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Efficacy and safety of a low-molecular-weight heparin (LMWH) were studied in 33 stable maintenance hemodialysis patients who had a bleeding tendency on unfractionated heparin. The optimal dose of LMWH for each patient was titrated before the study; the mean total LMWH dosage was 1,152 +/- 574 IU. No major bleeding or clot formation was noted in a total of 2,470 hemodialysis sessions during 6 months of LMWH administration. The mean value of plasma anti-factor Xa (anti-Xa) activity increased from 0.05 +/- 0.03 IU/ml before dialysis to 0.34 +/- 0.28 IU/ml after 2 h of dialysis and returned to 0.15 +/- 0.09 IU/ml after 4 h of dialysis; the mean activated partial thromboplastin time was 26.1 +/- 4.4 s before dialysis, 30.7 +/- 9.5 s (an 18% increase) after 2 h of dialysis, and 26.2 +/- 4.4 s after 4 h of dialysis. No significant change in serum antithrombin levels was noted throughout the whole study period. We conclude that a low dosage of LMWH is safe and effective in hemodialysis patients who have a risk of bleeding with unfractionated heparin. Serum anti-Xa activity is better than activated partial thromboplastin time and antithrombin in assessing the optimal dose of LMWH. A plasma anti-Xa activity of 0.37 IU/ml after 2 h of hemodialysis may represent an optimal dosage of LMWH for most patients.

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The association of mutant forms of Ras protein with a variety of human cancers has stimulated intense interest in therapies based on inhibiting oncogenic Ras signaling. Attachment of Ras proteins to the plasma membrane is required for effective Ras signaling and is initiated by the enzyme farnesyl protein transferase. We found that in the presence of potent farnesyl protein transferase inhibitors, Ras proteins in the human colon carcinoma cell line DLD-1 were alternatively prenylated by geranylgeranyl transferase-1. When H-Ras, N-Ras, K-Ras4A, and K-Ras4B were expressed individually in COS cells, H-Ras prenylation and membrane association were found to be uniquely sensitive to farnesyl transferase inhibitors; N- and K-Ras proteins incorporated the geranylgeranyl isoprene group and remained associated with the membrane fraction. The alternative prenylation of N- and K-Ras has significant implications for our understanding of the mechanism of action of farnesyl protein transferase inhibitors as anti-cancer chemotherapeutics.

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The mechanism of phospholipase D (PLD) activation by platelet-derived growth factor (PDGF) was examined using a NIH 3T3 fibroblast cell line (3T3-gamma 1) that stably overexpresses PLC-gamma 1 isozyme. Immunoblot analysis revealed that 3T3-gamma 1 cells contained about 10-fold more PLC-gamma 1 than a control cell line (3T3-C) transfected with expression vector lacking PLC-gamma 1 cDNA. PDGF-stimulated PLD activation was 10-fold greater in 3T3-gamma 1 cells than in 3T3-C cells, indicating that PLD activation is dependent upon the level of PLC-gamma 1. Phorbol 12-myristate 13-acetate (PMA) treatment increased PLD activity to a similar extent in both 3T3-gamma 1 cells and control cells. Pretreatment with tyrosine kinase inhibitors including staurosporine and genistein decreased PLD activity by 82.6% and 87.2%, respectively, and completely blocked tyrosine phosphorylation of PDGF receptor and PLC-gamma 1 in 3T3-gamma 1 cells stimulated with PDGF. Moreover, down-regulation of protein kinase C by pretreatment of PMA caused complete inhibition of PDGF- and PMA-stimulated PLD activation. Therefore, these results suggest that PDGF-induced PLD activation may be a consequence of primary stimulation of PLC-gamma 1 and that PLD may play a role downstream from PLC-gamma 1 in PDGF-triggered mitogenesis.

