Increased cellular internalization of amphiphiles in a multidrug-resistant CHO cell line.

The uptake of labeled palmitoyl carnitine and palmitoyl lysophosphatidylcholine by CHO cells was studied by measuring the extractability of these amphiphiles by bovine serum albumin. A multidrug-resistant cell line, CHRC5, showed a more rapid uptake, compared with the parental line, of these amphiphiles into a pool that was no longer susceptible to extraction with bovine serum albumin. The more rapid uptake by the drug-resistant cell line was reversed back to the rates observed with the parental cell line in the presence of verapamil, quinacrine or cyclosporin A. These latter three drugs also reverse the multidrug-resistant phenotype. These results demonstrate a relationship between the rate of amphiphile uptake and multidrug resistance.

Effect of staurosporine on the phorbol ester-induced activation of the Na+/H+ antiporter in smooth muscle cells.

Evidence is presented suggesting that the Na+/H+ antiporter activity of aortic smooth muscle cells is stimulated by protein kinase C activation. However, once the transporter has been activated, inhibitors of protein kinase C are not effective, supporting a model in which the Na+/H+ antiporter conserves memory of its activation by protein kinase C.

Dolichyl phosphate phosphatase in rat liver microsomes. Avoidance of the use of detergent in testing the effect of phospholipids on dolichyl phosphate phosphatase.

A system was developed for testing the effect of phospholipids on dolichyl phosphate phosphatase, a membrane-associated enzyme. This enzyme was solubilized, delipidated, stabilized and concentrated in such a way that minimal quantities of Triton X-100 were carried by enzyme extracts to the incubation mixture. Its substrate, dolichyl phosphate, could be kept in aqueous medium as suspended particles without addition of detergent. When dolichyl phosphate phosphatase was assayed using the substrate in this detergent-free form, values for Km, pH optimum and temperature optimum were different from those obtained with detergent-solubilized substrate. This assay of dolichyl phosphate phosphatase almost free of detergent allowed testing of the effect of specific phospholipids on enzyme activity with minimal interference produced by endogenous phospholipids or exogenous detergent. Sphingomyelin, phosphatidylethanolamine or phosphatidylcholine (zwitterionic phospholipids) acted as activators, whereas phosphatidic acid and phosphatidylinositol, negatively-charged phospholipids, were inhibitors of dolichyl phosphate phosphatase.

Hydrogen peroxide-and fetal bovine serum-induced DNA synthesis in vascular smooth muscle cells: positive and negative regulation by protein kinase C isoforms.

Hydrogen peroxide and fetal bovine serum stimulate DNA synthesis in growth-arrested smooth muscle cells with remarkably similar kinetics and cell density dependence. However, while stimulation with fetal bovine serum results in cell proliferation, that by H2O2 is followed by cell death. Depletion of conventional and novel protein kinase C isoforms, resulting from a long treatment with phorbol-12-myristate-13-acetate, further increases H2O2-induced DNA synthesis. On the other hand, the specific protein kinase C inhibitor calphostin C abolished the increased DNA synthesis promoted by fetal bovine serum or H2O2. H2O2 increases protein kinase C activity in smooth muscle cells. This effect is markedly reduced, but not abolished, by down-regulation of the alpha, delta and epsilon protein kinase C isoforms. Thus, the zeta isoform of protein kinase C, which is not down-regulated, may be responsible for the residual H2O2 stimulation of protein kinase C. In conclusion, the results obtained show that H2O2 stimulates protein kinase C activity and DNA synthesis in growth-arrested smooth muscle cells: these events are not followed by cell proliferation but rather by cell death. This H2O2 stimulated DNA synthesis appears to be negatively controlled by alpha, delta and epsilon isoforms and positively controlled by the zeta isoform of protein kinase C.

Inhibition of protein kinase C activity and vascular smooth muscle cell growth by d-alpha-tocopherol.

