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D M Raben

Publications and source records attributed to D M Raben.

33 records · Page 2Linked to original sources
PubMed1992

Alpha-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells.

The mechanism by which an agonist, binding to a cell surface receptor, exerts an effect on events in the nucleus is not known. We have previously shown (Leach, K. L., Ruff, V. A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266, 3215-3221) that alpha-thrombin treatment of IIC9 cells results in increased levels of cellular 1,2-diacylglycerol (DAG) and activation of protein kinase C (PKC). Here, we have examined whether changes in nuclear PKC and nuclear DAG also are induced following alpha-thrombin treatment. IIC9 cells were treated with 500 ng/ml alpha-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC alpha, but not PKC epsilon or zeta in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or alpha-thrombin. The increase in nuclear PKC alpha levels was accompanied by a 10-fold increase in nuclear PKC specific activity and stimulated phosphorylation of at least six nuclear proteins. The rise in nuclear PKC levels occurred rapidly and reached a maximum at 30-60 s, which was followed by a decline back to the control level over the next 15 min. In addition, alpha-thrombin treatment resulted in an immediate rise in DAG mass levels in the nuclear fractions. Kinetic analysis indicated that a maximum increase in DAG levels occurred 2.5-5 min after the addition of alpha-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, alpha-thrombin treatment induced an increase in radiolabeled nuclear diglycerides, suggesting that the stimulated nuclear DAGs are derived, at least in part, from phosphatidylcholine. Our results suggest that increases in both nuclear DAG levels and PKC activity following alpha-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.

Animals↗
PubMed1992

Activation of phospholipase D by alpha-thrombin or epidermal growth factor contributes to the formation of phosphatidic acid, but not to observed increases in 1,2-diacylglycerol.

The receptor-mediated activation of a phosphatidylcholine-hydrolysing phospholipase D (PLD) has recently been described. We investigated the effect of alpha-thrombin and epidermal growth factor (EGF) on cellular PLD activity in order to determine the role of this enzyme in mitogen-induced increases in phosphatidic acid and sn-1,2-diacylglycerol. In the presence of ethanol, stimulation of [3H]myristic acid-labelled quiescent IIC9 cells with alpha-thrombin or EGF resulted in a rapid increase in radiolabelled phosphatidyl-ethanol which reached a plateau at 1 min, indicating the rapid and transient activation of PLD. We observed a concomitant decrease in the mitogen-stimulated increase of radiolabelled phosphatidic acid. In contrast, ethanol did not significantly effect the elevation of sn-1,2-diacylglycerol levels stimulated by alpha-thrombin or EGF as determined by measurement of sn-1,2-diacylglycerol mass or the appearance of [3H]1,2-diacylglycerol. A novel lipid, detected by two-dimensional t.l.c. analysis, was generated in [3H]myristic acid-labelled cells stimulated with alpha-thrombin, but not EGF, in the presence of ethanol. Treatment in vitro of cellular lipids isolated from [3H]myristic acid-labelled cultures with PLD in the presence of ethanol also resulted in the generation of this novel lipid species, supporting the role of this enzyme in its production. These data indicate that in quiescent IIC9 cells: (a) alpha-thrombin or EGF rapidly and transiently activates a PLD; (b) although this activation is responsible for part of the mitogen-induced increases in phosphatidic acid, it does not contribute to induced increases in sn-1,2-diacylglycerol; and (c) activation of this enzyme appears to be involved in the formation of a novel lipid generated in response to alpha-thrombin, but not EGF, in IIC9 fibroblasts.

Animals↗
PubMed1991

Dissociation of protein kinase C activation and sn-1,2-diacylglycerol formation. Comparison of phosphatidylinositol- and phosphatidylcholine-derived diglycerides in alpha-thrombin-stimulated fibroblasts.

Diacylglycerols (DAGs) derived from phosphatidylcholine (PC) hydrolysis have been shown to activate protein kinase C (PKC) in vitro, but it is not known whether this event occurs in response to DAGs generated via agonist-induced PC hydrolysis in intact cells. In this report we have addressed this question directly, using alpha-thrombin stimulation of IIC9 fibroblasts. PKC activation in intact cells was assessed in two ways, by measuring: 1) PKC membrane association as determined by kinase activity and Western blot analysis and 2) the phosphorylation of an endogenous PKC substrate, an 80-kDa protein. Treatment with 500 ng/ml alpha-thrombin has been shown to stimulate both phosphoinositide and PC hydrolysis, whereas treatment with 100 pg/ml alpha-thrombin stimulates only PC breakdown. Using these two conditions, we show that DAG produced from phosphoinositide, but not PC hydrolysis, is associated with the activation of PKC.

