A Comparison of the Polypeptide Pattern Between a Brain Primary Culture and Fractions of Bulk-Isolated Glial Cells

Abstract: This paper reports on the electrophoretic protein/polypeptide pattern of a rat brain primary culture. For comparison, the polypeptide pattern of neuronal and glial enriched fractions from adult rat brain and cerebral hemispheres from newborn and adult rat have been analysed. Water-soluble and SDS-extractable polypeptide fractions appeared and/or increased in amount in the cultures until confluency. The polypeptide pattern of the cultures most resembled that of the glial cell fractions, showing some of this fraction's specificity. Removal of fetal calf serum and addition of 0.1 mM dibutyryl cyclic adenosine monophosphate (dB-cAMP) produced few changes in the electrophoretic pattern. The study thus provides evidence in favour of the astroglial nature of the brain primary culture. It also shows that the cells undergo some maturation in the culture.     

Enzyme activities of monoamine oxidase,cathechol-o-methyltransferase and γ-aminoubutyric acid transaminase in primary astroglial cultures and adult rat brain from different brain regions

The activities of monoamine oxidase (MAO), cathechol-O-methyltransferase (COMT) and -aminobutyric acid transaminase (GABA-T) were measured in primary cultures from newborn rat cultivated from 6 different brain regions. These primary cultures contained mostly astroglial cells, evaluated by the presence of the glial fibrillary acidic protein (GFAp, -albumin) and the S-100 protein. The enzyme activities in the corresponding brain areas from adult rat were also quantified. MAO activities were on the same level in 14-day old cultures and in adult rat brain homogenates, with significantly lower values in brain stem as compared to the other brain regions examined. COMT activities were on a higher level in the cultures than in adult rat brain homogenates. Astroglial cells from hippocampus were found to have the highest and those from brain stem the lowest COMT-activities. GABA-T activities were lower in the cultures than in adult rat homogenates. No significant differences were seen in the various astroglial cultures. Accumulation of [³H]dopamine and [³H]-aminobutyric acid (GABA) visualized by autoradiography showed only a slight uptake of dopamine in comparison with the uptake of GABA. It is concluded that astroglial cells in culture have enzymatic properties similar to those of astroglial cells in different brain regions of adult rat brain. Studies are in progress to evaluate if the regional heterogeneity observed among cultivated astroglial cells is affected by in vivo differentiation until cultivation and/or time in culture.     

THE EFFECTS OF METOPIRONE AND ADRENALECTOMY ON THE REGULATION OF THE ENZYMES MONOAMINE OXIDASE AND CATECHOL-O-METHYL TRANSFERASE IN DIFFERENT BRAIN REGIONS

Abstract— The role of glucocorticoids in the regulation of the enzymes monoamine oxidase (MAO) and catechol- O -methyltransferase (COMT) in brain regions has been studied. Glucocorticoids were blocked by Metopirone. The activities of MAO and COMT were determined in the hypophysis, hypothalamus, pineal gland and in the rest of brain. All the cerebral tissues except the pineal gland demonstrated highest MAO activity 8 h after Metopirone administration, when glucocorticoids were at the lowest level. Prolonged treatment for 10 days significantly augmented MAO activity in brain, hypophysis and hypothalamus, and COMT in the hypophysis increased by 56 per cent. The COMT activity in the rest of the brain did not change significantly with either short or prolonged administration. Complete ablation of the adrenal cortex resulted in a 167 per cent rise in MAO activity of the hypophysis. Metopirone and hydrocortisone inhibit MAO and COMT in vitro. The results suggest that glucocorticoids in the circulation of normal animals inhibit the activities of MAO and COMT. The inhibition or ablation of these hormones removes this rate-limiting control of catecholamine degradation resulting in higher activities of MAO and COMT. Metopirone, an inhibitor of MAO and COMT in vitro , acts in the opposite direction in vivo due to its inhibitory effects on corticoid biosynthesis.     

