Effect of trifluoperazine on DNA and LDL-receptor synthesis in smooth muscle cells exposed to hypercholesterolemic medium in vitro

We recently demonstrated that calmodulin and/or protein kinase C may play a crucial role in cholesterol-induced atherogenesis in experimental animal model system. The present study, which was undertaken to elucidate the effect of trifluoperazine (known as a potent inhibitor of calmodulin and protein kinase C) on DNA and LDL-receptor synthesis of aortic smooth muscle cells exposed to hypercholesterolemic medium, revealed that (a) trifluoperazine at a concentration of 25 microM caused an approximately threefold increase in the [35S]methionine-incorporated LDL-receptor protein as compared with values found in control cells; (b) the drug at concentrations greater than or equal to 0.1 microM caused inhibition of DNA synthesis as compared with values found in control cells. These results demonstrate that the preventive effect of trifluoperazine on the atherogenic activity of smooth muscle cells may be due to its ability to increase LDL-receptors synthesis as well as concomitant inhibitory action on DNA synthesis of smooth muscle cells exposed to hypercholesterolemic medium.     

Cholesterol metabolism. Effect of 26-thiacholesterol and 26-aminocholesterol, analogues of 26-hydroxycholesterol, on cholesterol synthesis and low-density-lipoprotein-receptor binding.

1. The effects of 26-aminocholesterol and 26-thiacholesterol on cholesterol synthesis and LDL (low-density lipoprotein)-receptor activity were compared with naturally occurring 26-hydroxycholesterol utilizing both human fibroblasts and hepatoma (Hep G2) cells. 2. At equimolar concentrations (0.625 microM), down-regulation of LDL-receptor activity and cholesterol synthesis was greater with human fibroblasts than with Hep G2 cells. 3. At much higher concentrations (5-20 microM) the 26-thia analogue had little effect on either cholesterol synthesis or LDL-receptor activity.     

Involvement of erythrocyte skeletal proteins in the modulation of membrane fluidity by phenothiazines

The effects of phenothiazines (chlorpromazine, chlorpromazine sulfoxide, and trifluoperazine) and antimitotic drugs (colchicine and vinblastine) on the erythrocyte membrane have been investigated. Chlorpromazine and trifluoperazine induced a dose-dependent increase in the freedom of motion of stearic acid spin-labels bound to both intact erythrocytes and ghosts, but did not affect the freedom of motion of stearic acids bound to vesicles depleted of spectrin and actin or of ghosts resealed with anti-spectrin antibodies. Further, chlorpromazine and trifluoperazine were able to eliminate a protein 4.1 dependent membrane thermal transition detected by stearic acid spin-labels at 8.5 +/- 1.5 degrees C. Antimitotic drugs and chlorpromazine sulfoxide did not change either the freedom of motion of stearic acid spin-labels or the 8.5 degrees C membrane thermal transition. Results indicate the involvement of skeletal proteins as possible membrane target sites of biologically active phenothiazines and suggest that the control of stearic acid spin-label freedom of motion is mediated by the spectrin-actin network and the proteins that link the skeletal network to the membrane.     

Effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of liposomes by Kupffer cells in culture

We investigated the effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of large unilamellar liposomes by rat Kupffer cells in maintenance culture. The phospholipid vesicles were labeled in the lipid moiety with phosphatidyl[14C]choline and contained [3H]inulin or [125I]iodoalbumin as nondegradable and degradable markers of the aqueous vesicle content, respectively. Cytochalasin B and dihydrocytochalasin B, inhibitors of microfilament function, reduced inert inulin label uptake by 75% maximally, but residual uptake was not followed by release of lipid degradation products from the cells. By contrast, colchicine, an inhibitor of microtubule assembly, reduced uptake of liposomal inulin by maximally 55% but could not inhibit release of lipid degradation products from the cells. It is concluded that the cytochalasins partly inhibit uptake but fully prevent the arrival of internalized liposomes in the lysosomal compartment, while the action of colchicine is to slow down the overall process of uptake and subsequent transportation to the lysosomes. Monensin reduced inulin uptake to an extent similar to that found with colchicine, but reversibly blocked degradation of liposomal lipid and encapsulated protein. The kinetics of degradation of liposomal constituents suggests that residual uptake in the presence of monensin represents accumulation in an intracellular compartment. Trifluoperazine did not affect binding, internalization or degradation of encapsulated protein at low concentration (6 microM), but completely inhibited release of liposomal lipid degradation products under these conditions. At intermediate concentration (14 microM), the drug also reduced the internalization, while a high concentration (22 microM) was required to inhibit protein degradation as well. We conclude that trifluoperazine has multiple sites of action in the uptake and processing of liposomal constituents by Kupffer cells.     

