Carbohydrate metabolism in transforming lymphocytes from the aged

There is an age-related decline in immune capacity which has been linked to a decreased response of lymphocytes to mitogens in vitro. During transformation, lymphocytes require a marked increase in energy production and biosynthesis which is supplied primarily by glycolysis. In the elderly, the glycolytic enzymes increase significantly in transforming lymphocytes at least 24 hr later than in the young and then at significantly reduced levels. Glucose utilization is also impaired in stimulated lymphocytes from the elderly but follows the impairment of glycolysis. In stimulated cells from the young, increases in glycolytic enzyme activity levels accompany sharp increases in blastogenesis while a delayed increase in glycolytic enzyme activity in the elderly is accompanied by a delay in blastogenesis. Maximal glycolytic enzyme activity levels are significantly reduced in transformed lymphocytes from the elderly though the number of transformed cells is also significantly reduced. However, glycolytic enzyme activity levels are significantly lower in the elderly than in the young even on a per transformed cell basis. Thus, this reduction cannot be attributed to the lower number of transformed cells that are present in the elderly. This defect in the increase of glycolysis in stimulated cells from the elderly suggests an intracellular mechanism which could be related to the impaired lymphocyte stimulation in vitro in the aged.     

Differential actions of insulin on enzyme activities in mammary organ culture

Mammary explants from midpregnant mice were cultured for 4, 24, 48, and 72 hours in the presence and absence of insulin. Changes in the activities of phosphoglucose isomerase (an enzyme of the glycolytic pathway for glucose metabolism) and of glucose-6-phosphate and 6-phosphogluconate dehydrogenases (enzymes of the pentose phosphate pathway) were assessed at each culture interval. During the first four hours of culture, no significant effect could be attributed to insulin on the activity of these enzymes. Moreover, insulin had no detectable stimulatory effect on phophoglucose isomerase until 48 hours, at which time the hormone caused a marked increase in the activity of this enzyme over the next 24 hours. In contrast, insulin stimulated only a small increase in dehydrogenase activity at 24 hours, after which this hormone acted mainly to maintain the activity that was present initially. These results indicate differential actions of insulin on two groups of enzymes catalyzing the same substrate.     

The effects of aging on phosphofructokinase induction during lymphocyte mitogenesis in relation to DNA and protein synthesis

The incorporation of radiolabeled leucine into phytohemagglutinin-stimulated human lymphocytes increases by 9 hours after mitogen addition in the young whereas this process is delayed by two-fold in the aged (18 hours). Once induced, the leucine incorporation is about 56% less in the aged as compared to the young. The induction of phosphofructokinase (PFK) catalytic activity mimics the induction of protein synthesis in both young (9 hours) and aged (18 hours) subjects also taking twice as long to induce in the aged and attaining much lower levels of induction with increasing subject age. The increase of thymidine incorporation in mitogen-stimulated cells does not occur until 12 hours after the increase in leucine incorporation in both the young (21 hours) and aged (30 hours) which also represents a 9 hour age-related delay in induction. The marked increase in protein synthesis rate occurs in a concerted manner with the induction of glycolysis and the delay and impairment in protein biosynthesis in the aged appears to relate to the similar age-related findings for glycolytic enzyme induction. The mitogen-induced increase in DNA synthesis is a later event and the age-related delay in DNA synthesis induction may be secondary to the delay in the induction of protein synthesis. Other enzyme-dependent processes besides DNA synthesis and glycolysis may also be secondary to a primary slowing of protein synthesis in the aged and related to the delayed cell cycle time frequently observed in aged subjects.     