Stimulation of Ornithine Decarboxylase Activity in Neural Cell Culture: Potential Role of Insulin

Abstract: Age-dependent decreases in the levels of ornithine decarboxylase activity were observed in the optic lobes, cerebral hemispheres, and midbrain-diencephalon of 6–17-day-old chick embryos. In dissociated cell cultures from chick embryonic brains a similar pattern of declining ornithine decarboxylase activity with time in culture was observed. Ornithine decarboxylase activity in the dissociated brain cell cultures was stimulated by changing the culture medium. The peak stimulatory effect was shown to occur 12 h after changing the medium. Although serum-free medium stimulated ornithine decarboxylase activity slightly, the presence of serum in the medium was the primary stimulatory factor. Both fetal calf serum and heat-inactivated fetal calf serum produced dose-dependent stimulation of ornithine decarboxylase activity. Dialyzed fetal calf sera stimulated ornithine decarboxylase, but to a lower level than that produced by nondialyzed sera. Insulin (0.5–10 μg/ml) stimulated ornithine decarboxylase activity in a dose-dependent manner in serum free medium. In addition, 10² M-L-asparagine stimulated ornithine decarboxylase activity in serum-free medium.     

THE POSTNATAL DEVELOPMENT OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Abstract— The postnatal development of three enzymes in the rat forebrain was studied. When expressed per tissue weight the catechol- O -methyl transferase (COMT) increased 2-fold from birth to adult age, the lactate dehydrogenase (LDH) 4-fold and the monoamine oxidase (MAO) 12-fold. Expressed per mg protein the increase in the enzyme activities in the subcellular fractions which contained the main part of the different enzymes was still 2–4-fold for COMT and LDH while for MAO it was 4-fold.
There was a relative increase in the COMT activity in the P₂ fraction (synaptosomes and mitochondria). This increase was identical with a corresponding increase in LDH activity and protein and was probably due to growth of nerve terminals. The COMT in the cytoplasm of the synaptosomes showed the same increase relative to the proteins as did the 'free' cytoplasmic enzyme.
The conclusion is drawn that the enzymes in the rat brain show a certain degree of development during brain growth. An additional increase of some enzymes is due to the development of specialized structures such as mitochondria and nerve terminals with synapses. COMT is not related to any such specialized structure.     

Nutrition and fetal brain maturation : I. Responses in vitro and in vivo

The glycolytic enzyme enolase increases during the perinatal period of brain development and was utilized as a marker for examining the effect of culture environment on differentiation of cells from 20-day fetal rat brain. Enolase activity in cell cultures increased from 0.91 +/- 0.03 (Day 0) to 2.11 +/- 0.10 mumol/min/mg protein (Day 6). Comparable levels were not reached in vivo until neonatal pups were 15 days old. The in vitro increase was inhibited by both cycloheximide and actinomycin D. Enolase activity in the cells responded to alterations in both incubation media and homologous serum. After 6 days in culture, cells incubated in rat serum (10%) added to MEM or RPMI produced twice as much enolase activity as cells incubated similarly in Ham's medium, i.e., 1.96 +/- 0.09 and 1.85 +/- 0.21 vs 1.02 +/- 0.09, P less than 0.001. Results of a comparable magnitude were obtained when fetal calf serum replaced adult rat serum, but enolase production was somewhat lower when newborn calf serum replaced adult rat or fetal calf serum. When cells were incubated for 6 days with graded concentrations of adult rat serum (2.5-15%), enolase activity increased progressively. The pattern of enolase response suggests that the fetal rat brain cell model described herein will provide a sensitive probe with which to gain insight into nutrition and fetal brain development.     