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The functional significance of phospholipase D (PLD) could most easily be investigated using selective inhibitors. We have isolated a family of fungal metabolites, ketoepoxides, that inhibit chemotactic peptide (formyl-Met-Leu-Phe)-stimulated PLD activation and superoxide generation in granulocytes in the low micromolar range (SCH 49210 having an IC50 of 1.6 microM). Unlike receptor-mediated PLD activation, ketoepoxides were poor inhibitors of phorbol ester-induced PLD activity in granulocytes (IC50 = 43 microM for SCH 49210). Ketoepoxides did not inhibit platelet-derived growth factor-stimulated PLD activity in fibroblasts at up to 50 microM. We also tested the effect of ketoepoxides on in vitro epidermal growth factor receptor and neu tyrosine kinase activities. SCH 49210 (and 49209) did not inhibit the tyrosine kinases at up to 100 microM. These results suggest that ketoepoxides do not inhibit PLD activation due to effects on tyrosine kinase activity. fMLP-induced phospholipase A2 (PLA2) activation is also inhibited by ketoepoxides in the low micromolar range (SCH 49210 having an IC50 of 3.2 microM), but the ketoepoxides were poorer inhibitors of Ca2+ ionophore A23187-induced PLA2 (SCH 49210 having an IC50 of 83 microM). As a measure of phospholipase C (PLC) activity, the generation of inositol-1,4,5 triphosphate in thrombin-stimulated platelets was measured. The ketoepoxides did not inhibit PLC activation indicating that, unlike the aminosteroid U73122, ketoepoxides exhibit some selectivity among receptor-linked phospholipases. The ketoepoxides were also effective inhibitors of tumor cell invasion, as measured by penetration of HT1080 human fibrosarcoma cells into a reconstituted basement membrane matrix. Interestingly, both PLD inhibition and anti-tumor invasion activity correlate closely. These ketoepoxides are, therefore, potential anti-metastatic compounds and may be useful probes to study the role of PLD in cell function.

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Rat 6 fibroblasts that stably overexpress cDNA for the beta 1 isozyme of protein kinase C (PKC3 cells) were used to determine the effect of protein kinase C (PKC) overexpression on hormonal stimulation of phospholipid hydrolysis. In control Rat 6 cells, inositol trisphosphate levels (InsP3) were increased 9-fold in 15 s in response to 10 nM alpha-thrombin, compared with only a 2-fold increase in PKC3 cells. PKC overexpression also inhibited thrombin-stimulated production of 1,2-diacylglycerol, the other product of phosphatidylinositol 4,5-bisphosphate hydrolysis, by 73% at 15 s. In permeabilized cells, PKC overexpression greatly reduced guanosine thiotriphosphate-stimulated InsP3 accumulation, but did not affect InsP3 stimulation by increased free calcium concentration. These data suggest that desensitization of thrombin-stimulated phosphoinositide-phospholipase C is enhanced by PKC-beta 1 overexpression and may involve modulation of G-protein/phospholipase C coupling. In contrast, thrombin was 4.5-fold more effective in stimulation of phosphatidylcholine-phospholipase D activity in PKC3 cells than in control cells, as determined by phosphatidylethanol formation. In permeabilized cells, guanosine thiotriphosphate also stimulated phospholipase D activity more effectively in PKC3 cells than in control cells, suggesting that upregulation of phospholipase D activity by PKC overexpression occurs distal to the thrombin receptor. These results suggest that PKC may act as a switch to up-regulate phosphatidylcholine-phospholipase D and down-regulate phosphoinositide-phospholipase C stimulations.

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The binding of a variety of agonists to their receptors leads to the breakdown of membrane phospholipids and the formation of intracellular second messengers. Hydrolysis of inositol phospholipids by phospholipase C results in the formation of two second messengers, inositol-1,4,5-trisphosphate which mobilizes intracellular calcium and the neutral lipid diacylglycerol (DAG) which binds to and activates protein kinase C (PKC). PKC is actually a family of homologous serine/threonine protein kinases which play a central role in regulation of growth, differentiation and secretion reactions in a variety of cell types. In addition to these feedforward roles of PKC, it is thought to play an important feedback role, regulating early events in signal transduction. To explore these feedback functions we have examined the effect of PKC inhibitors on second messenger formation in thrombin-stimulated human platelets (a rapidly responding system) and the effect of PKC overexpression on second messenger formation and mitogenesis in rat fibroblasts (a system where sustained signaling occurs). Treatment of platelets with inhibitors of PKC potentiates DAG mass formation in response to thrombin while prior activation of PKC with phorbol esters blocks DAG mass formation, consistent with PKC playing a negative feedback role, inhibiting inositol phospholipid breakdown. DAG can also be formed by the sequential hydrolysis of phosphatidylcholine by phospholipase D and phosphatidic acid phosphohydrolase. This is a minor reaction in the rapidly responding platelet system, but may play a role in sustained signaling events. We have found that fibroblasts which overexpress the beta 1 isozyme of PKC display greatly enhanced DAG formation and phospholipase D activation in response to phorbol ester treatment. Upon stimulation of fibroblasts with thrombin, phospholipase D activation is also enhanced by PKC overexpression while formation of inositol phosphates is suppressed. These data suggest that PKC may act as a switch, terminating inositol phospholipid hydrolysis and activating the hydrolysis of phosphatidylcholine. Furthermore, we have observed a strong correlation between activation of phospholipase D and mitogenesis, suggesting an important role for this enzyme in long-term cellular responses to activation.

