK₂ cells. Cell generation times were unchanged by this subculturing approach compared to standard subculturing procedure using trypsin to remove cells from surfaces. We have applied this technique to the production of vesicular stomatitis virus (VSV) from CHO-Kl cells. Viral yields were comparable (less than twofold difference) in microcarrier cultures which were subcultured via bead-to-bead transfer or by the standard means of removing cells from microcarriers with trypsin.     

Characteristics of arachidonic acid metabolism of human endothelial cells in culture

Human umbilical endothelial cells in culture retain differentiated morphological and functional characterization in primary culture and even in the early subcultures, after which they begin to degenerate. We have studied the morphological and biochemical characterization (ability to produce prostacyclin, prostaglandin E₂ and thromboxane A₂ in culture) of endothelial cells in the first seven subcultures. In addition the influence of serum and endothelial cell growth factor added to the culture medium have been evaluated. With 20% normal human serum, cell proliferation is faster than with the same concentration of human fetal or bovine fetal serum.After the 3rd passage, morphological and growth alterations become observable in the endothelial cells. However, prostacyclin, prostaglandin E₂ and thromboxane A₂ production showed no variations during the study.     

Interleukin-1 beta stimulates decidual stromal cell cyclo-oxygenase enzyme and prostaglandin production.

The cytokine interleukin-1 (IL-1 beta) increased prostaglandin production by decidual stromal cells in culture in a time and dose dependent manner. Optimum conditions for stimulation were found to be for 24 hours at a concentration of 100 pg IL-1 beta/ml. An apparent increase in cyclo-oxygenase enzyme synthesis accompanied the increase in prostaglandin production, and both changes were inhibited by the protein synthesis inhibitor cycloheximide. This implicates protein synthesis in the stimulatory effects of IL-1 beta, which may be mediated through the increase in cyclo-oxygenase enzyme. A pre-incubation period of 72 hours was found to be necessary to observe the stimulatory effect of IL-1 beta on prostaglandin production, but this did not seem to be due to any change in the sensitivity of the cells to IL-1 beta; the increase in the number of cyclo-oxygenase positive cells was the same if IL-1 beta was added on day 1, day 2 or day 3 of culture, even though prostaglandin production was not stimulated on day 1 or day 2. Cycloheximide increased prostaglandin production on the first two days of culture and had no effect on the third day of culture. This was interpreted as indicating that a factor inhibiting cyclo-oxygenase activity was synthesised during the initial period of culture, which prevented any increase in prostaglandin production following the increase in enzyme synthesis.     

Establishment of an in vitro culture system for chicken preblastodermal cells

To develop an alternative source for chicken pluripotent cells, we examined (1) whether undifferentiated preblastodermal cells could be subcultured in vitro for an extended period and (2) how subculturing affected the physiological properties of preblastodermal cells. The average number of preblastodermal cells was 2,397 in stage V embryos and 36,345 in stage VII embryos; stage X embryos had an average of 53,857 blastodermal cells. The average cell size decreased significantly (70.63-18.83 microm in diameter; P < 0.0001) as the embryo grew; this was closely related to a reduction in the size and number of lipid vesicles in the cell cytoplasm. The culture conditions were optimized for the stage V preblastodermal cells and the control stage X blastodermal cells. On STO feeder cells, the preblastodermal cells achieved stable growth in vitro only in HES medium or a mixed medium of the Knockout DMEM and HES media. However, more than 10 passages of preblastodermal cells at intervals of 3-4 days was possible only by using the Knockout/HES mixed medium and BRL cell-conditioned HES medium for the primary cultures and subcultures, respectively. Colony-forming preblastodermal cells had well-delineated cytoplasm, which was positively stained for stem cell-specific markers by anti-stage-specific embryo antigen-1 antibody, periodic acid-Schiff's solution, and alkaline phosphatase. When preblastodermal cells with or without culturing were transferred into the blastodermal cavity of stage X embryos, only in vitro-cultured preblastodermal cells at stage V (4/5 = 80%) and stage VII (2/8 = 25%) induced somatic chimerism in recipient chickens. In conclusion, undifferentiated preblastodermal cells could be subcultured, and only the colony-forming preblastodermal cells that stained positively for stem cell markers could induce somatic chimerism.     

Regulation of prostaglandin H synthase activity in dog urothelial cells.