Primary astroglial cultures

Conclusion Primary cultures from neonatal rat brain consist mainly of astroglial cells, immunohistochemically identified by GFAp and S-100. As other cells than astrocytes may survice in the culture, specific markers for the expected cells were used. Cells with phagocytic properties, endothelial-like cells, oligoblasts, ependymal cells and mesenchymal cells were identified. No neurons have so far been detected.The astroglial cells have a high-affinity uptake for glutamate, aspartate GABA, taurine and hypotaurine, while there is probably a non-saturable uptake of norepinephrine, dopamine and 5-HT. The enzymes MAO, COMT, GABA-T and GS have been demonstrated. It thus seems that astrocytes take part in the inactivation of neurotransmitters, although amino acids and monoamines are taken up with different mechanisms.The presence of receptors for different neurotransmitters and neuromodulators has been demonstrated on astrocytes.Astroglial-enriched cultures from various brain regions have shown that the cells express specialized functional properties concerning neurotransmitter uptake, metabolizing enzymes and receptor density.Astroglial cell differentiation in culture is shortly reviewed and one possibility to affect this maturation by co-cultivation with neuronal containing cultures is point out.     

Catecholamine-Metabolizing Enzymes of Bovine Brain Microvessel Endothelial Cell Monolayers

The activities of monoamine oxidase (MAO), catechol-O-methyltransferase (COMT), phenol sulfotransferase (PST), alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GT), and angiotensin converting enzyme (ACE) were quantitated in primary cultures of bovine brain microvessel endothelial cell monolayers and cerebral gray matter. Significant MAO-A and -B, cytosolic and membrane-bound COMT, PST, AP, GT, and ACE activities are demonstrated in bovine gray matter. By comparison, enzyme activities of the monolayers vary with the age of the monolayer and are generally higher in complete monolayers. Relative to gray matter enzyme activities, the monolayers are enriched with AP, GT, and ACE, enzymes considered to be markers for brain endothelium. Results also indicate that the activities of MAO-A and PST in the monolayers approach those found in the gray matter. Conversely, cytosolic COMT and MAO-B activities in the monolayers are negligible and much lower, respectively, compared to activities in gray matter. Additional studies with both tissues suggest that the PST of both tissues is the thermostable form of the enzyme.     

The effects of soman in vitro on catechol-O-methyltransferase and monoamine oxidase activities in rabbit tissues

Soman (pinacolyl methylphosphonofluoridate) not only increases acetylcholine levels by inhibiting cholinesterases, it also alters the levels of some other neurotransmitters including norepinephrine, dopamine, and serotonin. Soman also causes an alteration in the activities of the enzymes metabolizing norepinephrine when it is administered to animals. Because these alterations may result from indirect effects on the enzymes, the effects of in vitro application of soman on catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) activities in rabbit tissues were investigated. Enzyme activities were determined in rabbit lung, liver, cerebellum, cerebrum, brain stem, mesenteric artery, pulmonary artery, renal artery, central ear artery, thoracic aorta, and diaphragm. MAO and COMT activities were not affected by soman in any tissues tested, except the lung and liver, where the activity of COMT was increased (p less than 0.05). Thus, reported effects of soman in vivo on norepinephrine, dopamine, or serotonin concentrations, and MAO and COMT activities do not seem to result from direct effects on the activities of these amine-metabolizing enzymes.     

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.     

Nutrition and fetal brain maturation : II. Impact of maternal starvation on changing levels of acetylcholinesterase and enolase in vitro