Animals↗
PubMed1991

Differential dependence of early and late increases in 1,2-diacylglycerol on the presence of catalytically active alpha-thrombin: evidence for regulation at the level of 1,2-diacylglycerol generation.

alpha-Thrombin stimulates a biphasic increase in cellular 1,2-diacylglycerol mass in quiescent IIC9 fibroblasts. This report describes the use of hirudin, a high-affinity inhibitor of alpha-thrombin that renders it catalytically inactive, to investigate the dependence of elevated 1,2-diacylglycerol levels on the presence of catalytically active alpha-thrombin. When cultures were incubated in the presence of alpha-thrombin, 1,2-diacylglycerol levels remained elevated for greater than or equal to 4 h. Inactivation of alpha-thrombin after 15 s did not alter the kinetics of 1,2-diacylglycerol formation occurring over the next 1 h. However, sustained (1-4 h) increases in this lipid were eliminated. Inactivation of alpha-thrombin after 1 h of stimulation resulted in 1) an immediate and reversible decline in 1,2-diacylglycerol levels, 2) elimination of the sustained phase of 1,2-diacylglycerol production, 3) inhibition of the alpha-thrombin-stimulated generation of choline metabolites, and 4) a blunted mitogenic response to alpha-thrombin. These data indicate that early (0-1 h) and late (1-4 h) increases in 1,2-diacylglycerol are differentially dependent on the presence of catalytically active alpha-thrombin. Furthermore, sustained increases in 1,2-diacylglycerol in response to alpha-thrombin are regulated at least in part at the level of generation (via phosphatidylcholine hydrolysis). Our results also support a role for sustained 1,2-diacylglycerol levels in the mitogenic response.

Animals↗
PubMed1991

Carbachol stimulates a different phospholipid metabolism than nerve growth factor and basic fibroblast growth factor in PC12 cells.

We have examined 1,2-diglycerides (DGs) generated in PC12 cells in response to the muscarinic agonist carbachol and compared them with those generated in response to the differentiation factors nerve growth factor and basic fibroblast growth factor. Whereas carbachol stimulates a greater release of inositol phosphates, all three agonists generate similar levels of DGs. In this report, we have analyzed the molecular species of PC12 DGs generated in response to these three agonists. Additionally, we have analyzed the molecular species of PC12 phospholipids. The data indicate that 1) after 1 min of either nerve growth factor or basic fibroblast growth factor stimulation, DGs arise primarily from phosphoinositide hydrolysis; 2) in contrast, after 1 min of carbachol stimulation, DG are generated equally by both phosphoinositide and phosphatidylcholine hydrolysis; and 3) after 15 min of stimulation by any of these agonists, DGs are generated largely by phosphatidylcholine hydrolysis, with a smaller component arising from the phosphoinositides. These results suggest that at least part of the mechanism by which PC12 cells distinguish between different agonists is via alterations in phospholipid sources and kinetics of DG generation.

Animals↗
PubMed1990

Molecular species analysis of mitogen-stimulated 1,2-diglycerides in fibroblasts. Comparison of alpha-thrombin, epidermal growth factor, and platelet-derived growth factor.