Metabolic adaptation of the renal carbohydrate metabolism. III

Summary The adaptive response of renal metabolism of glucose was studied in isolated rat proximal and distal renal tubules after a high protein-low carbohydrate diet administration. This nutritional situation significantly stimulated the gluconeogenic activity in the renal proximal tubules (about 1.5 fold at 48 hours) due, in part, to a marked increase in the fructose 1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) activities. In this tubular fragment, FBPase activity increased only at subsaturating fructose 1,6-bisphosphate concentration (30% at 48 hours) which involved a significant decrease in the Km (31%) for its substrate without changes in the Vmax. This enzymatic behaviour is probably related to modifications in the activity of the enzyme already present in the renal cells. Proximal PEPCK activity progressively increased at all substrate concentrations (almost 2 fold at 48h of high protein diet) which brought about changes in Vmax without changes in Km. These changes are in agreement with variations in the cellular concentration of the enzyme. Neither gluconeogenesis nor the gluconeogenic enzymes changed in the distal fractions of the renal tubules. On the other hand, a high protein diet did not apparently modify the glycolytic ability in any fragment of the nephron, although a significant increase in the phosphofructokinase (PFK) and pyruvate kinase (PK) activities was found in the distal renal tubules. This short term regulation involved a significant decrease from 24 hours in the Km value of distal PFK (almost 40%) without changes in Vmax. The kinetic behaviour of distal PK was mixed. In the first 24h after high protein diet a significant decrease in the Km for phosphoenolpyruvate was found (30%) without variation in the Vmax, however during the second 24 hours the activity of this glycolytic enzyme increased significantly (almost 1.3 fold) without modifications in its Km value. On the contrary, this nutritional state did not modify the kinetic behaviour of any glycolytic enzyme in the proximal regions of the renal tubules.     

Stimulation of ornithine decarboxylase synthesis and its control by polyamines in regenerating rat liver and cultured rat hepatoma cells.

Ornithine decarboxylase has been induced in log phase hepatoma cells grown in suspension culture. Induction with N6, O2'-dibutyryl cyclic adenosine 3':5'-monophosphate produced a 4-fold increase in enzyme activity by 3 hours which was followed by a return to base levels by 6 hours. Induction with dexamethasone, a potent synthetic glucocorticoid, exhibited a slow steady rate of increase in enzyme activity, reaching a plateau level of approximately 5- to 6-fold stimulation by about 12 hours. Induced cell and regenerating rat liver ornithine decarboxylase were shown to be indistinguishable by titration with antibody monospecific to the latter and by heat stability. L-[14C]Leucine incorporation into immunoprecipitable enzyme protein after induction in vitro or partial hepatectomy showed an increase which, when coupled with the increase in enzymatic activity, indicated de novo synthesis of enzyme protein. Physiological concentrations of the naturally occurring polyamines, spermidine and spermine, abolish cyclic AMP induction whereas they have no effect on dexamethasone induction. Both inductions were abolished by cycloheximide; in contrast, inhibition by actinomycin D was complete for dexamethasone induction and only partial with respect to cyclic AMP induction. The different time pattern of induction seen with cyclic AMP and dexamethasone, the partial inhibition of the cyclic AMP induction seen with actinomycin D, as well as the absence of inhibition of the dexamethasone induction by polyamines, indicate that these inducers might affect different aspects of the control of the same enzyme.     

Increased phosphofructokinase content during chronic hypoxia in cultured skeletal muscle (L8) cells

Chronic hypoxia results in increased measured activity of all of the glycolytic enzymes and is associated with an increase in glycolytic capacity. Phosphofructokinase, a rate-limiting glycolytic enzyme, was measured under normoxic and hypoxic conditions to determine the relationship between increased activity and enzyme content. Monoclonal antibodies were used to isolate pure enzyme in rat skeletal muscle cells (L8) cultured hypoxically (PO2 = 14 torr) and normoxically (PO2 = 142 torr). Phosphofructokinase content per cell in cultures maintained under chronic (96 h) hypoxic conditions was twice that of cells cultured under normoxic conditions (0.0675 +/- 0.008 (S.E.) and 0.0345 +/- 0.003 micrograms enzyme protein/microgram DNA, P less than 0.01). Phosphofructokinase activity increased proportionately (hypoxia, 0.020 +/- 0.003; normoxia, 0.010 +/- 0.001 units/microgram DNA). The specific activity (units/mg enzyme protein) of phosphofructokinase in the hypoxic (296 +/- 32) versus the normoxic (290 +/- 15) cultures was not significantly different, indicating that the increased activity was accounted for by an increase in enzyme content. Glycolytic rate appears to be regulated at the level of enzyme content.     