TPA regulation of prostaglandin H synthase activity in primary and subcultured dog urothelial cells was investigated. Previous studies have demonstrated an early (0-2 hr) increase in PGE2 synthesis mediated by TPA which is dependent upon release of endogenous arachidonic acid by a phospholipase-mediated pathway. In this study, prostaglandin H synthase activity was assessed directly with microsomes and indirectly after addition of exogenous arachidonic acid at a maximum effective concentration (100 microM) to media. PGE2 synthesis, measured by radioimmunoassay, served as an index of prostaglandin H synthase activity. After a 24-hr incubation with 0.1 microM TPA or 1.0 microM A23187, arachidonic acid elicited significantly more PGE2 synthesis in agonist-treated cells than it did in control cells in primary culture. Microsomes from 24-hr TPA-treated cells exhibited significantly more prostaglandin H synthase activity than did those from control cells. In addition, the PGE2 content of overnight media was approximately 10-fold greater in TPA-treated cells than in control cells. The late (24 hr) response was more sensitive to lower concentrations of TPA than was the earlier (0-2 hr) response. TPA at 0.1 microM was a maximum effective dose for both responses. The 24-hr response was blocked by cycloheximide and staurosporine, inhibitors of protein synthesis and protein kinase C, respectively. Pretreatment of cells with aspirin, an irreversible inhibitor of prostaglandin H synthase, prior to addition of TPA did not prevent the late TPA-mediated increase in PGE2 synthesis. Subcultured cells exhibited both an early and a late TPA response. Only the early response was inhibited by aspirin pretreatment. Results suggest that the late response with TPA is caused by de novo synthesis of prostaglandin H synthase. Thus, primary and subcultured dog urothelial cells possess two distinct mechanisms for regulating signal transduction by arachidonic acid metabolism. This study provides a basis for assessing these mechanisms of signal transduction in urothelial cell lines and transformed cells.     

Expression of inducible nitric oxide synthase in human umbilical vein endothelial cells during primary culture

The adaptive response of endothelial cells to stress may lead to the upregulation of nitric oxide (NO) production. Herein, we report inducible nitric oxide synthase (iNOS) induction in primary cultures of human umbilical vein endothelial cells (HUVEC). The enzyme expression was earlier observed in 12-h cultures, reaching maximal levels after 3 days and decreasing when cells become confluent. The time course of NO production by HUVEC paralleled iNOS expression during the whole culture period, indicating that enzyme was functionally active. Conversely, iNOS induction could not be further detected in HUVEC subcultures passed once from cells presenting maximal levels of iNOS expression in the primary culture. Induction of iNOS in HUVEC was not related to lipopolysaccharide contamination, since the enzyme expression was not affected in the presence of polymyxin B added to primary cultures. Further analysis showed that aminoguanidine, a specific iNOS inhibitor, did not affect cell proliferation, suggesting that the NO produced by HUVEC may not be directly related to cell growth. Platelet endothelial cell adhesion molecule-1 expression was upregulated during cell confluence, in contrast to the decrease of iNOS expression and activity. The data suggest that iNOS expression may be a molecular mechanism mediating the adaptive response of endothelial cells to culture environment.     

The influence of cell population density and serum on the (Na+K)-ATPase of 3T3 cells

The (Na+K)-ATPase of 3T3 cells has been measured as a function of culture density and the type of serum in which the cells were grown. When 3T3 cells were grown in medium containing 10% newborn calf serum their (Na+K)-ATPase activity increased as the culture density increased and the cells became quiescent at densities of about 17.5 X 10(4) cells/cm2. When these cells are subcultured into medium containing 10% foetal bovine serum the activity of the enzyme decreases as the culture density increases and the cells attain final densities of about 5 X 10(4) cells/cm2.     

Cell suspension as a tool to study the biosynthesis of pilocarpine in Jaborandi

Jaborandi (Pilocarpus microphyllus) is a species that naturally occurs in the North and Northeast of Brazil, whose leaves produce pilocarpine (an imidazole alkaloid that has been used to treat glaucoma and xerostomy), the biosynthesis of which is still uncertain. The aim of this work was to establish cell lineages and select them according to an alkaloid profile similar to the one from Jaborandi leaves. The induction of callus was done in different culture media and growth regulators. Calluses from primary cultures or those subcultured several times were used as explants for the obtainment of six cell lineages. Alkaloids content analyses and growth curves showed that lines obtained from primary cultures produced more alkaloids and a better development. Cell lines from 12 subcultures presented a decrease in pilocarpine and pilosine production. After 24 subcultures, the production of alkaloids remained constant. ESI-MS analysis showed that cell culture extracts have the same alkaloid composition as extracts made from leaves. The results indicate that cell suspensions can be used as a model to study the biosynthesis of the imidazole alkaloid in P. microphyllus.     