Trifluoperazine Inhibits 45Ca2+ Uptake and Catecholamine Secretion and Synthesis in Adrenal Medullary Cells

Abstract: In isolated adrenal medullary cells, carbamyl-choline and high K⁺ cause the calcium-dependent secretion of catecholamines with a simultaneous increase in the synthesis of ¹⁴C-catecholamines from [¹⁴C]tyrosine. In these cells, trifluoperazine, a selective antagonist of calmodulin, inhibited both the secretion and synthesis of catecholamines. The stimulatory effect of carbamyl-choline was inhibited to a greater extent than that of high K⁺. The inhibitory effect of trifluoperazine on carbamylcholine-evoked secretion of catecholamines was not overcome by an increase in either carbamylcholine or calcium concentration, showing that inhibition by trifluoperazine occurs by a mechanism distinct from competitive antagonism at the cholinergic receptor and from direct inactivation of calcium channels. Doses of trifluoperazine that inhibited catecholamine secretion and synthesis also inhibited the uptake of radioactive calcium by the cells. These results suggest that trifluoperazine inhibits the secretion and synthesis of catecholamines mainly due to its inhibition of calcium uptake. Trifluoperazine seems to inhibit calcium uptake by uncoupling the linkage between cholinergic receptor stimulation and calcium channel activation.     

Modulating effects of phenothiazines on mast cells deformities induced by colchicine. Importance of calcium

The shape changes of peritoneal rat mast cells induced by colchicine are completely inhibited by trifluoperazine (10(-4) M), known to inhibit calmodulin, and by EDTA (2 X 10(-3) M). Promethazine and chlorpromazine increase these modifications up to 10(-4) M and inhibit them at higher concentrations.     

Effect of intracellular free Ca2+ on leucine transport in Chang liver cells

Addition of Ca2+ ionophore (A23187) to the medium stimulated the Na+-independent leucine transport in Chang liver cells, increasing the cytoplasmic free Ca2+ concentration, irrespective of the presence or absence of extracellular Ca2+. Anticalmodulin drugs, such as chlorpromazine, trifluoperazine, and W-7, significantly inhibited the leucine transport in the cells. The stimulatory effect of A23187 on leucine transport was completely blocked in the presence of the anticalmodulin drug. Two microtubule disrupting drugs, colchicine and colcemid, significantly stimulated leucine transport. On the other hand, taxol, a microtubule stabilizing agent, decreased the stimulatory effect of colchicine on the leucine transport. These results strongly suggest the involvement of Ca2+ and calmodulin in regulation of Na+-independent leucine transport, possibly through control of assembly and disassembly of the microtubule network.     

Effect of trifluoperazine on DNA synthesis during liver regeneration

An intraperitoneal injection of the calcium-calmodulin blocker trifluoperazine into rats at 4 hr after a partial hepatectomy produced a strong inhibition of DNA synthesis observed at 24 hr after surgery; but when injection was administered at 20 hr after hepatectomy, it did not produce any effect on DNA replication. These observations indicate that trifluoperazine acted by blocking one or more events involved in triggering DNA replication but it did not affect on-going DNA synthesis. A more detailed study indicated that when trifluoperazine was injected at 4 hr after surgery, a 12 hr delay in the cytosolic calmodulin surge observed between 6 and 12 hr after partial hepatectomy (previous to initiation of DNA replication) and also in the starting of DNA synthesis was produced. These findings suggest that the pre-replicative surge of cytosolic calmodulin could be involved in triggering DNA synthesis observed after partial hepatectomy.     