Mechanism of corticotropin and cAMP induction of mitochondrial cytochrome P450 system enzymes in adrenal cortex cells

We studied the kinetics of corticotropin (ACTH) induction of mitochondrial cytochromes P450scc and P450c11 and their electron transport proteins, adrenodoxin and adrenodoxin reductase, in bovine adrenal cortex cells in primary culture. The mRNA levels of these enzymes increase and reach a peak within 3-12 h after ACTH addition. The protein levels of adrenodoxin reductase and P450scc show an increase only nearly 24 h after ACTH addition. After ACTH addition, the intracellular level of cAMP reaches maximal levels within 5 min, and then decreases gradually over 60 min. Hence, we examined the effect of a pulse of ACTH or cAMP analogs on enzyme and mRNA levels. Exposure of the cells to ACTH for 1-2 h was sufficient for maximal induction of the enzymes and P450scc mRNA. In contrast, the induction of the enzymes and the mRNA by cAMP analogs or forskolin required the continuous presence of these agents for over 12 h. But, these agents stimulated cortisol secretion to the medium quickly, indicating that they can activate some intracellular processes while not showing any effect on enzyme induction. The absence of any effect of prolonged cAMP pulses on enzyme and mRNA levels weakens the previous hypothesis that cAMP is the sole second messenger for the ACTH induction of steroidogenic enzymes in adrenal cortex cells. The inductive ability of a brief pulse of ACTH indicates that ACTH can rapidly initiate a series of reactions that result in enzyme induction many hours later.     

Density-dependent changes in hexose transport, glycolytic enzyme levels, and glycolytic rates, in uninfected and murine sarcoma virus-transformed rat kidney cells

In normal rat kidney (NRK) cell cultures, increased cell density results in a decrease in the rates of hexose transport, glucose utilization, and lactate production and an increase in the level of hexokinase activity. A murine sarcoma virus (Kirsten)-transformed cell line (KNRK) showed little or no density-dependent variation in sugar uptake, glucose consumption, or lactate production. On the other hand, hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities were elevated in dense transformed cultures as compared to sparse or uninfected cultures. In another virus-transformed cell line (ts339/NRK) exhibiting temperature-dependent morphology, growth pattern, and transport of 2-deoxy- -glucose, the levels of glycolytic enzyme activity were related to cell density but not to the culture temperature. The lack of correlation between glycolytic enzyme activity and lactate production by either uninfected or murine sarcoma virus-transformed cultures supports the suggestion that enhanced growth and/or hexose transport capacity rather than elevated glycolytic enzyme activity are responsible for the increased rate of lactate production by virus-transformed NRK cells.     

Induction of ornithine decarboxylase, RNA, and protein synthesis in macrophage cell lines stimulated by immunoadjuvants

Early biochemical changes associated with adjuvant stimulation of macrophage protein synthesis were studied using two murine macrophage cell lines, PU5-1.8 and J774.1. An induction of ornithine decarboxylase (ODC) was detected 2 hours after exposure of PU5-1.8 and J774.1 cells to two crude immunoadjuvants, BCG cell walls (BCGcw) and lipopolysaccharides from Escherichia coli (LPS). The chemically defined immunoadjuvant glycopeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDPL) also promoted an increase in ODC activity at 2 hours that was maximal after 4 hours, while little or no effect was observed with the D-alanyl analog (MDPD) that is devoid of adjuvant activity. The increase in ODC activity promoted by BCGcw in PU5-1.8 and J774.1 cells returned toward control levels by 6 to 8 hours. BCGcw also stimulated RNA and protein synthesis which remained elevated for at least 24 hours and was associated with a decrease in DNA synthesis and cell proliferation. ODC induction by BCGcw and MDPL was enhanced by the addition of PGE2 in both cell lines. Indomethacin slightly depressed the magnitude of ODC stimulation by BCGcw in J774.1 cells but failed to alter the response of PU5-1.8 cells. Additional observations indicated that the induction of ODC by BCGcw in both cell lines was preceded by an activation of cyclic AMP-dependent protein kinase. These observations suggest that a cyclic AMP-mediated induction of ODC may be an early biochemical marker of adjuvant stimulation in macrophages.     

