Enhanced separation of antidepressant drugs using a polymerized nonionic surfactant as a transient capillary coating

PURPOSE: To investigate the role of the P-glycoprotein (P-gp) drug efflux pump in the intracellular disposition of colchicine and vinblastine. METHODS: Uptake and efflux kinetics were studied in vitro in human lymphocytes and in HL-60 cells with or without the P-gp modulator, verapamil. RESULTS: In human lymphocytes, colchicine was slowly taken up (uptake half-life was 18.9+/-1.1 hr.) and verapamil increased colchicine uptake by 37%, whereas it did not modify colchicine efflux from cells. In HL-60 cells, colchicine uptake was non-linear and slower than that of vinblastine, the colchicine uptake half-life (11.1+/-0.5 hr.) being 25-fold longer than that of vinblastine at 25 nM. Verapamil did not significantly modify colchicine uptake half-life, but increased its intracellular accumulation by 23% and that of vinblastine by 81%. Immuno-flow cytometry showed that P-gp expression in HL-60 cells increased significantly from 24 hr. following colchicine or vinblastine exposure. The significant increase in colchicine uptake induced by verapamil at 24 hr. was correlated with this enhanced P-gp expression. The drug efflux half-life was 11.5-fold higher for colchicine (23+/-0.9 hr) than vinblastine, indicating a much slower elimination of colchicine from cells that could be related to its longer dissociation half-life from the tubulin receptor. Verapamil treatment did not modulate either colchicine or vinblastine efflux kinetics, suggesting that the intracellular drugs are not available to the transmembrane P-gp binding sites. CONCLUSIONS: P-gp may not be the main reason for the slowness of colchicine uptake. It may be more efficient at controlling entry of colchicine and vinblastine through the plasma membrane than at mediating their efflux from HL-60 cells.     

Microtubules and pancreatic amylase release by mouse pancreas in vitro

The effects of vinblastine and colchicine on pancreatic acinar cells were studied by use of in vitro mouse pancreatic fragments. Vinblastine inhibited the release of amylase stimulated by bethanechol, caerulein, or ionophore A23187. Inhibition required preincubation with vinblastine,and maximum inhibition was observed after 90 min. Inhibition was relatively irreversible and could not be overcome by a high concentration of stimulant. Inhibition could also be produced by colchicine although longer preincubation was required and inhibition was only partial. Uptake of [3H]vinblastine and [3H]colchicine by pancreatic fragments was measured and found not to be responsible for the slow onset of inhibition by these drugs. In incubated pancreas, microtubules were present primarily in the apical pole of the cell and in association with the Golgi region. Vinblastine, under time and dose conditions that inhibited the release of stimulated amylase, also reduced the number of microtubules. The only other consistent structural effects of vinblastine were the presence of vinblastine-induced crystals and an increased incidence of autophagy. The remainder of cell structure was not affected nor were overall tissue ATP and electrolyte contents or the stimulant-induced increase in 45Ca++ efflux. It is concluded that the antisecretory effects of vinblastine and colchicine are consistent with a microtubular action, but that acinar cell microtubules are more resistant to the drugs than many other cell types.     

Functionality of protective colloids affecting the formation, size uniformity and morphology of drug-free polylactic acid microspheres

Colchicine is a microtubule depolymerizing agent used extensively in the study of cytoskeleton-dependent cell functions. In studying the possible functional interaction between the GABA(A) receptor and the cytoskeleton, we found that colchicine inhibits GABA(A) receptor function by mechanisms independent of microtubule depolymerization. Human GABA(A) receptor alpha1beta2gamma2L subunits were co-expressed in Xenopus oocytes and the effects of colchicine on GABA(A) receptor function was assessed using the two-electrode voltage-clamp technique. Co-application of GABA (10 microM) with colchicine (100 microM) resulted in a 59.9% inhibition of GABA-gated chloride currents. This effect was instantaneous in onset with no pre-incubation required and reversed within seconds. Other depolymerizing agents, such as nocodazole (20 microM) and vinblastine (200 microM), did not affect GABA(A) receptor function using the same co-application protocol used with colchicine. The polymerizing agent taxol (10-50 microM) did not affect colchicine inhibition of the GABA responses and did not itself alter GABA-gated chloride currents. The inhibitory effect of colchicine was present under conditions in which the oocyte microtubules had been depolymerized by cold temperature. These results indicate that colchicine inhibits the GABA(A) receptor via mechanisms unrelated to microtubule depolymerization. To further examine the inhibitory effect of colchicine on the GABA response, GABA (10-3000 microM) concentration-response curves were performed in the absence or presence of various concentrations of colchicine (30-300 microM). In the presence of colchicine, the GABA concentration-response curve was shifted to the right in a parallel fashion. A Schild plot of this data yielded a linear slope indicating that colchicine acts as a competitive antagonist at the GABA binding site. We conclude that colchicine is a competitive antagonist at the GABA(A) receptor and that studies using colchicine to examine the functional interaction between GABA(A) receptors and microtubules should be interpreted with caution.     