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Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.

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The human m1 and m2 muscarinic acetylcholine receptor (AChR) genes were subcloned, permanently expressed in HeLa cells and analyzed for their pharmacological and biochemical profiles. Both subtypes displayed saturable, high affinity binding of [3H]-quinuclidinyl benzilate (QNB) which was displaced by muscarinic agonists and antagonists. Stimulation of intact HeLa cells expressing the human m1 AChR gene by the muscarinic agonist oxotremorine-M, in the presence of ethanol, resulted in the activation of phospholipase D (PLD) and the formation of phosphatidylethanol (PEt). In contrast, oxotremorine-M did not activate PLD in the HeLa cells expressing the human m2 AChR subtype. These data suggest that the human m1 AChR is linked to the signal transduction mechanism of PLD activation, whereas the human m2 AChR interacts with a different guanine nucleotide regulatory binding protein (G-protein) which does not cause the activation of PLD or the formation of PEt.

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We are using a Rat-6 fibroblast cell line that stably overexpresses the beta 1 isozyme of protein kinase C (PKC) to study regulation of phospholipid hydrolysis by PKC. Stimulation of control (R6-C1) or overexpressing (R6-PKC3) cells with phorbol ester results in an increase in diacylglycerol (DAG) mass with no increase in inositol phosphates, indicating that DAG is not formed by inositol phospholipid breakdown. A more dramatic DAG increase occurs in R6-PKC3 cells (4.0-fold over basal) compared to R6-C1 cells (1.5-fold over basal). To further define the source of DAG, phosphatidylcholine (PC) pools were labeled with [3H]myristic acid or with [3H]- or [32P]alkyllyso-PC and formation of labeled phosphatidylethanol, an unambiguous marker of phospholipase D activation, was monitored. Phorbol ester-stimulated phosphatidylethanol formation is 5-fold greater in the R6-PKC3 cell line. Formation of radiolabeled phosphatidic acid (PA) is also enhanced by PKC overexpression. In cells double-labeled with [3H]- and [32P]-alkyl-lysoPC, the 3H/32P ratio of PA and PC are identical 15 min after stimulation, suggesting that a phospholipase D mechanism predominates. In support of this, the PA phosphohydrolase inhibitor propranolol decreased phorbol 12-myristate 13-acetate-stimulated DAG formation by 72%. Increases in DAG and phosphatidylethanol were inhibited by the PKC inhibitors K252a and staurosporine. These results indicate that phospholipase D is regulated by the action of PKC. Enhanced phospholipase D activity may contribute to the growth abnormalities seen in PKC-overexpressing cells.

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The phorbol ester 12-O-tetradecanoylphorbol-13-acetate activates the phospholipase D pathway in bovine lymphocytes, leading to a synthesis of phosphatidylethanol (PEt) from exogenous alcohol. Concomitant treatment of the cells with 10(-8) M etiocholanolone, dehydroepiandrosterone or 16 alpha-bromo-epiandrosterone results in the production of phosphatidylethanols that carry metabolically altered forms of arachidonic acid at position 2. The observed steroid response appears to be mediated by a receptor mechanism in that it depends on the synthesis of new RNA and protein. The spectrum of steroids producing the response suggests that the postulated receptor system may be distinct from the well-studied glucocorticoid, progesterone, estrogen and androgen specific receptors. The possible relevance of these new metabolites of ethanol to the problem of alcoholism in humans and to the field of carcinogenesis in general is discussed.