The inhibition by d-alpha-tocopherol of protein kinase C activity has been studied in synchronised A7r5 rat smooth muscle cells during the cell cycle. Cell protein kinase C activity has been found to oscillate, with a minimum in the G0 phase, a maximum in the late G1 phase and a new minimum in the S phase. An inhibition of protein kinase C activity by d-alpha-tocopherol appears to be at the basis of cell growth inhibition. Nevertheless, the amount of the different protein kinase C isoenzymes present in smooth muscle cells, measured by their specific antibodies, does not change during the cell cycle in both untreated and d-alpha-tocopherol-treated cells. The possible mechanisms of protein kinase C modulation during the cell cycle and of its inhibition by d-alpha-tocopherol are discussed.

Dimerization of the P-glycoprotein in membranes.

Plasma membranes from a CHO cell line, CHRC5, which exhibits multidrug resistance was studied using radiation inactivation analysis. The P-glycoprotein content of the membrane was determined by Western blots. Irradiation resulted in the loss of P-glycoprotein. The dependence of this loss on radiation dose corresponded to a target size of 250 kDa which is the molecular mass of a dimer of the P-glycoprotein. This is strong evidence to indicate that the P-glycoprotein self associates in the membrane.

Inhibition of smooth muscle cell proliferation and protein kinase C activity by tocopherols and tocotrienols.

alpha-Tocopherol, the most active form of vitamin E, causes a dose-dependent inhibition of serum-induced proliferation of smooth muscle cells (A7r5) in culture. Some tocopherol-related compounds exhibiting various degrees of antioxidant potency have also been tested on cellular proliferation. No direct correlation between the antioxidant activity of these compounds and their effect on smooth muscle cell growth could be observed. While most of the derivatives employed were not effective in inhibiting protein kinase C, in the case of alpha-tocopherol the antiproliferative effect was found to be parallel to the inhibition of protein kinase C activity, as measured in streptolysin-O permeabilized cells.

The role of hydrogen peroxide and RRR-alpha-tocopherol in smooth muscle cell proliferation.

Oxidants can be considered early growth signals, since they have been shown to activate a number of pathways that are also stimulated by growth factors. In particular, H(2)O(2) activates the protein kinase C signal transduction pathway in smooth muscle cells. These events certainly play a role in the activation of the DNA synthesis machinery although it is still unclear whether they can also regulate the lethal response. Evidence exists of an oxidant-mediated increase in tyrosine protein phosphorylation as an early event in the signal transduction cascade of growth factor receptors, leading to augmentation of cell proliferation. Oxidants can also induce transcription of enzymes, such as ornithine decarboxylase and the phosphatase CL-100. CL-100 is the first example of a new class of protein phosphatases responsible for modulating the activation of MAP kinase following exposure of quiescent cells to growth factors and further implicates MAP kinase activation/deactivation in the cellular response to hydrogen peroxide. Moreover H(2)O(2) activates the MAP kinase cascade by stimulating the tyrosine kinase and protein kinase C pathways. JNK1, a relative of the MAP kinase group, is activated by dual phosphorylation at Thr and Tyr during the UV response. RRR-alpha-tocopherol and RRR-beta-tocopherol have different and competing effects on smooth muscle cell proliferation, indicating that they do not act as antioxidants. The earliest event brought by RRR-alpha-tocopherol in the signal transduction cascade contolling receptor mediated cell growth is the inhibition of the transcription factor AP-1, activated by phorbol esters. RRR-beta-tocopherol alone is without effect but in combination with RRR-alpha-tocopherol prevents the AP-1-inhibiting effect of the latter. Protein kinase C is inhibited by RRR-alpha-tocopherol and not by RRR-beta-tocopherol, which also in this case prevented the effect of RRR-alpha-tocopherol. The inhibition of RRR-alpha-tocopherol of protein kinase C is not the consequence of a direct interaction but is due to a diminution, produced by RRR-alpha-tocopherol of the kinase phosphorylation. A tocopherol binding protein appears to be at the basis of the RRR-alpha-tocopherol, that discriminates between RRR-alpha-tocopherol and RRR-beta-tocopherol and initiates a cascade of events at the level of cell signal transduction leading to cell proliferation inhibition.

d-alpha-tocopherol control of cell proliferation.