The effect of colchicine on the induction of ornithine decarboxylase by 12-O-tetradecanoyl-phorbol-13-acetate

The induction of mouse epidermal ornithine decarboxylase, 1 of the earliest and largest phenotypic changes following treatment of mouse skin with the tumor-promoting agent, 12-O-tetradecanoyl-phorbol-13-acetate, can be inhibited by prior administration of colchicine. Maximal inhibition of this enzyme induction was observed when colchicine was injected i.p. 90 or 120 min before promoter treatment, although time intervals up to 20 hr between colchicine and promoter treatment were effective. The effect of colchicine was dose dependent, with a dose as low as 25 nmoles/mouse causing an inhibition of 35%. Other microtubule-disrupting agents, vinblastine, vincristine, and Colcemid, had a similar effect on ornithine decarboxylase activity. However, beta, gamma-lumicolchicine, a photochemical derivative of colchicine with no antimitotic or microtubule-disrupting ability, and cytochalasin B, an inhibitor of microfilament-dependent processes, had no effect. N6, O2'-dibutyryl 3',5'-cyclic adenosine monophosphate, when administered just before colchicine, blocked the inhibitory action of colchicine. The results of these studies suggest that colchicine-sensitive structures, most likely containing microtubules, may be mediating elements between the binding of tumor promoters, perhaps to specific cell surface receptors, and the subsequent induction of ornithine decdaboxylase.     

Role of P-glycoprotein in colchicine and vinblastine cellular kinetics in an immortalized rat brain microvessel endothelial cell line

Uptake and efflux of colchicine and vinblastine, whose effects are related to their high-affinity binding to tubulin, were studied in the immortalized rat brain microvessel endothelial cell line RBE4. At 10 nM extracellular drug concentration, uptake equilibrium was approached at 45 hr for colchicine, but at only 3.5 hr for vinblastine. After 1 hr preincubation with 200 nM colchicine or vinblastine, drug efflux fitted biexponential kinetics with an initial fast phase (half-life = 2.2 min and 9.6 min, respectively) and a later slow phase (half-life = 3.6 hr and 1.8 hr, respectively). After 6 hr preincubation with 200 nM colchicine, only the slow phase (half-life = 3.6 hr) could be observed. The colchicine and vinblastine uptake rate was increased by cyclosporin A, an inhibitor of the drug efflux pump P-glycoprotein, which is expressed at the blood-brain barrier. Whereas cyclosporin A decreased vinblastine efflux, its effect on colchicine efflux was apparent after only 13 hr washout and was associated with the re-uptake by cells of colchicine molecules. Differences in uptake kinetics of colchicine and vinblastine could be related to differences in their lipid solubility, and mainly in their binding affinities to tubulin. Differences in efflux kinetics could in addition be explained by the involvement of P-glycoprotein in the efflux of vinblastine, whereas efflux of colchicine was not influenced by this pump. Indeed, binding of colchicine to tubulin would imply that most intracellular colchicine may be inaccessible to P-glycoprotein. In the case of a cytotoxic drug such as colchicine, which is tightly bound to intracellular receptors, the role of P-glycoprotein within the blood-brain barrier would be more to protect the brain against entry of this drug than to detoxify the brain by its extraction.     

Effect of glucagon on pinocytosis by the yolk sac of the rat

The uptake of macromolecular markers by fluid pinocytosis in the rat yolk sac was inhibited by glucagon, with half-maximal effect at a hormone concentration of approximately 3 X 10(-8) M. Glucagon had no effect on the cellular distribution of the marker subsequent to its uptake. Rates of uptake promptly returned to normal when the yolk sacs were transferred from a glucagon-containing to a glucagon-free medium. Epinephrine also inhibited, but only at much higher concentrations. The effect of the latter was augmented by theophylline. Insulin (10(-6) M) had no effect when added alone or with an inhibitory level of glucagon (10(-7) M). The presumption that the hormone effect was mediated by cyclic AMP was supported by the findings that the cellular levels of cyclic AMP were elevated in the presence of glucagon and that dibutyryl cyclic AMP could replace glucagon as an effective inhibitor. The conclusion that the hormone effect was on uptake rather than on subsequent regurgitation was based on the linearity of accumulation in both the presence and absence of glucagon and the inability of glucagon to stimulate loss of invertase from preloaded cells. Colchicine and vinblastine also inhibited uptake. This finding and those of others which are discussed suggest the possibility that effects of cyclic nucleotides on certain cell functions may involve their regulation of microtubular status.     