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Stimulation of platelets with thrombin leads to rapid degradation of inositol phospholipids, generation of diacylglycerol (DAG) and subsequent activation of protein kinase C (PKC). Previous studies indicated that prior activation of PKC with phorbol myristate acetate (PMA) desensitizes platelets to thrombin stimulation, as indicated by a decreased production of inositol phosphates and decreased Ca2+ mobilization. This suggests that PKC activation generates negative-feedback signals, which limit the phosphoinositide response. To test this hypothesis further, we examined the effects of PKC activators and inhibitors on thrombin-stimulated DAG mass formation in platelets. Pretreatment with PMA abolishes thrombin-stimulated DAG formation (50% inhibition at 60 nM). Pretreatment of platelets with the PKC inhibitors K252a or staurosporine potentiates DAG production in response to thrombin (3-4-fold) when using concentrations required to inhibit platelet PKC (1-10 microM). K252a does not inhibit phosphorylation of endogenous DAG or phosphorylation of a cell-permeant DAG in unstimulated platelets, indicating that DAG over-production is not due to inhibition of DAG kinase. Sphingosine, a PKC inhibitor with a different mechanism of action, also potentiates DAG formation in response to thrombin. Several lines of evidence indicate that DAG formation under the conditions employed occurs predominantly by phosphoinositide (and not phosphatidylcholine) hydrolysis: (1) PMA alone does not elicit DAG formation, but inhibits agonist-stimulated DAG formation; (2) thrombin-stimulated DAG formation is inhibited by neomycin (1-10 mM) but not by the phosphatidate phosphohydrolase inhibitor propranolol; and (3) no metabolism of radiolabelled phosphatidylcholine was observed upon stimulation by thrombin or PMA. These data provide strong support for a role of PKC in limiting the extent of platelet phosphoinositide hydrolysis.

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It has recently been demonstrated that the chemotactic peptide N-formyl-Met-Leu-Phe activates phospholipase D (PLD) in dimethyl sulfoxide-differentiated HL-60 granulocytes to produce phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) (Pai, J.-K., Siegel, M. I., Egan, R. W., and Billah, M. M. (1988) J. Biol. Chem. 263, 12472-12477). We now report that biologically active phorbol esters, a cell-permeable diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), and calcium ionophore A23187 are also potent inducers of PLD in these HL-60 granulocytes. HL-60 granulocytes have been selectively labeled in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P by incubating the cells with alkyl-[32P]lyso-phosphatidylcholine (PC). When these labeled cells are treated with phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate, OAG, or A23187, alkyl-[32P]PA is formed. Because cellular ATP has not been labeled with 32P, the formation of alkyl-[32P]PA conclusively demonstrates PLD activation by these agents. In the presence of 0.5% ethanol, phorbol esters, OAG, and A23187 also induce formation of alkyl-[32P]PEt, demonstrating that the activated PLD catalyzes transphosphatidylation between the phosphatidyl moiety of the alkyl-[32P]PC and ethanol. Formation of alkyl-[32P]PA and alkyl-[32P]PEt in response to these various agents occurs in a time- and dose-dependent manner and exhibits differential Ca2+ requirements. Based on experiments with both [3H]alkyl-PC and alkyl-[32P]PC, it is concluded that alkyl-PA and alkyl-PEt formed in response to PMA, OAG, or A23187 are derived exclusively from PLD action on alkyl-PC. Furthermore, subthreshold concentrations of PMA (0.5-2.0 nM) or OAG (1.0-25 microM) combined with subthreshold levels of A23187 (15-60 nM) induce the formation of alkyl-[32P]PA and alkyl-[32P]PEt, suggesting that receptor-mediated activation of PLD might involve cooperative interactions between Ca2+ and diglyceride. Although PLD is activated by agents that also activate protein kinase C, the protein kinase C inhibitor, K252a, inhibits PMA-induced protein phosphorylation but causes only partial inhibition of PLD activation. We conclude that phorbol esters, OAG, and A23187 activate PLD in HL-60 granulocytes via protein kinase-independent as well as protein kinase-dependent mechanisms.