The impact of maternal starvation during Days 17-20 of gestation was examined in 20-day fetal rat brain tissue cultured for 6 days in MEM and 10% adult rat serum. Acetylcholinesterase (AChE) activities were consistently greater in fetal brain cell cultures from starved mothers. When fetal tissues from starved mothers were continuously exposed to 72-h fasted serum, AChE activities increased from 1.03 +/- 0.14 to 1.59 +/- 0.21 mumol/h/mg protein (P less than 0.001). In fetal tissues from fed mothers, lower AChE activities were increased from 0.78 +/- 0.09 to 1.04 +/- 0.07 mumol/h/mg protein (P less than 0.05) when 72-h fasted serum was used to replace the fed serum during incubation. When fetal brain cell cultures from fed mothers were exposed for 6 days to graded concentrations of fed serum (2.5-15%), the activities of AChE fell reciprocally from 1.34 +/- 0.10 to 0.82 +/- 0.12 mumol/h/mg protein (P less than 0.05). The levels of AChE activity in tissues exposed to fasted serum were consistently greater, but fell similarly from 1.62 +/- 0.10 to 0.97 +/- 14 mumol/h/mg protein (P less than 0.01), when serum concentrations were increased from 2.5 to 15%. AChE activities were 30% higher in tissues incubated with cycloheximide 10(-3) M (P less than 0.02). Unlike AChE, fetal brain enolase activities were unaffected by maternal starvation. In fetal brain cell cultures from fed mothers, enolase fell from 1.85 +/- 0.10 to 1.37 +/- 0.12 mumol/min/mg protein following exposure to fasted instead of fed serum (P less than 0.02). In fetal cultures from starved mothers, enolase activities were depressed similarly from 1.76 +/- 0.08 to 1.41 +/- 0.09 mumol/min/mg protein when fasted replaced fed serum (P less than 0.02). Thus, the fetal brain cell cultures appear to maintain enzymatic realignments imposed by maternal starvation for at least 6 days. In addition, serum from fasted animals has significant growth inhibiting properties manifested by heightened activities of AChE and lower activities of enolase.     

A study of selected catecholamine metabolizing enzymes: a comparison of depressive suicides and alcoholic suicides with controls

—The regional distributions of monamine oxidase (MAO) (EC 1.4.3.4), catechol-O-methyltransferase (COMT) (EC 2.1.1.6), tyrosine hydroxylase (TH) (EC 1.14.3.2), and dopamine-β-hydroxylase (DBH) (EC 1.14.2.1) have been examined in human brains obtained at autopsy from persons who died of natural causes (controls), and from persons who committed suicide and were further categorized as suffering from affective disorder (depression) or from alcoholism. Post mortem animal studies showed no changes in MAO or COMT activities in rabbit brain or in DBH activity in rat brain when the intact bodies were left at room temperature up to 24 h. TH activity in rabbit brains, however, began to decline immediately after death and after 24 h at room temperature it was approximately 48 per cent of the fresh brain level. There was no significant variation in activity of COMT, TH and DBH in human brain attributable to age or sex. MAO activities in the 60–70 yr decade were 34 per cent higher than in the 30–40 yr decade. MAO activities were highest in the hypothalamus and substantia nigra, TH activities were highest in substantia nigra, putamen and head of caudate, and DBH activities were greatest in tegmentum of pons and hypothalamus. Only minimal regional differences in COMT activities were observed. No significant differences were found between enzyme activities in brain areas of controls and suicides with the possible exception of TH in the substantia nigra, where the depressive suicides (but not the alcoholics) showed greater activity (P < 0·02). These findings appear not to support the catecholamine hypothesis of affective disorder.     

Glutamyl transferase and acetylcholinesterase activity in various areas of the rat brain in alcoholic intoxication

Albino mongrel rats were used for the determination of the gamma-glutamyl transferase (gamma-GTF) and acetylcholine esterase (AChE) activities in various brain areas (cerebral hemispheres, cerebellum, hippocampus, brain stem) during acute (1.5; 4 and 6 g/kg i. p.) and chronic (15 months) alcoholic intoxication and alcohol withdrawal (24-48 h, 4 and 8 days). An increase or a decrease in the activity of these two enzymes in the various rat brain areas depends on the dose of ethanol and the time of its action. The activity of gamma-GTF grew in all brain areas during chronic ethanol intoxication; the activity of AChE was also enhanced in three brain areas but it was diminished in cerebral hemispheres. Alcohol withdrawal caused diverse changes in the activities of these two enzymes in various areas of the brain. A tendency to normalization of the gamma-GTF and AChE activities is manifested 4-8 days after alcohol withdrawal.     