Recent studies have implicated the hydrolysis of phosphoinositides and phosphatidylcholine in agonist-stimulated events. The potent mitogen, alpha-thrombin, stimulates the generation of diglycerides in a biphasic and sustained manner in IIC9 fibroblasts (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). Using measurements of radiolabeled headgroup release and molecular species analysis, we previously determined that alpha-thrombin generates diglycerides through the hydrolysis of both the phosphoinositides and phosphatidylcholine at early times (15 s), and at later times (greater than or equal to 5 min) through the hydrolysis of primarily, if not exclusively, phosphatidylcholine (Pessin, M. S., and Raben, D. M. (1989) J. Biol. Chem. 264, 8729-8738). In contrast, IIC9 fibroblasts respond to the mitogenic treatments of (a) alpha-thrombin following chymotrypsin pretreatment or (b) epidermal growth factor by increasing their levels of diglycerides in a monophasic and sustained manner (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). In this report, we have analyzed the molecular species of the diglycerides generated by these two different treatments and have also examined the lipid response of IIC9 fibroblasts to platelet-derived growth factor. Based on both the molecular species analyses and the release of radiolabeled head-groups, all three of these different mitogenic treatments generate diglycerides primarily through the stimulation of phosphatidylcholine hydrolysis. However, while similar, the molecular species profiles of the diglycerides generated by these three treatments are not identical to the molecular species profile of total cellular phosphatidylcholine. In addition, the molecular species profiles of the diglycerides generated by these three mitogenic treatments greatly resemble each other, with significant differences between any two profiles occurring in at most one molecular species. This finding differs from that seen with alpha-thrombin stimulation alone, where the molecular species profile of the diglycerides generated following 5 min of alpha-thrombin stimulation is nearly identical to the molecular species profile of total cellular phosphatidylcholine. These data support the possibility of hormone-sensitive phosphatidylcholine pools or selective diglyceride metabolism.

Animals↗
PubMed1990

Sustained increase in 1,2-diacylglycerol precedes DNA synthesis in epidermal-growth-factor-stimulated fibroblasts. Evidence for stimulated phosphatidylcholine hydrolysis.

A property common to many growth factors is that they must be present for several hours before the commitment to DNA synthesis and cell division occurs. The intracellular signals that are relevant during this period are poorly defined. We examined the formation of 1,2-diacylglycerol in IIC9 fibroblasts after stimulation with epidermal growth factor (EGF), and found that the mass of this lipid remained elevated for at least four hours. The concentration-dependence of EGF-stimulated 1,2-diacylglycerol production and [3H]thymidine incorporation were similar. Studies of phospholipid metabolism strongly suggested that phosphatidylcholine was the source of the 1,2-diacylglycerol generated in response to EGF. EGF did not stimulate the hydrolysis of other phospholipids, including the phosphoinositides, nor did it increase synthesis de novo of 1,2-diacylglycerol. This pattern of sustained 1,2-diacylglycerol formation from phosphatidylcholine may be important in the mitogenic signalling of EGF and potentially other growth factors.

Animals↗
PubMed1990

Kinetic and molecular species analyses of mitogen-induced increases in diglycerides: evidence for stimulated hydrolysis of phosphoinositides and phosphatidylcholine.

A wide variety of agonist-induced events appear to be mediated through an increase in cellular diglyceride levels. With regard to the ability of diglycerides to mediate these events, three important parameters must be considered: a) the kinetics of diglyceride generation, b) the absolute mass levels, and c) their molecular species. While this increase is often due to a stimulated hydrolysis of phosphoinositides, there is increasing evidence that the stimulated hydrolysis of phosphatidylcholine also contributes to agonist-induced increases in diglyceride levels. The kinetics of mass increases in diglyceride levels stimulated in cultured fibroblasts are agonist-dependent. High concentrations of alpha-thrombin stimulate a biphasic increase in diglyceride levels with the first phase peaking at 15 s and the second phase peaking at 5 min. In contrast, stimulation with epidermal growth factor, or platelet-derived growth factor, results in a monophasic increase in cellular diglyceride levels. Furthermore, the molecular species and phospholipid source of the stimulated diglycerides are also agonist-dependent. While the hydrolysis of phosphoinositides is major source of diglycerides initially generated in response to some agonists (15 s with alpha-thrombin at 500 ng/ml), phosphatidylcholine is hydrolyzed as well. Following longer incubations, or at all times following stimulation by epidermal growth factor or platelet-derived growth factor, phosphatidylcholine hydrolysis is the principal source of the stimulated diglycerides.

Cells, Cultured↗
PubMed1989

Molecular species analysis of 1,2-diglycerides stimulated by alpha-thrombin in cultured fibroblasts.