Microtubule-interfering agents activate c-Jun N-terminal kinase/stress-activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways

The essential cellular functions associated with microtubules have led to a wide use of microtubule-interfering agents in cancer chemotherapy with promising results. Although the most well studied action of microtubule-interfering agents is an arrest of cells at the G2/M phase of the cell cycle, other effects may also exist. We have observed that paclitaxel (Taxol), docetaxel (Taxotere), vinblastine, vincristine, nocodazole, and colchicine activate the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathway in a variety of human cells. Activation of JNK/SAPK by microtubule-interfering agents is dose-dependent and time-dependent and requires interactions with microtubules. Functional activation of the JNKK/SEK1-JNK/SAPK-c-Jun cascade (where JNKK/SEK1 is JNK kinase/SAPK kinase) was demonstrated by activation of a 12-O-tetradecanoylphorbol-13-acetate response element (TRE) reporter construct in a c-Jun dependent fashion. Microtubule-interfering agents also activated both Ras and apoptosis signal-regulating kinase (ASK1) and coexpression of dominant negative Ras and dominant negative apoptosis signal-regulating kinase exerted individual and additive inhibition of JNK/SAPK activation by microtubule-interfering agents. These findings suggest that multiple signal transduction pathways are involved with cellular detection of microtubular disarray and subsequent activation of JNK/SAPK.     

Metabolism of methanol by Rhodopseudomonas acidophila

The mechanisms whereby neutrophils become cytotoxic to chicken erythrocyte (CRBC) target cells were investigated in a system of lectin-dependent neutrophil-mediated cytotoxicity (LDNMC). Through the use of drugs and specific metabolic inhibitors, LDNMC was found to be dependent on energy supplied by anaerobic glycolysis and on other active metabolic functions of the neutrophil. 2-Iodoacetamide, 2-deoxy-D-glucose, di-isopropyl-fluorophosphate, colchicine, cytochalasin B, and dibutyryl cyclic AMP all caused dose-dependent inhibition of cytotoxicity, while inhibitors of protein and nucleic acid synthesis were without effect. Cell surface membrane-active agents, such as chloroquine, hydrocortisone and chlorpromazine inhibited cytotoxicity, while vitamin A caused enhancement. Lectins which agglutinated neutrophils, but not necessarily CRBC, such as phytohaemagglutinin (PHA-P), concanavalin A (Con A), soybean agglutinin (SBA), and Ricinus communis agglutinin (RCA), mediated cytotoxicity while lectins which did not cause agglutination, such as pokeweed mitogen (PWM), did not mediate cytotoxicity. Preincubation of neutrophils, but not CRBC with PHA-P, resulted in time-dependent enhancement of cytotoxicity, while pre-incubation with Con A yielded progressive inhibition of cytotoxicity. These studies suggest that lectin binding to the cell surface causes alterations of the membrane, that LDNMC requires cell to cell surface contact, and that cytotoxicity depends on active metabolic processes.     

Modulation of membrane drug permeability in Chinese hamster ovary cells

The kinetics of colchicine uptake into Chinese hamster ovary cells have been investigated and found to be consistent with an unmediated diffusion mode. A variety of compounds such as local anesthetics and non-ionic detergents as well as drugs such as vinblastine, vincristine, daunomycin and actinomycin D potentiate the rate of colchicine uptake into these cells and into colchicine resistant mutants. In all cases, higher concentrations of these compounds were required to stimulate colchicine uptake in the colchicine resistant mutants than in the cells of the parental line. This stimulation was observed also in the uptake of puromycin, a structurally and functionally different drug. These stimulatory agents did not, however, cause the cells to become nonspecifically leaky since the uptake of 2-deoxy-D-glucose was unaffected. In addition, the activation energy of colchicine uptake was unaltered in the presence of stimulating agents, implying that they were not causing colchicine to enter the cells via a different mechanism. The results are compatible with the view that these compounds are membrane-active, and are able to stimulate an increased rate of unmediated diffusion of colchicine into the cells. It appears that a mechanism for the regulation of passive permeability is modified in the resistant mutants.     