Characterization of "fetal-type" acetylcholinesterase in hemin-treated K562 cell culture

The alteration of acetylcholinesterase (ACHE) activity, a marker enzyme of erythroid differentiation, was studied during the hemin-induced erythroid differentiation of K562 human leukemia cells in suspension culture. The kinetics of postinduction differentiation was followed by determining the hemoglobin (Hb) content and the ACHE activity of cells. Embryonic hemoglobins as well as small quantities of fetal Hb (HbF) were synthetized by stimulated cells. The peaks of ACHE activity preceded the highest level of Hb content and, following induction, reached their pinnacles at 72 and 120 hours, respectively. These data indicate that ACHE activity is an earlier and more sensitive marker for hemin-induced erythroid differentiation of K562 cells than is elevated Hb content. Electrophoretic mobility of ACHE from hemin-treated cells proved to be the fetal type, but after incubation with neuraminidase, the rate of migration decreased to the level of the adult type enzyme.     

Regulation of aryl hydrocarbon (benzo-(A)-pyrene) hydroxylase activity in mammalian cells. Induction of hydroxylase activity by N6,O2'-dibutyryl8 adenosine 3':5'-monophosphate and aminophylline.

Treatment of hamster BHK cells with N6,O2'-dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP), aminophylline, theophylline, or papaverine increased the level of aryl hydrocarbon (benzo(a)pyrene) hydrolxylase activity. The highese increase, 100-fold, was obtained with Bt2cAMP plus aminophylline or theophylline. N2,O2-Dibutyryl guanosine 3':5'-monophosphate gave a lower induction than Bt2cAMP. The level of hydroxylase activity started to decrease 6 hours after treatment with the inducer and was reduced to almost the uninduced level after 24 hours. Repeated addition of Bt2cAMP and aminophylline did not prevent this decrease. The hydroxylase can also be induced by treating cells with benz(a)anthracene, and the level of this induced activity was maintained for 24 hours. Aminophylline gave a 2- to 8-fold stimulation of the induction by benz(a)anthracene. The enzyme activity induced by Bt2cAMP, aminophylline, and benz(a)anthracene converted benzo(a)pyrene to similar alkali-extractable metabolities with a fluorescence spectra similar to that of 3-hydroxybenzo(a)pyrene. These induced enzyme activities also showed a similar heat stability. Induction by Bt2cAMP and aminophylline, like induction by benz(a)anthracene, required continued protein synthesis and only an initial period of RNA synthesis. Compared to the benz(a)anthracene-induced hydroxylase with a Km of 4.3 muM, the hydroxylase induced by Bt2cAMP and aminophylline showed a Km of 0.14 muM, and was 100-fold more sensitive to inhibition by 7,8-benzoflavone. Increasing the serum concentration in the culture medium stimulated the induction by aminophylline but did not stimulate induction by benz(a)anthracene. The results indicate that aryl hydrocaarbon (benzo(a)pyrene) hydroxylase can be induced by compounds that increase the level of adenosine 3':5'-monophosphate and that this induction and induced enzyme activity differs from that caused by benz(a)anthracene.     

Regulatory role of endogenous regucalcin in the enhancement of nuclear deoxyribonuleic acid synthesis with proliferation of cloned rat hepatoma cells (H4-II-E)

The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.     