Uncontrolled cell growth is at the basis of neoplastic proliferation and arteriosclerotic lesions. In vitro proliferation of vascular smooth muscle cells, Balb c/3T3 fibroblasts, retinal neuroepithelial cells and neuroblastoma cells is inhibited by d-alpha-tocopherol. On the contrary Chinese hamster ovary cells, osteosarcoma cells and macrophages are not sensitive. PDGF-BB activated proliferation is highly d-alpha-tocopherol sensitive while lysophosphatidic acid induced growth is poorly inhibited. d-beta-Tocopherol, an analogue of d-alpha-tocopherol, with similar antioxidant properties, does not inhibit proliferation. Protein kinase C activity is inhibited by d-alpha-tocopherol but not by d-beta-tocopherol, suggesting a central role of this enzyme in the control of cell proliferation by d-alpha-tocopherol. Activation of the transcription activation complex AP-1 (but not NFKB) is prevented by d-alpha-tocopherol and not by d-beta-tocopherol.

Suramin, an anti-cancer drug, inhibits protein kinase C and induces differentiation in neuroblastoma cell clone NB2A.

Protein kinase C purified from rat brain was found to be inhibited by suramin, a substance used originally in the therapy of antitrypanosomic infections and more recently proposed as antineoplastic agent. The inhibition of suramin was competitive with one of the substrates of the enzyme, ATP with a Ki of 10 microM. At concentrations adequate to inhibit the isolated enzyme, suramin was shown to slow the rate of proliferation of neuroblastoma NB2A cells in vitro and to induce their differentiation as evidenced by typical morphological changes.

The phosphorylation state of MAP-kinases modulates the cytotoxic response of smooth muscle cells to hydrogen peroxide.

Micromolar concentrations of hydrogen peroxide induced the phosphorylation of mitogen-activated protein (MAP) kinases and a lethal response in growth-arrested smooth muscle cells (A7r5). The H202-induced phosphorylation of MAP-kinases was markedly lower in the presence of protein tyrosine kinase (PTK) inhibitors or in protein kinase C (PKC) down-regulated cells. Similarly, the toxicity of H202 was diminished by concomitant addition of either PKC or PTK inhibitors and was also lower in PKC down-regulated cells. These results are consistent with the possibility that phosphorylation of MAP-kinases is a critical event in the toxic response of cultured smooth muscle cells to H202.

d-alpha-Tocopherol inhibits low density lipoprotein induced proliferation and protein kinase C activity in vascular smooth muscle cells.

Native and malondialdehyde modified low density lipoproteins have been shown to stimulate smooth muscle cell proliferation (A7r5) in vitro. The stimulation is associated with an increase of protein kinase C activity. d-alpha-Tocopherol, at physiological concentrations, has been found to inhibit both protein kinase C activity and cell proliferation.

Vitamin E: a sensor and an information transducer of the cell oxidation state.

We studied the effects of RRR-alpha-tocopherol and RRR-beta-tocopherol in smooth muscle cells from rat (line A7r5) and human aortas. RRR-alpha-Tocopherol, but not RRR-beta-tocopherol, inhibited smooth muscle cell proliferation in a dose-dependent manner at concentrations in the range from 10 to 50 mumol/L. RRR-beta-Tocopherol added simultaneously with RRR-alpha-tocopherol prevented growth inhibition. The earliest event brought about by RRR-alpha-tocopherol in the signal transduction cascade controlling receptor-mediated cell growth was the activation of the transcription factor AP-1. RRR-beta-tocopherol alone was without effect but in combination with RRR-alpha-tocopherol prevented the AP-1 activating effect of the latter. Protein kinase C was inhibited by RRR-alpha-tocopherol and not by RRR-beta-tocopherol, which also in this case prevented the effect of RRR-alpha-tocopherol. Calyculin A, a protein phosphatase inhibitor, prevented the effect of RRR-alpha-tocopherol on protein kinase C. The data can be rationalized by a model in which a tocopherol-binding protein discriminates between RRR-alpha-tocopherol and RRR-beta-tocopherol and initiates a cascade of events at the level of cell signal transduction that leads to the inhibition of cell proliferation.

Ciprofibrate stimulates protein kinase C-dependent phosphorylation of an 85 kDa protein in rat Fao hepatic derived cells.