Role of P-glycoprotein in the blood-brain transport of colchicine and vinblastine

Classically, drug penetration through the blood-brain barrier depends on the lipid solubility of the substance, except for some highly lipophilic drugs, like colchicine and vinblastine, both substrates of P-glycoprotein, a drug efflux pump present at the luminal surface of the brain capillary endothelial cells. Colchicine and vinblastine uptake into the brain was studied in the rat using the in situ brain perfusion technique and two inhibitors of P-glycoprotein, verapamil and SDZ PSC-833. When rats were pretreated with PSC-833 (10 mg/kg, intravenous bolus), colchicine and vinblastine uptake was enhanced 8.42- and 9.08-fold, respectively, in all the gray areas of the rat brain studied. The mean colchicine distribution volume was increased from 0.67 +/- 0.41 to 5.64 +/- 0.70 microliters/g and vinblastine distribution volume from 2.74 +/- 1.15 to 24.88 +/- 4.03 microliters/g. When rats were pretreated with verapamil (1 mg/kg, intravenous bolus), colchicine distribution volume was increased 3.70-fold. The increase in colchicine and vinblastine did not differ between the eight brain gray areas. PSC-833 and verapamil pretreatment had no influence on the distribution volume of either drug in the choroid plexus. Nevertheless, distribution volumes remained small, considering the highly lipophilic nature of the substances. We suggest that P-glycoprotein is either only partially inhibited (difficulty of fully saturating P-glycoprotein, especially under in vivo conditions) or not the only barrier to these two drugs.     

Involvement of P1 receptors in the effect of forskolin on cyclic AMP accumulation and export in PC12 cells

In PC12 cells, forskolin as well as the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased intracellular adenosine-3',5'-cyclic monophosphate (cyclic AMP) levels, which peaked at 45-60 minutes and declined thereafter. Maximum levels were 3000 and 1700 pmol/10(6) cells during treatment with 10 microM forskolin or 0.1 microM NECA, respectively. Extracellular cyclic AMP rose with time, at mean rates of 24.7 (forskolin) and 11.3 (NECA) pmol/min/10(6) cells. With either drug, a linear correlation was obtained between the calculated time integral of intracellular cyclic AMP and the measured extracellular cyclic AMP levels, indicating that the outflow of cyclic AMP was sustained by a nonsaturated transport system. The ability of forskolin to increase intracellular and extracellular cyclic AMP levels was hindered in a concentration-dependent manner by 8-(p-sulfophenyl)theophylline (8-SPT). A similar inhibition was exerted by other two adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine and 3,7-dimethyl-1-propargylxanthine. The concentration-response curve to adenosine was shifted to the right by 25 microM 8-SPT, whereas that of forskolin was shifted downwards. Adenosine deaminase (ADA, EC 3.5.44, 1 U/mL) reduced the intracellular cyclic AMP response to forskolin by 68%, whereas the adenosine transport inhibitor, dipyridamole (10 microM), significantly increased 1 and 10 microM forskolin-dependent cyclic AMP accumulation. Erythro-9-(2-hydroxy-3-nonyl)adenine (10 microM), an inhibitor of ADA, and alpha,beta-methyleneadenosine 5'-diphosphate (100 microM), an inhibitor of ecto-5'-nucleotidase, did not alter forskolin activity. These results demonstrate that a cyclic AMP extrusion system operates in PC12 cells during adenylyl cyclase stimulation by forskolin and that this stimulation involves a synergistic interaction with endogenous adenosine. However, extruded cyclic AMP does not appear to significantly contribute to the formation of the endogenous adenosine pool.     

Effect of the microtubular inhibitor vinblastine on ferritin clearance and release in the rat

We have previously demonstrated that colchicine inhibits ferritin clearance from the circulation of normal and iron-loaded rats and stimulates endogenous ferritin release into both the serum and bile of iron-loaded rats. The aim of the present study was to determine the effect of vinblastine on ferritin clearance and release in normal and iron-loaded rats. Vinblastine was administered at either 1 or 10 mg/kg to both normal and iron-loaded rats, infused over a 5 h period with either a rat liver ferritin or saline solution. Serum and biliary ferritin levels were determined every 30 min. After 5 h, 90% of the infused ferritin was cleared from the circulation in the absence of vinblastine. Low-dose vinblastine decreased ferritin uptake 10-20% in iron-loaded rats. High-dose vinblastine inhibited ferritin clearance by 25% in normal rats and 20-40% in iron-loaded rats. Vinblastine administration caused a 2-3-fold increase in the serum ferritin concentration and a 3-5-fold peak in biliary ferritin levels. Thus, vinblastine caused the release of endogenous ferritin into both the serum and bile of iron-loaded rats in the presence of a ferritin load. We therefore conclude that disturbed microtubule function accounts for the observed inhibition of ferritin uptake and intracellular transport; however, the mechanism of increased ferritin release remains unclear.     