Regulation of Cerebroside and Sulfatide Metabolism in Glia Cells

Mouse oligodendroglioma cells, G-26 clone 20 and 24, contain galactosylceramide (cerebroside) and sulfogalactosylceramide (sulfatide) as determined by an HPLC technique. The synthesis of both these lipids was stimulated by 10(-6) M hydrocortisone (cortisol) and also by the removal of serum from the culture medium. Forty-eight hours after the addition of cortisol the incorporation of H235SO4 into sulfatide, the level of sulfatide and the specific activity of the enzyme 3'-phosphoadenosine 5'-phosphosulfate:galactosylceramide sulfotransferase in the cells increased three- to fourfold. The level of cerebroside and the specific activity of UDP-galactose:hydroxyacyl sphingosine galactosyltransferase also increased threefold in the cells on treatment with cortisol. The effect of the hormone on the synthesis of cerebroside preceded the increase in sulfatide synthesis. Experiments with cycloheximide and actinomycin D showed that the effect of the hormone on glycolipid synthesis in these cells were mediated through de novo messenger RNA and protein synthesis. Removal of serum from the culture medium resulted in an approximately twofold enhancement of H235SO4 incorporation into sulfatide within 24 h. The levels of sulfatide and cerebroside and the specific activity of the galactosyltransferase and sulfotransferase also increased significantly after serum removal. However, in contrast to the effect of the steroid, the sulfotransferase activity and the level of sulfatide increased prior to elevations in galactosyltransferase and cerebroside. The effect of serum removal was also found to be mediated by de novo RNA and protein synthesis. The effects of cortisol and serum removal on the synthesis of cerebroside and sulfatide were strictly additive.     

Serum levels of parathormone, calcium, magnesium and phosphorus in the perinatal period

Two groups of newborns on different diets were studied: group A was given artificial milk, while group B was breast fed. Blood samples from the cord artery and vein were taken at birth and further blood samples after 1, 6, 12, 24, 48, 72, 96 and 168 hours. No baby underwent more than three blood samples. Calcium (Ca), magnesium (Mg), phosphorus (P) and parathormone (PTH) were measured in all samples. There was a rapid increase in PTH levels in all subjects in the first hours after birth. At 48th the babies in group A had significantly higher serum levels of P than the group B subjects, and their serum Ca and Mg levels were significantly lower. At 72th PTH serum levels were significantly higher in group A, while Ca and Mg levels were significantly higher in group B. In conclusion, the subjects demonstrated a normal secretion of PTH at birth, and the type of diet notably influenced the homeostasis of calcium in the perinatal period.     

Activities of glucose metabolic enzymes in human preantral follicles: in vitro modulation by follicle-stimulating hormone, luteinizing hormone, epidermal growth factor, insulin-like growth factor I, and transforming growth factor beta1

Modulation of glucose metabolic capacity of human preantral follicles in vitro by gonadotropins and intraovarian growth factors was evaluated by monitoring the activities of phosphofructokinase (PFK) and pyruvate kinase (PK), two regulatory enzymes of the glycolytic pathway, and malate dehydrogenase (MDH), a key mitochondrial enzyme of the Krebs cycle. Preantral follicles in classes 1 and 2 from premenopausal women were cultured separately in vitro in the absence or presence of FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF-I), or transforming growth factor beta1 (TGFbeta1) for 24 h. Mitochondrial fraction was separated from the cytosolic fraction, and both fractions were used for enzyme assays. FSH and LH significantly stimulated PFK and PK activities in class 1 and 2 follicles; however, a 170-fold increase in MDH activity was noted for class 2 follicles that were exposed to FSH. Although both EGF and TGFbeta1 stimulated glycolytic and Krebs cycle enzymes for class 1 preantral follicles, TGFbeta1 consistently stimulated the activities of both glycolytic enzymes more than that of EGF. IGF-I induced PK and MDH activities in class 1 follicles but negatively influenced PFK activity for class 1 follicles. In general, only gonadotropins consistently stimulated both glycolytic and Krebs cycle enzyme activities several-fold in class 2 follicles. These results suggest that gonadotropins and ovarian growth factors differentially influence follicular energy-producing capacity from glucose. Moreover, gonadotropins may either directly influence glucose metabolism in class 2 preantral follicles or do so indirectly through factors other than the well-known intraovarian growth factors. Because growth factors modulate granulosa cell mitosis and functionality, their role on energy production may be related to specific cellular activities.     

Comparative studies of glucose-fed and glucose-starved hamster cell cultures: responses in galactose metabolism.