Relationship Between Catechol-O-Methyltransferase and Phenolsulfotransferase in the Metabolism of Dopamine in the Rat Brain

The relationship between phenolsulfotransferase (PST) and catechol-O-methyltransferase (COMT) in the metabolism of free 3,4-dihydroxyphenylethylamine (DA, dopamine) in the rat brain was studied. In rats not pretreated with a monoamine oxidase (MAO) inhibitor a huge increase of free DA in the brain, following an intraperitoneal injection of L-3,4-dihydroxyphenylalanine (L-DOPA) or an intraventricular injection of free DA, did not lead to any noticeable change in DA sulfate or 3-methoxytyramine (3-MT), which remained undetectable by the present HPLC method. However, in rats previously treated with the MAO inhibitors pargyline or tranylcypromine, the same L-DOPA or free DA treatment resulted in significant increases in both 3-MT and DA sulfate in the hypothalamus, brainstem, and striatum. This response of COMT and PST was not affected by prior treatment of the rats with 6-hydroxydopamine, which suggests that O-methylation and sulfoconjugation occur outside adrenergic neurons not destroyed by the neurotoxin. Inhibition of COMT activity did not lead to any increase in DA sulfate, which showed that despite their common mode of action (both enzymes react preferentially at the same hydroxyl group in the DA molecule), the two enzymes are not competitive. After MAO inhibition there were strong correlations between an increase in DA sulfate and 3-MT on the one hand, and between free DA and 3-MT on the other. Because 3-MT is a marker of central DA release, these data suggest that inhibition of MAO activity not only affects DA metabolism by this enzyme but also influences DA release in the rat brain.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

Retardation of fetal brain cell growth during maternal starvation: Circulating factors versus altered cellular response

Maternal starvation inhibits fetal brain development during late gestation in the rat. To determine whether intrinsic or extrinsic factors might be the principal contributor to altered growth, brain cells from 20 day fetuses were cultured in a 96 well plate with MEM and 10% adult rat serum. Tissue growth was monitored by spectrophotometric measurement of the mitochondrial reduction of a chromagen 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT). After 1, 4 or 6 days incubation, MTT activity in non confluent cultures was shown to be directly related to tissue mass. When fetal brain cell cultures were incubated with 1% and 10% concentrations of adult rat serum, an 11-fold increase in MTT activity paralleled a 15-fold increase in tritiated thymidine incorporation. The impact of maternal starvation on fetal brain cell growth was examined by measuring MTT activity in fetal brain cells from fed and starved mothers. When cultures were incubated for 6 days with graded concentrations of fed adult serum (1.25–10%), the MTT response was slightly but consistently lower in cells from starved when compared with cells from fed mothers. By contrast, a marked difference in MTT activity which was paralleled by a lower DNA content became apparent when fetal rat brain cells were incubated with starved adult serum. Fetal serum and adult male serum were found to support growth equally well, while incubation of fetal brain cells with maternal sera resulted in lower MIT values than with the corresponding fetal sera. When cells were incubated with fetal sera pooled from starved mothers, MTT activity was decreased by 42 to 45%. A relative decrease in MTT activity was also apparent when cells were exposed to sera from starved mothers. Graded concentrations of starved fetal serum (2.5–10%) produced an increase in MTT activity that was consistently lower than similar concentrations of fed fetal serum, a finding suggesting a decrease in growth factors. Mixing fasted with fed serum did not correct the diminished growth, and indicated that an inhibitor might also be functioning to restrict growth. These findings therefore suggest that the principal determinants of diminished fetal brain growth during maternal starvation are not only intrinsic to the cells but are importantly related to the altered extrinsic factors in the fetal circulation.     

Development of gamma-glutamyl transpeptidase activity in primary cultures of neurones and glial cells derived from the cerebral hemispheres of chick embryos

The development of gamma-glutamyl transpeptidase (GGT) activity in neurones and glial cells was studied in primary cell cultures derived from the cerebral hemispheres of chick embryos. GGT activity was found in both basic types of nervous tissue cells. It was always higher in glial cell cultures, where it was up to 2.3-fold the values in neurone-enriched cultures. If the culture medium contained foetal calf serum, the GGT activity of both types of nerve cells was higher than in the presence of inactivated calf serum. Comparison with the in vivo situation showed that the level of GGT activity in nerve cell cultures was significantly lower. Between the seventh day of embryogenesis and the third day of postnatal development of the nerve cells, there were marked differences between the GGT activity of cells maintained under in vitro conditions and cells of the same age in brain tissue homogenate. GGT activity in cerebral hemisphere homogenates from a 17-day-old embryos amounted to 4-fold the activity in a primary glial cell culture and to 16-fold the value in a neurone-enriched culture from hemispheres at the same stage of development.     