Diglycerides derived from the phospholipase C-mediated hydrolysis of phosphoinositides are implicated as important mediators of agonist-induced responses, including the stimulation of cell division. alpha-Thrombin-stimulated proliferation of fibroblasts is associated with a sustained increase in cellular diglycerides, while the hydrolysis of phosphoinositides is transient (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). A rigorous assessment of this apparent discrepancy requires an analysis of the molecular species of the lipids involved. In this report, we have analyzed the molecular species of 1,2-diglycerides present in quiescent and alpha-thrombin-stimulated IIC9 Chinese hamster embryo fibroblasts. The molecular species profiles of the stimulated diglycerides were compared to the profiles of molecular species contained in cellular phospholipids. We demonstrate that 1) stimulation of IIC9 cells by alpha-thrombin results in an increase in the levels of diglyceride molecular species already present in control, quiescent cultures, without the addition of new species or the complete loss of existing species; 2) the diglycerides present in control cultures as well as in cultures stimulated with alpha-thrombin are all ester-linked; and 3) while the phosphoinositides contribute a significant proportion of the diglycerides generated 15 s following alpha-thrombin addition, phosphatidylcholine contributes most of the diglycerides generated after 5 min and 1 h.

Animals↗
PubMed1988

Kinetic analysis of 1,2-diacylglycerol mass levels in cultured fibroblasts. Comparison of stimulation by alpha-thrombin and epidermal growth factor.

We have examined the kinetics of 1,2-diacylglycerol production in quiescent IIC9 fibroblasts. alpha-Thrombin and epidermal growth factor (EGF) both stimulate an increase in the mass of cellular 1,2-diacylglycerol. The generation of 1,2-diacylglycerol is biphasic when stimulated by a high concentration of alpha-thrombin (500 ng/ml), with an early phase peaking at 15 s and a late phase peaking at 5 min. Production of 1,2-diacylglycerol is monophasic when stimulated by: (a) a low concentration of alpha-thrombin (100 pg/ml); (b) a high concentration of alpha-thrombin added to cultures which had been pretreated with chymotrypsin; or (c) EGF. In all cases the stimulation of 1,2-diacylglycerol was sustained for at least 30 min. In a previous report (Raben, D. M., Yasuda, K., and Cunningham, D. D. (1987) Biochemistry 26, 2759-2765), it was demonstrated that alpha-thrombin stimulates lipid metabolism in fibroblasts via two coupling mechanisms designated R1 and R2. We now present evidence that the early phase of alpha-thrombin-stimulated 1,2-diacylglycerol production is related to R1, which is characterized by: 1) increased release of arachidonic acid, 2) hydrolysis of polyphosphoinositides, and 3) inhibition by pretreating cultures with chymotrypsin. The late phase is related to R2 which is characterized by 1,2-diacylglycerol production in the absence of stimulated phosphoinositide hydrolysis and arachidonic acid release. In addition, EGF activates an R2-like mechanism in that it does not stimulate the release of arachidonic acid or hydrolysis of polyphosphoinositides but does stimulate a 2-fold increase in 1,2-diacylglycerol mass.

Animals↗
PubMed1987

Modulation of thrombin-stimulated lipid responses in cultured fibroblasts. Evidence for two coupling mechanisms.

Treatment of cultured fibroblasts with thrombin results in the stimulation of cell division and lipid metabolism. Proteolytically active alpha-thrombin rapidly stimulates (a) release of arachidonic acid, (b) generation of inositol phosphates, and (c) increase in cellular diacylglycerol levels. Pretreatment of the fibroblasts with chymotrypsin before alpha-thrombin prevented the first two responses, (a) and (b), and reduced response c. Treatment of fibroblasts with gamma-thrombin, a proteolytic derivative of alpha-thrombin, produced a response indistinguishable from the alpha-thrombin treatment when preceded by chymotrypsin. These data support a model, similar to one for platelets [McGowan, E. B., & Detwiler, T. C. (1986) J. Biol. Chem. 261, 739-746], that fibroblasts possess two coupling mechanisms for the stimulation of lipid metabolism by thrombin. Similar to platelets, one mechanism, R1, mediates the stimulated release of arachidonic acid and is capable of activating Ni, a GTP-binding protein. R1 is inactivated by chymotrypsin and does not respond to gamma-thrombin. The other mechanism, R2, responds to gamma-thrombin and is not activated by chymotrypsin. In contrast to the mechanisms proposed for platelets, we demonstrate that the phospholipase C responsible for the hydrolysis of phosphoinositides is not activated by R2 but is activated via R1. Importantly, stimulation of either mechanism results in the elevation of cellular diacylglycerol. This indicates that the stimulated elevation of diacylglycerol, or those events dependent upon the elevation of diacylglycerol, is not a reliable indicator for establishing the hydrolysis of phosphoinositides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Relationship of thrombin-stimulated arachidonic acid release and metabolism to mitogenesis and phosphatidylinositol synthesis.