The effect of ciprofibrate on early events of signal transduction was previously studied in Fao cells. Protein kinase C (PKC) assays performed on permeabilized cells showed a more than two-fold increase in PKC activity in cells treated for 24 h with 500 microM ciprofibrate. To show the subsequent effect of this increase on protein phosphorylation, the in vitro phosphorylation on particulate fractions obtained from Fao cells was studied. Among several modifications, the phosphorylation of protein(s) with an apparent molecular mass of 85 kDa was investigated. This modification appeared in the first 24 h of treatment with 500 microM ciprofibrate. It was shown to occur on Ser/Thr residue(s). It was calcium but not calmodulin-dependent. The phosphorylation level of this/these protein(s) was reduced with kinase inhibitors and especially with 300 nM GF-109203X, a specific inhibitor of PKC. All these results suggest that the phosphorylation of the 85 kDa protein(s) is due to a PKC or to another Ser/Thr kinase activated via a PKC pathway. A possible biochemical candidate for 85 kDa protein seems to be the beta isoform of phosphatidylinositol 3-kinase regulatory subunit.

Phosphorylation of peroxisome proliferator-activated receptor alpha in rat Fao cells and stimulation by ciprofibrate.

The basic mechanism(s) by which peroxisome proliferators activate peroxisome proliferator-activated receptors (PPARs) is (are) not yet fully understood. Given the diversity of peroxisome proliferators, several hypotheses of activation have been proposed. Among them is the notion that peroxisome proliferators could activate PPARs by changing their phosphorylation status. In fact, it is well known that several members of the nuclear hormone receptor superfamily are regulated by phosphorylation. In this report, we show that the rat Fao hepatic-derived cell line, known to respond to peroxisome proliferators, exhibited a high content of PPARalpha. Alkaline phosphatase treatment of Fao cell lysate as well as immunoprecipitation of PPARalpha from cells prelabeled with [32P] orthophosphate clearly showed that PPARalpha is indeed a phosphoprotein in vivo. Moreover, treatment of rat Fao cells with ciprofibrate, a peroxisome proliferator, increased the phosphorylation level of the PPARalpha. In addition, treatment of Fao cells with phosphatase inhibitors (okadaic acid and sodium orthovanadate) decreased the activity of ciprofibrate-induced peroxisomal acyl-coenzyme A oxidase, an enzyme encoded by a PPARalpha target gene. Our results suggest that the gene expression controlled by peroxisome proliferators could be mediated in part by a modulation of the PPARalpha effect via a modification of the phosphorylation level of this receptor.

Molecular basis of alpha-tocopherol inhibition of smooth muscle cell proliferation in vitro.

The molecular events responsible for the inhibition of cell proliferation by alpha-tocopherol have been investigated. Smooth muscle cells in vitro have been shown to be specifically inhibited by alpha-tocopherol with a concomitant inhibition of protein kinase C activity. beta-Tocopherol was inactive, despite its similar radical scavenging activity. The point of inhibition of alpha-tocopherol relative to the cell cycle was localized in the late G1 phase. A second effect of alpha-tocopherol observed with smooth muscle cells was the stimulation of protein kinase C biosynthesis in both the S and G2 phases of the cell cycle. The implications of these findings for the onset of arteriosclerosis are discussed.

Relationship between signal transduction and PPAR alpha-regulated genes of lipid metabolism in rat hepatic-derived Fao cells.

The goal of this study was to characterize phosphorylated proteins and to evaluate the changes in their phosphorylation level under the influence of a peroxisome proliferator (PP) with hypolipidemic activity of the fibrate family. The incubation of rat hepatic derived Fao cells with ciprofibrate leads to an overphosphorylation of proteins, especially one of 85 kDa, indicating that kinase (or phosphatase) activities are modified. Moreover, immunoprecipitation of 32P-labeled cell lysates shows that the nuclear receptor, PP-activated receptor, alpha isoform, can exist in a phosphorylated form, and its phosphorylation is increased by ciprofibrate. This study shows that PP acts at different steps of cell signaling. These steps can modulate gene expression of enzymes involved in fatty acid metabolism and lipid homeostasis, as well as in detoxication processes.

Alpha-tocopherol as a modulator of smooth muscle cell proliferation.