Effect of Trifluoperazine On Dna Synthesis During Liver Regeneration

Abstract. An intraperitoneal injection of the calcium-calmodulin blocker trifluoperazine into rats at 4 hr after a partial hepatectomy produced a strong inhibition of DNA synthesis observed at 24 hr after surgery; but when injection was administered at 20 hr after hepatectomy, it did not produce any effect on DNA replication. These observations indicate that trifluoperazine acted by blocking one or more events involved in triggering DNA replication but it did not affect on-going DNA synthesis. A more detailed study indicated that when trifluoperazine was injected at 4 hr after surgery, a 12 hr delay in the cytosolic calmodulin surge observed between 6 and 12 hr after partial hepatectomy (previous to initiation of DNA replication) and also in the starting of DNA synthesis was produced. These findings suggest that the pre-replicative surge of cytosolic calmodulin could be involved in triggering DNA synthesis observed after partial hepatectomy.     

Trifluoperazine Stimulates Acetylcholine Receptor Synthesis in Cultured Chick Myotubes

Acetylcholine receptor appearance rate in the presence of the phenothiazines trifluoperazine and chlorpromazine was measured in cultured embryonic chick myotubes by means of 125I-alpha-bungarotoxin. At drug concentrations of 5 to 10 X 10(-6) M, receptor appearance rate was significantly enhanced while receptor half-life, cellular protein, net protein synthesis rate, and acetylcholinesterase levels were not similarly affected. The sulfoxide derivatives were without effect. At concentrations of 3 X 10(-5) M and above, both trifluoperazine and chlorpromazine caused myotube contracture and cell loss. Drug combination experiments revealed that receptor stimulation caused by phenothiazines is overcome by low concentrations of veratridine and ryanodine, but not by membrane depolarization with 20 mM KCl. These results lend support to the role of calcium as an intracellular messenger in acetylcholine receptor synthesis regulation, but are difficult to reconcile with the notion that cytosolic calmodulin serves as the calcium receptor in this signaling pathway. Since the trifluoperazine effect resembles that caused by the calcium antagonist D-600, phenothiazines may stimulate receptor synthesis by blocking a voltage-gated calcium channel.     

Inhibition of phototropism inPhycomyces sporangiophores by calmodulin antagonist and antimicrotubular agents

A simple method was devised to measure growth and phototropism in isolated sporangiophores fromPhycomyces blakesleeanus. With this method, the effects of a number of substances that affect different metabolic reactions were tested. A large number of compounds inhibited or stimulated sporangiophore growth, but did not affect the phototropic response. Only colchicine, thiabendazole, and specially trifluoperazine affected both sporangiophore growth and phototropism. These results suggest that microtubules and calmodulin are involved in the response of the fungus to light.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Development, food intake, and ethinylestradiol influence hepatic triglyceride lipase and LDL-receptor mRNA levels in rats

The influence of development and ethinylestradiol on low density lipoprotein (LDL)-receptor mRNA and hepatic triglyceride lipase (HTGL) activity and mRNA levels was studied in rat liver and intestine. Intestinal LDL-receptor mRNA levels are maximal in the perinatal period, whereas liver LDL-receptor and HTGL mRNA levels are highest after weaning in adult life. All mRNA levels reach a maximum between day 15 and 20 when rats still consume a lipid-rich diet, and increase twofold during weaning. Liver and intestinal LDL-receptor mRNA levels are not influenced by ovariectomy, but increase after ethinylestradiol treatment. Liver LDL-receptor mRNA shows a dose-dependent increase after ethinylestradiol and a sevenfold rise in liver LDL-receptor mRNA is attained with a dose of 2000 micrograms/day. Intestinal LDL-receptor mRNA increases slightly more than twofold after ethinylestradiol and this increase is not dose-dependent. Changes in LDL-receptor mRNA are independent of changes in food intake induced by ethinylestradiol treatment, since they are still observed after pair-feeding. The ethinylestradiol-induced increases in LDL-receptor mRNA levels are reflected by decreased serum apoB levels. HTGL mRNA levels increase after ovariectomy and show a dose-dependent decrease after ethinylestradiol. Pair-feeding abolishes the increase seen after ovariectomy, while the estrogen-mediated decrease is attenuated. These alterations in HTGL mRNA are reflected by similar changes in liver HTGL activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discovery of novel and orally active FXR agonists for the potential treatment of dyslipidemia & diabetes