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There exists circumstantial evidence for activation of phospholipase D (PLD) in intact cells. However, because of the complexity of phospholipid remodeling processes, it is essential to distinguish PLD clearly from other phospholipases and phospholipid remodeling enzymes. Therefore, to establish unequivocally PLD activity in dimethyl sulfoxide-differentiated HL-60 granulocytes, to demonstrate the relative contribution of PLD to phospholipid turnover, and to validate the hypothesis that the formation of phosphatidylethanol is an expression of PLD-catalyzed transphosphatidylation, we have developed methodologies to label HL-60 granulocytes in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P without labeling cellular ATP. These methodologies involve (a) synthesis of alkyl-lysoPC containing 32P by a combination of enzymatic and chemical procedures and (b) incubation of HL-60 granulocytes with this alkyl-[32P] lysoPC which enters the cell and becomes acylated into membrane-associated alkyl-[32P]PC. Upon stimulation of these 32P-labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) is formed rapidly. Because, under these conditions, cellular ATP has not been labeled with 32P, alkyl-[32P]PA must be formed via PLD-catalyzed hydrolysis of alkyl-[32P]PC at the terminal phosphodiester bond. This result conclusively demonstrates fMLP-induced activation of PLD in HL-60 granulocytes. These 32P-labeled HL-60 granulocytes have also been stimulated in the presence of ethanol to produce alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Formation of alkyl-[32P]PEt parallels that of alkyl-[32P]PA with respect to time course, fMLP concentration, inhibition by a specific fMLP antagonist (t-butoxycarbonyl-Met-Leu-Phe), and Ca2+ concentration. These results strongly support the hypothesis that in HL-60 granulocytes, PEt is formed via PLD-catalyzed transphosphatidylation. Moreover, using HL-60 granulocytes double-labeled by incubation with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC, it has been established that the early (30 s) appearance of alkyl-PA is due primarily to PLD, not phospholipase C as previously thought, and that alkyl-PEt is formed exclusively by PLD. These results constitute the first direct evidence for receptor-linked activation of PLD, leading to the generation of PA and PEt in an intact cell system.

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Activation of phospholipase D (PLD) has been investigated in dimethylsulfoxide differentiated HL-60 granulocytes labeled in endogenous 1-0-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) by incubation with [3H]alkyl-lysoPC. Stimulation of these labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), induces rapid generation of [3H]phosphatidic acid (PA) and slower formation of [3H]diglyceride, suggesting hydrolysis of alkyl-PC by PLD. A unique feature of PLD is its ability to transfer the phosphatidyl moiety of phospholipids to alcohols (transphosphatidylation). This characteristic has been exploited to identify PLD activity. For example, when ethanol is present during stimulation of the HL-60 cells, [3H]phosphatidylethanol (PEt) is formed with a concomitant decrease in [3H]PA. Cells incubated with [32P]orthophosphate to label the terminal phosphate of ATP do not incorporate 32P into PEt, consistent with the [3H]PEt not being synthesized from [3H]diglyceride. In contrast, [3H]PA arises from both PLD and diglyceride kinase activities. Furthermore, PEt synthesis closely parallels PA formation and both are inhibited by an fMLP receptor antagonist, suggesting that both PA and PEt are derived from agonist-stimulated PLD action. These observations are consistent with phospholipase D-catalyzed breakdown of alkyl-PC in fMLP- stimulated granulocytes.

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Tumor-promoting phorbol esters acutely activate a pathway in lymphocytes leading to the synthesis and accumulation of phosphatidylethanol, using exogenous ethanol as a precursor. This product is a representative of a unique class of acidic glycerophospholipids in which the head group is a primary alcohol. The formation of this lipid, in response to different phorbol ester derivatives, correlates with their activity as tumor promoters and inducers of growth changes in a variety of animal cells. Since phosphatidylethanol represents an unusual metabolite of ethanol, it is proposed that studies of its synthesis and biological functions may also provide new perspectives on the biology of alcohol addiction as well as the role of this biological pathway in tumor promotion.

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Treatment of bovine lymphocytes with phorbol esters activates an oxygen-dependent system for the formation of a class of arachidonic acid metabolites which appears distinct from prostaglandins, prostacyclins, thromboxanes and leukotrienes. A full activation of the system is achieved within minutes by 10(-7) M 12-O-tetradecanoylphorbol-13-acetate. This activation can be largely prevented by the lipoxygenase inhibitors, 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid.

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