Glutamate decarboxylase and GABA-aminotransferase activity in brain mitochondria under conditions of ethanol exposure in postnatal ontogenesis

The activity of the enzymes of GABA- GDK and GABA-T metabolism in the brain mitochondria in 6 periods of postnatal development under the conditions of high dose of ethanol was studied. It has been revealed that after the impact of high dose of ethanol (3.5 g/kg of 25% solution, intraperitoneally) the enzymes' activities in initial mitochondrial fractions of cerebral cortex, cerebellum, hypothalamus and brain stem increases. The activity of GABA-T in 10- and 21-days animals significantly decreases, while in other periods on the contrary it increases.     

γ-VINYL GABA: EFFECTS OF CHRONIC ADMINISTRATION ON THE METABOLISM OF GABA AND OTHER AMINO COMPOUNDS IN RAT BRAIN

Rats were given γ-vinyl GABA (4-amino-hex-5-enoic acid), a new irreversible inhibitor of GABA aminotransferase (GABA-T), by daily subcutaneous injection (100mgkg) for 11 days. Amino acids were quantitated in the brains of the γ-vinyl GABA-treated and control animals 24 h after the last injection, and enzyme activities of GABA-T and glutamic acid decarboxylase (GAD) were measured. Chronic administration of γ-vinyl GABA produced a 150% increase in brain GABA content, along with marked increases in the contents of B-alanine and homocarnosine. Brain GABA-T activity was reduced by 26%, and GAD activity was reduced by 22%. In addition, γ-vinyl GABA caused a marked increase in hypotaurine content in rat brain, suggesting that it acts as an inhibitor of hypotaurine dehydrogenase, and it produced significant decreases in brain contents of glutamine and threonine. Although it is an effective GABA-T inhibitor, γ-vinyl GABA apparently affects several other brain enzymes as well, and it may not be an ideal drug for elevating brain GABA levels in man.     

Stimulation of DNA synthesis in primary cultures of adult rat hepatocytes by rat platelet-associated substance(s)

Summary Experiments in whole animals have shown that normally quiescent adult rat hepatocytes are induced to proliferate by blood borne substances, which we are now probing in primary monolayer cultures. Under our conditions, freshly isolated adult hepatocytes do not proliferate actively in a defined medium, but are stimulated to synthesize DNA — an essential first step — by either serum or an EGF-hormone combination. Stimulation of [³H]thymidine incorporation into hepatocyte DNA by addition of dialyzed mouse, human, horse, or bovine (fetal, newborn, or calf) serum, whose activities are all similar, is regularly surpassed by an EGF-insulin mixture without serum. This, in turn, is exceeded by dialyzed normal rat serum, which is several times more potent than the other sera tested. Removal of blood platelets reduces the activity of normal rat serum by over 50%. Heat inactivation (56° C) causes a similar loss, but heat treatment of platelet-poor serum fails to cause further reduction. The activity of mouse and human serum is not reduced by platelet removal. Serum from partially hepatectomized rats is not significantly more stimulatory than normal rat serum, and its activity is depressed in the same way by platelet deprivation and heat inactivation. Lack of enhancement by partial hepatectomy is not consonant with whole animal studies and requires further investigation. The heat-labile portion of the DNA synthesis-stimulating activity of rat serum appears to derive from platelets. This activity differs from the well-characterized heat-stable human PDGF. Its relation to other reported platelet-associated growth factors is still undetermined. This work was supported by USPHS Grants CA-02146 and AM-19435.