Splenic cultures from rainbow trout,Oncorhynchus mykiss: establishment and characterisation

This study describes the culture conditions and the phenotypic features of different types of splenic cultures established from explants. Using the same culture technique it was possible to grow splenic explants from which monolayers of reticular origin, long-term haematopoietic cultures, and subcultures were obtained. The cultures were characterised by light and electron microscopy, cytochemical and immuno-cytochemical analyses, phagocytic activity and susceptibility to virus. The cultures comprised multilayers of epithelioid and fibroblastoid cells with haemopoietic foci, melanomacrophages and eosinophilic granular cells. The cytochemical and immuno-cytochemical analyses revealed that the stromal cells were always positive for ANAE activity. The stromal cells in primary cultures were negatively or weakly stained by antibodies directed against cytokeratins and S-100, but in the subcultures they were strongly stained by these antibodies. The stromal cells had very poor phagocytic activity and were susceptible to VHS virus.     

On the metabolism of prostaglandins by human gastric fundus mucosa.

1.Specific radioimmunoassays for the prostaglandins E2, F2alpha and A2 and the metabolites 13,14-dihydro-15-keto-prostaglandin E2, 15-keto-prostaglandin F2alpha and 13,14-dihydro-15-keto-prostaglandin F2alpha were used to study the metabolism of prostaglandins by gastroscopically obtained small biopsy specimens of human gastric fundus mucosa. 2.Three prostaglandin-metabolizing enzymes were found in the 100 000 X g supernatant of human gastric fundus mucosa, 15-hydroxy-prostaglandin-dehydrogenase, delta13-reductase and delta9-reductase. The specific activity was highest for 15-hydroxy-prostaglandin-dehydrogenase and lowest for delta9-reductase. 3.Formation of prostaglandin A2 (or B2) was not observed under the same conditions. 4.None of the three enzyme activities detected in the 100 000 X g supernatant was found in the 10 000 X g and 100 000 X g pellets of human gastric fundus mucosa. 5.The results indicate that high speed supernatant derived from human gastric mucosa can rapidly metabolize prostaglandin E2 and prostaglandin F2alpha to the 15-keto and 13,14-dihydro-15-keto-derivatives. Furthermore, prostaglandin E2 can be converted to prostaglandin F2alpha, the biological activity of which, on gastric functions, differs from that of prostaglandin E2.     

Increasing GPI-anchored protein harvest concentrations from suspension and porous microcarrier CHO cell cultures

Chinese hamster ovary (CHO) cells expressing the human melanoma tumour antigen, p97, were used to develop a controlled release process for the production of recombinant glycosyl-phosphatidylinositol (GPI) anchored proteins. The cells were cultured either in suspension or immobilized on porous microcarriers and p97 was selectively cleaved from the cell surface by the bacterial enzyme, phosphatidylinositol-phospholipase C (PI-PLC). The kinetics of p97 cleavage from the cell surface by PI-PLC was shown to be approximated by Michaelis-Menten kinetics. The recovered p97 concentrations were increased by reusing the PI-PLC enzyme solution to harvest multiple batches of cells. A convenient PI-PLC assay was developed to monitor the harvesting process and to determine the stability of PI-PLC under harvesting conditions. Although the Pl-PLC was stable under harvesting conditions, it rapidly adsorbed to the cell surface and was depleted from the reused enzyme solution. In order to maintain PI-PLC activity, it was necessary to add fresh PI-PLC to the reused enzyme solution before harvesting a fresh batch of cells. The maximum p97 concentration that could be obtained from harvesting CHO cells cultured on porous microcarriers was limited by the dilution effects of sample removal, adding fresh PI-PLC and liquid associated with settled microcarriers. A model was developed that adequately predicted the p97 concentration after each harvest and the maximum p97 concentration that could be achieved by this harvesting method. The dilution effects were minimized by harvesting from centrifuged suspension culture cells and the harvested p97 concentration was increased by over sixfold to 0.64 mg/mL. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 136-147, 1997.     