The effects of alpha-tocopherol and beta-tocopherol have been studied in rat and human aortic smooth muscle cells. Alpha-tocopherol, but not beta-tocopherol, inhibited smooth muscle cell proliferation and protein kinase C in a dose-dependent manner, at concentrations ranging from 10 to 50 microM. Beta-tocopherol added simultaneously with alpha-tocopherol prevented both proliferation and protein kinase C inhibition. Protein kinase C inhibition was cell cycle-dependent and it was prevented by okadaic acid, a protein phosphatase inhibitor. Protein kinase C activity measured from aortas of cholesterol-fed rabbits was also inhibited by alpha-tocopherol. By using protein kinase C (PKC) isoform-specific inhibitors and immunoprecipitation reactions it was found that PKC-alpha was selectively inhibited by alpha-tocopherol. Further, an activation of protein phosphatase 2A by alpha-tocopherol was found, which caused PKC-alpha dephosphorylation and inhibition. Ultimately, this cascade of events at the level of cell signal transduction leads to the inhibition of smooth muscle cell proliferation.

Vitamin E mediated response of smooth muscle cell to oxidant stress.

Oxidant stress is associated with diminution of antioxidant molecules, such as alpha-tocopherol. Alpha-tocopherol specifically decreases, in a concentration dependent way, the proliferation of vascular smooth muscle cells. At the same concentrations (10-50 microM) it induces inhibition of protein kinase C (PKC) activity. The latter event is not due to a decrease in PKC level or to alpha-tocopherol binding to PKC, but it results from increase of protein phosphatase 2A1 activity. In vitro data, as well as at a cellular level, demonstrates that protein phosphatase 2A1 is activated, in its trimeric structure--but not as a dimer by alpha-tocopherol. This activation is followed by PKC-alpha dephosphorylation. The activation of protein phosphatase 2A1 and deactivation of PKC-alpha affect the AP1 transcription factor, resulting in a change in the composition and the binding of this factor to DNA. By transfecting smooth muscle cell with a construct containing three TRE (TPA responsive elements), the promoter thymidine kinase and the reporter gene chloramphenicol-acetyl-transferase a modulation of gene expression by alpha-tocopherol is observed. Beta-tocopherol does not cause any of the responses observed with alpha-tocopherol and R,R,R-alpha-tocopherol is twice as potent as all-rac-alpha-tocopherol. When added together, beta-tocopherol prevents the effects of alpha-tocopherol indicating that the mechanism involved is not related to the radical-scavenging properties of these two molecules, which are essentially equal. By differential display analysis it has been found that several genes of smooth muscle cells are differentially transcribed in the presence of alpha-tocopherol but not beta-tocopherol. In particular, the gene of alpha-tropomyosin shows a transient enhancement of transcription as a function of the cell cycle time. Alpha-tropomyosin translation is also increased by alpha-tocopherol and not by beta-tocopherol. Because no changes of mRNA stability can be observed in the presence of alpha-tocopherol, the data supports the conclusion of a transcriptional control exerted by alpha-tocopherol on alpha-tropomyosin. Generally, the data strongly suggests the existence of a ligand/receptor type of mechanism at the basis of alpha-tocopherol action. It is concluded that an oxidative stress-induced diminution of alpha-tocopherol in smooth muscle cell activates a reaction cascade leading to changes in gene expression and increase in cell proliferation by a non-antioxidant mechanism.

The effect of dolichol on the permeability properties of phosphatidylcholine bilayers.

The permeability of liposomes to water, glucose, Ca2+ and alkaline cations was monitored by recording the change in absorbance at 450 nm using a rapid reaction stopped-flow spectrophotometer. Liposomes were prepared with egg phosphatidylcholine and concentrations of dolichol ranging from 0.1% to 9% (w/w). Net permeability of phosphatidylcholine bilayers to alkaline cations was induced by the incorporation of dolichol. This effect was not observed in the case of non-charged solutes like glucose or in that of alkaline earth cations such as calcium. Permeation of K+ was significantly increased above the phase transition temperature. These results suggest that dolichols may play a role in biological membranes, besides the well-known glycosyl carrier function in the biosynthesis of glycoproteins.