Thrombin and certain prostaglandins are both capable of stimulating the proliferation of cultured cells. Since thrombin stimulates the release and metabolism of arachidonic acid, the precursor of prostaglandins, we examined the relationship between this release and metabolism and the stimulation of cell division in cultured fibroblasts. We also examined the role of prostaglandin synthesis in thrombin-stimulated phosphatidylinositol synthesis. The data in this report demonstrate that the release and metabolism of arachidonic acid are not necessary for thrombin-stimulated cell division. The presence of a low concentration of chymotrypsin prevented thrombin-stimulated arachidonic acid release and metabolism without affecting the stimulation of cell division. Furthermore, thrombin-stimulated cell division occurred in the presence of indomethacin concentrations that prevented cyclooxygenase-mediated metabolism of arachidonic acid. The following experiments showed that thrombin-stimulated phosphatidylinositol synthesis was brought about by a cyclooxygenase-mediated metabolite(s) of arachidonic acid. Indomethacin inhibited the cyclooxygenase-mediated metabolism of arachidonic acid without affecting the thrombin-stimulated release of arachidonic acid. Indomethacin also inhibited thrombin-stimulated phosphatidylinositol synthesis. The dose dependence of this inhibition paralleled the inhibition by indomethacin of cyclooxygenase-mediated metabolism of arachidonic acid. In addition, prostaglandin F2 alpha stimulated phosphatidylinositol synthesis in the presence of indomethacin concentrations which prevented thrombin-stimulated phosphatidylinositol synthesis.

Animals↗
PubMed1985

Effects of EGF and thrombin on inositol-containing phospholipids of cultured fibroblasts: stimulation of phosphatidylinositol synthesis by thrombin but not EGF.

The effects of growth factors on inositol-containing phospholipids were investigated to test the hypothesis that alterations in their metabolism are involved in mitogenic stimulation. Thrombin and EGF stimulated comparable increases in the synthesis (30-50%) and degradation (20-40%) of phosphatidylinositol 4-monophosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) in a cell line which is mitogenically responsive to both growth factors. The increases in synthesis were time and dose dependent in a manner which was consistent with their involvement in mitogenesis; the increases were observed only under conditions where a mitogenic response occurred. While it has been suggested that an increased synthesis of phosphatidylinositol (PI) is coupled to the stimulation of DPI and TPI synthesis, we found that thrombin stimulated an early synthesis PI but EGF did not. To further evaluate the involvement of PI in thrombin-stimulated cell division we determined the time and dose dependence of the stimulated PI synthesis and found that it also occurred in a manner which was consistent with its involvement in thrombin-stimulated cell division. Furthermore, the stimulated PI synthesis was not observed with nonmitogenic proteases or in cell lines which were not responsive to thrombin. These results demonstrate that the metabolism of DPI and TPI appears closely related to the mitogenic response generated by EGF and thrombin. However, an early stimulation of PI synthesis is not coupled to this metabolism and is not necessary for mitogenic stimulation by EGF. Thus, a stimulation of PI synthesis is not a valid measure of alterations in inositol-containing phospholipids and what has been termed the "PI response."

Animals↗
PubMed1979

Effect of plasma membranes on solute transport in 3T3 cells.

Sparse cultures of Swiss 3T3 cells are arrested early in the G1 phase of growth by the addition of a plasma membrane fraction obtained from confluent 3T3 cells. We have examined whether the changes in solute transport which are usually associated with cessation of growth at confluency also take place when cell growth is arrested by the addition of plasma membranes. We find that the rate of uptake of alpha-aminoisobutyric acid and uridine is decreased after the addition of plasma membranes to 3T3 cells, but the rate of uptake of 2-deoxyglucose and phosphate is not. We conclude from these observations that uptake of uridine and alpha-aminoisobutyric acid are related to contact inhibition of growth, while the decline in the rate of uptake of 2-deoxyglucose and phosphate observed at high cell density must be due to changes other than cell to cell contact.

Aminoisobutyric Acids↗