Herein we describe the synthesis and structure activity relationship of a new class of FXR agonists identified from a high-throughput screening campaign. Further optimization of the original hits led to molecules that were highly active in an LDL-receptor KO model for dyslipidemia. The most promising candidate is discussed in more detail.     

Heterogeneity of human lipoprotein Lp[a]: cytochemical and biochemical studies on the interaction of two Lp[a] species with the LDL receptor

Human Lp[a] can be fractionated into two species with different affinities for lysine-Sepharose. Forty to 81% of the total Lp[a] in the density fraction 1.055-1.15 g/ml from five individuals was retained by this affinity column. The remaining unretained Lp[a] species with no apparently functional lysine binding site was similar to the retained species in its electrophoretic mobility, lipid, protein, and apolipoprotein composition, and the heterogeneity was not related to apo[a] size polymorphism. Interaction of the two species with the low density lipoprotein (LDL) receptor was studied in human fibroblasts. Using gold-labeled lipoproteins and an immunochemical procedure, both Lp[a] species could be located in clusters on the cell surface, but the extent of labeling was far lower than that seen with LDL. Both Lp[a] variants were less effective than LDL in 1) down-regulation of LDL-receptor activity; 2) suppression of cellular sterol synthesis; and 3) stimulation of cholesteryl ester formation in human fibroblasts. Although degradation of both species of Lp[a] by the perfused rat liver was stimulated after estrogen induction of hepatic LDL-receptor activity, the stimulation amounted to only a quarter of that seen with LDL. The heterogeneity of Lp[a] with respect to the ability to bind epsilon-aminocarboxylic acid will need to be considered in studying the physiological role of this lipoprotein. Both Lp[a] species exhibited a similar interaction with the LDL-receptor in vitro, and confirmed previous investigations that Lp[a] is only a poor ligand for the LDL-receptor.     

Colchicine stimulation of hyaluronate synthesis and secretion in bone organ culture

Parathyroid hormone (PTH) is known to have a number of effects on bone tissue in vitro, including the stimulation of calcium release and the synthesis and turnover of hyaluronate. PTH-stimulated calcium release is inhibited by colchicine. Since hyaluronate may play a role in demineralization, calcium release into the media as a measure of bone resorption was correlated with the synthesis and secretion of ³H-glucosamine-labeled macromolecules. Newborn mice were labeled with ⁴⁵Ca, and the calvaria removed and incubated in vitro in media containing ³H-glucosamine. Addition of colchicine to the culture media inhibited the release of ⁴⁵Ca into the media while stimulating the synthesis and secretion of ³H-glucosamine labeled macromolecules. DEAE-cellulose chromatography resolved the labeled macromolecular material into four peaks, of which the third peak containing hyaluronate demonstrated approximately twice the amount of radioactivity. PTH stimulation of calcium release was inhibited likewise by colchicine, while ³H-glucosamine incorporation into labeled macromolecules was stimulated. Short term labeling studies emphasized the marked stimulatory effect that both PTH and colchicine have on the incorporation of ³H-glucosamine into hyaluronate. PTH stimulated the incorporation of ³⁵S-sulfate, while colchicine markedly inhibited its incorporation into non-dialyzable material. Both PTH and colchicine inhibited protein synthesis. Based on the observations, colchicine appears to stimulate the synthesis and secretion of hyaluronate and alter a number of metabolic pathways. Hyaluronate does not appear to be directly involved in the demineralization process.     