Role of microtubules in the adaptive response to low phosphate of Na/Pi cotransport in opossum kidney cells

The role of microtubules and actin microfilaments in adaptive changes of the apical Na-dependent transport of phosphate (Pi) was investigated in opossum kidney (OK) cells. Up-regulation of Na/Pi cotransport was achieved by incubating OK cells in a medium containing 0.1 mM Pi; down-regulation of Na/Pi cotransport was provoked by refeeding adapted cells with 2 mM Pi. Up-regulation of Na/Pi cotransport was found to be inhibited by approximately 50% after a pretreatment of the cells with the microtubule disrupting agents nocodozole and colchicine; indirect immunofluorescence indicated complete depolymerization of the microtubular network. No inhibition of the adaptive response was observed after treatment of the cells with cytochalasin B to depolymerize actin microfilaments. In adapted cells, depolymerization of microtubules by nocodozole led to a reversibility of Na/Pi cotransport similar to that observed after refeeding adapted cells with 2 mM Pi. No effects of the microtubule disrupting drugs were observed on Na/L-glutamic acid transport. Depolymerization of microtubules did not prevent parathyroid-hormone-mediated inhibition of Na/Pi cotransport. It is concluded that microtubules are (at least in part) involved in the correct insertion of newly synthesized apical Na/Pi cotransport systems and that microtubules are not involved in the internalization of Na/Pi cotransport systems.     

Prostatic intraepithelial neoplasia. Development of a Bayesian belief network for diagnosis and grading

The nucleotide regulation of a calcium-activated nonselective cation (Ca-NS+) channel has been investigated in the rat insulinoma cell line CRI-G1. The activity of the channel is reduced by both AMP and ADP (1-100 microM) in a concentration-dependent manner, with AMP being more potent than ADP. At lower concentrations (0.1-5 microM), both ADP and AMP activate the channel in some patches. Examination of the nucleotide specificity of channel inhibition indicates a high selectivity for AMP over the other nucleotides tested with a rank order of potency of AMP > UMP > CMP > or = GMP. Cyclic nucleotides also modulate channel activity in a complex, concentration-dependent way. Cyclic AMP exhibits a dual effect, predominantly increasing channel activity at low concentrations (0.1-10 microM) and reducing it at higher concentrations (100 microM and 1 mM). Specificity studies indicate that the cyclic nucleotide site mediating inhibition of channel activity exhibits a strong preference for cyclic AMP over cyclic GMP, with cyclic UMP being almost equipotent with cyclic AMP. Cyclic IMP and cyclic CMP are not active at this site. The cyclic nucleotide site mediating activation of the channel shows much less nucleotide specificity than the inhibitory site, with cyclic AMP, cyclic GMP and cyclic IMP being almost equally active.     

Stabilization of microtubule dynamics by estramustine by binding to a novel site in tubulin: a possible mechanistic basis for its antitumor action

The cellular targets for estramustine, an antitumor drug used in the treatment of hormone-refractory prostate cancer, are believed to be the spindle microtubules responsible for chromosome separation at mitosis. Estramustine only weakly inhibits polymerization of purified tubulin into microtubules by binding to tubulin (Kd, approximately 30 microM) at a site distinct from the colchicine or the vinblastine binding sites. However, by video microscopy, we find that estramustine strongly stabilizes growing and shortening dynamics at plus ends of bovine brain microtubules devoid of microtubule-associated proteins at concentrations substantially below those required to inhibit polymerization of the microtubules. Estramustine strongly reduced the rate and extent both of shortening and growing, increased the percentage of time the microtubules spent in an attenuated state, neither growing nor shortening detectably, and reduced the overall dynamicity of the microtubules. Significantly, the combined suppressive effects of vinblastine and estramustine on the rate and extent of shortening and dynamicity were additive. Thus, like the antimitotic mechanisms of action of the antitumor drugs vinblastine and taxol, the antimitotic mechanism of action of estramustine may be due to kinetic stabilization of spindle microtubule dynamics. The results may explain the mechanistic basis for the benefit derived from combined use of estramustine with vinblastine or taxol, two other drugs that target microtubules, in the treatment of hormone-refractory prostate cancer.     

Tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle

Tubulin is the biochemical target for several clinically used anticancer drugs, including paclitaxel and the vinca alkaloids vincristine and vinblastine. This review describes both the natural and synthetic agents which are known to interact with tubulin. Syntheses of the more complex agents are referenced and the potential clinical use of the compounds is discussed. This review describes the biochemistry of tubulin, microtubules, and the mitotic spindle. The agents are discussed in relation to the type of binding site on the protein with which they interact. These are the colchicine, vinca alkaloid, rhizoxin/maytansine, and tubulin sulfhydryl binding sites. Also included are the agents which either bind at other sites or unknown sites on tubulin. The literature is reviewed up to October 1997.     

Exit transport of a cyclic nucleotide from mouse L-cells

At a concentration of 30 mum, 1,4,5,6,8-pentazaacenaphthylene, 3-amino-1,5-dihydro-5-methyl-1-beta-D-[5-14C]ribofuranosyl (NSC-154020), a tricyclic, 7-deazapurine nucleoside (TCN) is rapidly taken up by cultured mouse L-cells and converted to intracellular TCN-monophosphate, but not further metabolized. The TCN-monophosphate is also excreted by the cells into the medium. It is released by a saturable process against a concentration gradient and the release is inhibited by various inhibitors of energy production. This inhibition correlates with a depletion of the cells of ATP. Thus TCN-monophosphate excretion probably involves an active transport system. This transport system is highly temperature-dependent (the Q10 falls between 3 and 4) and is inhibited by papaverine, theophylline, Persantin, Probenecid, phenethyl alcohol and p-chloromercuribenzoate, but not by 500 muM cyclic AMP, AMP, or adenosine. Significant amounts of various natural phosphorylated intermediates (AMP, ATP, UTP, UMP, UDP-hexoses, and phosphorylcholine) are not released from the cells under similar experimental conditions either in the absence or presence of 30 muM TCN.     

Wide-spectrum antibiotic activity of synthetic, amphipathic peptides

Transbilayer phosphatidylethanolamine (PtdEtn) movements in the plasma membrane of Saccharomyces cerevisiae are regulated by an ATP-dependent, protein-mediated process(es). To examine whether this process is influenced by the actin cytoskeleton, we have studied the PtdEtn translocation in S. cerevisiae cells after treatment with microfilament disrupting and microtubule-disrupting agents. PtdEtn translocation was studied by measuring the external PtdEtn levels, using fluorescamine as the external membrane probe, in the ATP-depleted, ATP-depleted and repleted, and N-ethylmaleimide-treated cells. The microfilaments and microtubules were disrupted by treatment with various cytochalasins and colchicine (or benomyl) respectively PtdEtn translocation became abnormal in the cytochalasin-treated cells but not in cells that were treated with microtubule-disrupting agents, such as colchicine or benomyl. These results have been interpreted to suggest that the actin cytoskeleton is involved in regulating the PtdEtn translocase activity in the yeast cell plasma membrane.     

Suggestibility in hospitalized schizophrenics

Tissue factor activity in suspension cultures of WISH amnion cells is modulated by pharmacologic doses of agents which alter membrane structure and function. Lysosomal stabilizing steroids (hydrocortisone, dexamethasone, aldosterone, prednisolone, and estradiol) suppress the change in activity which follows subculture; lytic steroids (testosterone and progesterone) are ineffective. Chloroquine both increases the specific activity and extends the time before return to the basal level. Dimethyl sulfoxide and ouabain suppress the complete expression of activity but do not inhibit the subsequent decay. The effect of cytochalasin B is complex, the drug being either suppressive or slightly stimulatory depending on the time of addition. Cyclic nucleotides (AMP or GMP) or insulin do not regulate the expression of tissue factor in these cells. A dramatic increment and prolongation of activity occurs when colchicine or vinblastine is added to the cell suspension shortly after subculture; there is much less stimulation by griseofulvin. Lumicolchicine has no effect while deuterium oxide is inhibitory. From these experiments, we conclude that increased membrane fluidity or altered secretory processes resulting from microtubule disruption stabilize tissue factor in cultured cells. Since contradictory results were obtained with agents which stabilize lysosomes or inhibit transport, the role of these cellular functions in tissue factor production or decay is unclear.