Specific inhibition by prostaglandin D2 and its metabolites of lysozyme synthesis in mouse macrophage-like cell line, Mm-1

The cultured mouse macrophage-like cell line Mm-1 synthesizes and secretes lysozyme continuously like normal macrophages. Culture of the cells in the presence of prostaglandin D2 for 3 days strongly inhibited their production of lysozyme activity. Prostaglandin D2 caused dose-dependent inhibition of the activity: 1 X 10(-6) M prostaglandin D2 caused about 50% inhibition. Inhibition by prostaglandin D2 was not related to cytotoxicity and was reversible. The rate of synthesis of lysozyme protein was measured by culturing Mm-1 cells with radioactive amino acids and then immunoprecipitating the protein. At the concentrations used, prostaglandin D2 inhibited the synthesis of lysozyme dose-dependently, but did not suppress the synthesis of total protein. Of the various types of prostaglandin, only prostaglandin D2 inhibited the production of lysozyme in Mm-1 cells. Moreover, prostaglandin D2 did not inhibit the production of other lysosomal enzymes, such as acid proteinase, acid phosphatase and beta-glucuronidase, and did not affect Fc receptors on the cell surface, adherence of cells to the culture dish or the cell morphology. These results indicate that prostaglandin D2 specifically inhibits the synthesis of lysozyme in Mm-1 cells. When Mm-1 cells were cultured for 3 days in the presence of the ethyl acetate extract from the culture medium in which Mm-1 cells had been cultured with prostaglandin D2 for 3 days, the production of lysozyme activity of Mm-1 cells was also markedly inhibited by the extract. After the incubation of prostaglandin D2 for 3 days with Mm-1 cells, less than 10% of the initial prostaglandin D2 remained and two major metabolites appeared. These results suggest that the metabolites of prostaglandin D2 were involved in the inhibitory action of prostaglandin D2 in Mm-1 cells.     

Glucocorticosteroid regulation of prostaglandin biosynthesis in human myometrial smooth muscle cells in monolayer culture

In the present investigation, we evaluated the production of prostaglandins by human myometrial smooth muscle cells maintained in monolayer culture in the absence or presence of glucocorticosteroids. In the presence of cortisol (10(-7) M) or dexamethasone (10(-8) M), the rate of production of prostacyclin (PGI2) by these cells was decreased significantly. The glucocorticosteroid-mediated inhibition of prostaglandin production was attenuated when cortisol-21-mesylate (10(-6) M), a glucocorticosteroid antagonist, was present in the culture medium. The rate of conversion of radiolabeled arachidonic acid to radiolabeled prostaglandins as determined by use of sonicates of myometrial cells and optimal assay conditions, however, was not affected significantly by treatment with cortisol or dexamethasone in concentrations sufficient to inhibit prostaglandin formation by more than 80%. These findings are suggestive that glucocorticosteroids act in human myometrial smooth muscle cells in culture to inhibit prostaglandin formation by way of a receptor-mediated process that does not involve inhibition of enzyme activities that are involved in the biosynthesis of prostaglandins, i.e. the conversion of arachidonic acid to prostaglandin.     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.     

Isolation and characterization of guinea-pig tracheal smooth muscle cells that retain differentiated function in long-term subculture

Summary A simple 30-min enzyme digestion procedure has been used to release guinea-pig tracheal smooth muscle cells that retain differentiated function in long-term subculture. Primary cell cultures initially consist of numerous epithelial colonies and 70–1000 morphologically differentiated smooth muscle cells per 600 mg (wet weight) tracheal tissue depending on the age of the animal. Both cell types proliferate to form a confluent monolayer within 5–17 days. Pure subcultures of tracheal smooth muscle cells are obtained by limited trypsin digestion of the primary culture. Eighty percent of these subcultured smooth muscle cells retain the ability to contract in response to histamine (10^-6 M) and to form reaggregates even after 20 or more passages. Examination of these cells by electron microscopy reveals both biosynthetic and contractile components of smooth muscle. Analysis of this dual phenotype may provide valuable information about the regulation of tracheal smooth muscle cell growth and differentiation.This research was supported in part by a grant from the Foundation for Research in Bronchial Asthma and Related Diseases, Worcester, Massachusetts 01604, USA     