Effect of trifluoperazine on cholesterol metabolism of smooth muscle cells exposed to hypercholesterolemic medium in vitro

We recently demonstrated that the preventive effect of trifluoperazine (a potent inhibitor of calmodulin, protein kinase C, and phospholipase A2) on cholesterol-induced atherogenic activity of smooth muscle cells was mediated through its ability to inhibit smooth muscle cellular DNA synthesis coupled with stimulation of LDL receptor synthesis. The present study addressed the effect of trifluoperazine on cholesterol metabolism of aortic SMCs enriched with cholesterol through the nonreceptor pathway and revealed that (a) TFP caused inhibition of cholesterol synthesis compared with control cells bathed with hypercholesterolemic medium alone. (b) The drug also caused inhibition of free cholesterol and cholesteryl ester accumulation within smooth muscle cells compared to control cells. These results demonstrate that the preventive effect of TFP on atherogenic activity of smooth muscle cells may also be due to its ability to affect the altered/modified cholesterol metabolism of smooth muscle cells exposed to hypercholesterolemic medium in vitro.     

Alteration of hormone-stimulated cyclic AMP synthesis in guinea pig peritoneal macrophages.

The decrease of PGE-stimulated cyclic AMP synthesis due to pretreatment of intact cells with PGE (hormone-specific desensitization) was shown to be a rapid process in macrophages. Desensitization was found to be extensive after 5-min treatment of macrophages with PGE₂ and almost complete after 20 min. Furthermore, incubation of intact macrophages with colchicine caused a two- to sixfold increase in the rate of PGE₁-stimulated cyclic AMP synthesis in intact macrophages. Colchicine alone did not alter cyclic AMP levels. The enhancing effect of colchicine is related to its ability to disrupt microtubules. Vinblastine, another microtubule-disrupting agent, caused similar enhancement of PGE-stimulated cyclic AMP synthesis; no enhancement was found when lumicolchicine was used. Hormonestimulated cyclic AMP synthesis by colchicine-treated macrophages was also measured after cell homogenization. The enhancement of hormone sensitivity by colchicine was found to be lost upon homogenization. These findings suggest that colchicine acts at the interior of the cell to reversibly affect adenylate cyclase.     

Interference with the transport of heparin-releasable lipoprotein lipase in the perfused rat heart by colchicine and vinblastine.

The effect of pretreatment with colchicine or vinblastine on the lipoprotein lipase activity of rat heart was studied. Administration of colchicine or vinblastine 4 h prior to perfusion of the heart caused a very marked reduction in lipoprotein lipase activity released into the perfusate within 1 min of heparin perfusion. At the same time an increase in residual heart lipase occurred so that total lipoprotein lipase content of the heart (heparin releasable plus residual) did not change. The colchicine effect was dose and time dependent; no decrease in heparin-releasable enzyme activity occurred after only 30 min of pretreatment or upon addition of colchicine into the perfusate. These results indicate that colchicine did not impede enzyme synthesis or its release from the cell surface, but may have interfered with the transport of lipoprotein lipase from the site of its synthesis to the endothelial cell surface.     

Cytoskeleton and differentiation: effects of cytochalasin B and colchicine on expression of the differentiated phenotype of rabbit costal chondrocytes in culture

Cytochalasin B changed the shape of cultured rabbit costal chondrocytes from polygonal to nearly spherical and stimulated glycosaminoglycan synthesis, which is a differentiated phenotype of chondrocytes, whereas colchicine changed them from polygonal to flattened and inhibited glycosaminoglycan synthesis. These morphological changes occurred parallel with the changes in glycosaminoglycan synthesis. Induction of ornithine decarboxylase by parathyroid hormone, which is a good marker of differentiated chondrocytes, was markedly potentiated in the spherical cells which had been pretreated with cytochalasin B, whereas pretreatment with colchicine inhibited the induction of the enzyme. Both cytochalasin B and colchicine inhibited DNA synthesis. The inhibitions were observed after the appearance of changes in the morphology of the cells and glycosaminoglycan synthesis. These findings suggest that intactness of microtubules and disruption of microfilaments are involved in regulating the expression of the differentiated phenotype of chondrocytes in culture.