The metabolic flow of trace amounts of D-[14C]-galactose was followed in cultures of transformed and untransformed hamster cells over a period ranging from five minutes to two hours. The results of chromatographic and enzymatic analyses of the soluble pools are described. Non-glycolytic cells(previously deprived of sugar periods of up to 24 hours) convert D-galactose to galactose-1-phosphate and uridine diphosphoglucuronic acid in 10 to 20 minutes. In the same short assay time, glycolytic cells which have been maintained for 24 hours in media containing glucose or galactose convert D-galactose to uridine diphsphogalactose and uridine diphosphoglucose (ratio 1.4:1). Long term diprivation of sugar also results in 3- to 4-fold increases in the uptake of galactose. In addition, the incorporation of galactose label into chloroformethanol soluble material appears to be influenced by the culture conditions of the untransformed cells while incorporation in the transformed cells appears unaffected. When cycloheximide is included in the maintenance medium for extended periods, the non-glycolytic cells also show increases in galactose uptake rates but the glucose-fed, glycolytic cells llose uptake ability. UDPhexose is the main galactose metabolic peak in the soluble pools of the cycloheximide-treated, glycolytic and the cycloheximide-treated, non-glycolytic cells. The results of these experiments suggests that uptake of galactose and its subsequent metabolism are under separate control.     

Polyribosome Formation in Relation to Cytokinin-induced Cell Division in Suspension Cultures of Glycine max [L.] Merr

We have investigated the relationship between cell proliferation and protein synthetic capacity in a cytokinin-requiring strain of cultured soybean cells (Glycine max [L.] Merr. cv. Sodifuri, of cotyledonary origin) in suspension culture. When transferred to a defined medium lacking cytokinin, very little cell division or cell enlargement took place over the course of a 6-day culture period. Cells transferred to medium of the same composition, but containing 0.5 mum zeatin, exhibited rapid initial growth, with maximum mitotic activity occurring after 24 hours in culture, and a doubling of the cell population within the first 36 hours of the culture period. The polyribosomal RNA content of the cells decreased over the course of the first 24 hours of the growth cycle while the polyribosome to monoribosome (P/M) ratio increased. The increase in the P/M ratio was greater in the cytokinin-treated cells. This apparent relationship between cytokinin-induced cell proliferation and polyribosome formation was examined further. Polyribosome formation was stimulated when zeatin was added directly to cell populations which had been cultured for 24 hours in medium lacking a cytokinin. Transfer to fresh medium alone also stimulated polyribosome formation, whether this medium contained a cytokinin or not. The magnitude of transfer-induced polyribosome formation depended upon the initial cell density (number of cells/ml of medium). Regardless of the initial cell density and independent of the P/M ratios attained, the cytokinin-treated cell populations divided while the cytokinin-deprived cell populations did not. In vivo labeling with [(35)S]methionine and slab gel electrophoretic separation of sodium dodecyl sulfate derivatives of the labeled polypeptides demonstrated qualitative changes in the spectrum of proteins synthesized by the cytokinin-treated cells. These qualitative changes were independent of the cell density (and hence, independent of the P/M ratio) but they preceded cytokinin-induced cell division.     

In vitro studies of the effect of intermittent compressive forces on cartilage cell proliferation.

Intermittent compressive (IC) forces (96 mm Hg, 0.3 Hz) inhibit by 35–60% the serum stimulated increase in ornithine decarboxylase activity (ODC) in chick embryo epiphyseal cartilage cells and rat chondrosarcoma cells. IC had no effect on mouse fibroblast L-cells ODC. The dose-response pattern of the IC effect indicated an all-or-none response with a threshold at 80 mm Hg, a pressure roughly equivalent to the in vivo weight bearing force. The k_m of the cartilage cell ODC, measured at four hours, was about 0.1 mM and was not affected by IC. The V_max, on the other hand, was significantly reduced by IC which is consistent with less enzyme or non-competitive inhibition. IC also produced a significant increase in cAMP levels in both cartilage explants and isolated cells in the presence and absence of serum and a significant reduction in ³H-thymidine incorporation into DNA. The findings show that cellular cAMP, on one hand, and ODC and DNA synthesis, on the other hand, change in opposite directions following exposure to serum and/or IC. Investigation of the IC effect on DNA synthesis in serum-deprived synchronized cartilage cells revealed that IC reduced the number of cells going into S but did not lengthen the G₁ phase. Exposure to IC early in G₁ (0–13 hours) produced the full effect, whereas IC application between 13 to 24 hours (pre S) had no effect. IC had no effect on ³H-thymidine incorporation in L-cells.