Evolution of cAMP phosphodiesterase activity in cultured myometrial cells: Effects of steroids and of successive subcultures

Cyclic AMP phosphodiesterase (PDE) activity was characterized in culture of ewe myometrial cells and its sensitivity to steroid hormones was tested. Cultured myometrial cells were maintained from the first to the 20th subculture in the presence of 2% of serum in a medium supplemented with 1 μM of insulin. It was found that myometrial cells possess a PDE activity with atypical kinetics. The nonlinear responses in Lineweaver-Burke plots suggest the presence of high- and low-affinity PDE activities. In cell culture, apparent Km values were similar to those obtained from the original myometrium. Vmax values increased with successive subcultures, revealing an increase in the capacity of the cells to degrade cAMP; in parallel, the growth rate decreased. The PDE specific activity in cultured myometrial cells was inhibited by estra-diol or progesterone. When added together, no synergistic effect was obtained. The rate of inhibition for both steroids was constant during successive passages for both low- and high-affinity conditions. Results obtained in myometrial cell long-term culture were compatible with reports in other species in vivo. Considering the role of cAMP in the regulation of uterine functions, subcultured myometrial cells provided us a useful experimental system with which to study the cAMP metabolism process.     

Serum-free culture of stromal and functionally polarized epithelial cells of guinea-pig endometrium: a potential model for the study of epithelial-stromal paracrine interactions.

Stromal and glandular epithelial (GE) cells were isolated from guinea-pig endometrium and growth to near confluency (6-8 days) in primary culture on plastic surfaces in a serum-supplemented medium (SSM). The stromal cells were subcultured on plastic dishes and maintained for 72 h in SSM. Then SSM was replaced by a chemically defined medium (CDM) and the stromal cells grown to confluency (5-7 days). The GE cells were subcultured in CDM, on a basement membrane matrix (Matrigel) applied to permeable Millicell-PC filters, and grown to confluency (5 days). Homogeneity of the subcultured endometrial cell populations was ascertained immunocytochemically. The filter-cultured GE monolayers were polarized morphologically, and displayed epithelial-specific specialized structures. These monolayers had functional tight junctions as verified by a measurable transepithelial resistance. The subcultured cell populations were distinguished by an analysis of their cellular and secretory proteins after labelling with [35S]-methionine and analysis by polyacrylamide gel electrophoresis. The filter-cultured GE monolayers allowed identification of the proteins released vectorially in the apical or the basal secretory compartment, thus demonstrating the functional polarization of GE cells in this bicameral culture system. Within the defined conditions of this culture system, the paracrine factors released by the two endometrial cell populations as well as the interplay of stromal-epithelial interactions and ovarian hormones could be investigated.     

Normal rat kidney proximal tubule cells in primary and multiple subcultures

Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.     

Influence of culture conditions on monoamine oxidase A and B activity in rat astrocytes

Astroglial cells dispersed from newborn rat hemispheres were established in medium supplemented with 20 per cent fetal calf serum (FBS) and then grown to a confluent monolayer in the presence of 10 per cent FBS or charcoal-stripped FBS (CS). Type 1 astrocytes were subcultured and either maintained under the same conditions of the primary cultures or converted to serum-free chemically defined medium (CDM). No differences were found in either MAO A or MAO B activity of astrocytes grown in the presence of FBS or CS after 15 and 21 days in vitro (day 1 and 6 of subculture). In contrast, on day 21 both MAO A and MAO B activities were markedly higher in astrocytes subcultured in CDM compared with cells maintained in serum-supplemented medium. This difference appeared to be due to increased number of enzyme molecules, since kinetic analysis showed an increase in V_max of both MAO isoenzymes in serum-free medium, but no change in K_m. Consistently, the recovery of MAO A and MAO B activity after irreversible enzyme inhibition by clorgyline and deprenyl was faster in CDM than in FBS-supplemented medium, indicating enhanced enzyme synthesis under serum-free condition. Estimates of half-lives for the recovery of MAO A and MAO B activity indicated that, under both culture conditions, type A activity had a higher turnover rate than type B. The effect of CDM on astrocyte MAO does not appear to be due to selection of a subpopulation of cells, but rather linked to a morphological change (differentiation) with increased synthesis of both MAO isoenzymes.