Modulation of passive permeability by external ATP and cytoskeleton-attacking agents in cultured mammalian cells

External ATP causes a passive permeability change in several transformed cells, but not in untransformed cells. We previously demonstrated that in CHO-K1 cells, a transformed clone of Chinese hamster ovary cells, the external ATP-dependent permeability change was induced when the intracellular ATP concentration was reduced by a mitochondrial inhibitor (Kitagawa, T. and Akamatsu, Y. (1981) Biochim. Biophys. Acta 649, 76–82). A permeability change with similar characteristics was also observed when the CHO cells were treated with external ATP and a cytoskeleton-attacking agent such as vinblastine or cytochalasin B. Just like mitochondrial inhibitors, vinblastine could increase the sensitivity of transformed 3T3 cells to external ATP but showed no effect on passive permeability of normal 3T3 cells. However, in contrast with the effect of the mitochondrial inhibitors, the cytoskeleton drugs caused the permeability change with little reduction of intracellular ATP concentration, suggesting different actions of these two kinds of drug on the permeability change. The present results suggest an important role of cytoskeletal structures in controlling the external ATP-dependent permeability change in transformed cells. Possible effects of intracellular ATP on cytoskeletal structures are also discussed.     

External ATP-induced passive permeability change and cell lysis of cultured transformed cells: action in serum-containing growth media

External ATP causes a marked increase in the passive permeability to phosphorylated metabolites in several types of transformed cells in alkaline medium containing low concentrations of Ca2+, but not in untransformed cells. Such increased membrane permeability with external ATP was also observed in B16 melanoma cells at pH 7.4-7.5 in both Tris-buffered saline and a growth medium containing 10% calf serum and divalent ions at normal concentrations, although a higher concentration of ATP was required. The permeability change in the growth medium was significantly enhanced by calmodulin-interacting drugs, such as trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and chlorpromazine (CPZ). As expected, prolonged exposure of the cells to ATP in the serum-containing medium led to cell lysis. This ATP-dependent cell lysis was observed only in several transformed cell lines, and not in untransformed mouse fibroblasts. These results indicate that the effect of ATP on the membrane permeability in transformed cells is elicited under the physiological conditions and this would be useful in some limited way for cancer chemotherapy management.     

Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.     

Control of membrane permeability by external ATP in mammalian cells: isolation of an ATP-resistant variant from Chinese hamster ovary cells

External ATP causes a great increase in the passive permeability of the plasma membrane for phosphorylated metabolites and other small molecules in cultured mammalian cells. We previously demonstrated that in CHO-K1 cells an ATP-dependent permeability change was induced in the presence of a mitochondrial inhibitor (KCN or rotenone), a cytoskeleton-attacking agent (vinblastine) and a calmodulin antagonist (trifluoperazine). These permeability changes were reversible but long exposure, for 30-60 min, to ATP together with a mitochondrial inhibitor significantly reduced the cell viability of the treated cells. Since this cell lysis was shown to be due to the ATP-dependent permeability change, we could isolate several clones resistant to the action of the external ATP from CHO-K1 cells after repeated treatment with ATP and rotenone. In 9.1 cells, one of the isolated clones, little or no ATP-dependent permeability change was observed in the presence of either a mitochondrial inhibitor, vinblastine or trifluoperazine. This CHO variant could be specifically resistant as to the change in membrane permeability induced by external ATP, since the permeabilities for the 2-deoxyglucose and drugs used in the present studies were similar to those in the case of the parent cells. These results suggest that a specific defect or alteration in the plasma membrane is involved in the ATP-dependent permeability change. It is also reported that Mg2+-dependent ATPase activity was found on the cell surface of both CHO-K1 and 9.1 cells, and this activity was shown to be not involved in the permeability change controlled by external ATP.     

Modulation of drug permeability in Chinese hamster ovary cells. Possible role for phosphorylation of surface glycoproteins.

We have previously reported that the uptake of colchicine and other drugs in Chinese hamster ovary (CHO) cells can be greatly enhanced by the addition of metabolic inhibitors such as cyanide (See, Y.P., Carlsen, S.A., Till, J.E. and Ling, V. (1974) Biochim. Biophys. Acta 373, 242-252). This has led us to postulate the presence of an active drug permeability barrier in these cells. In this paper we provide evidence for the dependence of this permeability barrier on intracellular ATP levels. Colchicine-resistant mutants of CHO cells exhibiting a reduced drug permeability, however, can maintain this drug permeability barrier at much lower ATP levels, suggesting that they possess an altered active drug permeability barrier. We have also observed a membrane-associated protein kinase-phosphoprotein phosphatase system in the isolated membranes of mutant and wild-type cells. Differences in the intrinsic protein phosphorylation patterns between the membranes of these cells have led us to conclude that the control of the drug permeability barrier may be mediated via the phosphorylation of at least two high molecular weight surface glycoproteins.     

Antiasthmatic drugs targeting the cysteinyl leukotriene receptor 1 alleviate central nervous system inflammatory cell infiltration and pathogenesis of experimental autoimmune encephalomyelitis

Cysteinyl leukotrienes (CysLTs) are potent proinflammatory mediators and are considered to play a key role in inflammatory diseases such as asthma. Antagonists targeting the receptor of CysLTs (CysLT1) are currently used as antiasthmatic drugs. CysLTs have also been implicated in other inflammatory reactions. In this study, we report that in experimental autoimmune encephalomyelitis animals, CysLT1 is upregulated in immune tissue and the spinal cord, and CysLT levels in the blood and cerebrospinal fluid are also higher than in normal mice. Two clinically used antiasthma drugs, montelukast and zafirlukast, both targeting CysLT1, effectively block the CNS infiltration of inflammatory cells and thus reduce the incidence, peak severity, and cumulative clinical scores. Further study indicated that CysLT1 signaling does not affect the differentiation of pathogenic T helper cells. It might affect the pathogenesis of experimental autoimmune encephalomyelitis by increasing the secretion of IL-17 from myelin oligodendrocyte glycoprotein-specific T cells, increasing the permeability of the blood-brain barrier and inducing chemotaxis of T cells. These effects can be blocked by CysLT1 antagonists. Our findings indicate that the antiasthmatic drugs against CysLT1 can also be used to treat multiple sclerosis.     

Verapamil enhances high-density lipoprotein processing in Hep G2 cells preloaded with cholesterol

The effects of the calcium channel blocker of the arylalkylamine series verapamil have been investigated on high-density lipoprotein (HDL3) catabolism in the human hepatoma cell line Hep G2. It was found that verapamil markedly enhanced HDL3 binding, uptake and degradation in Hep G2 cells preloaded with nonlipoprotein cholesterol. This effect was dose-dependent, and a 1.5-2-fold increase of the three studied parameters was observed in cells pretreated 24 h with 100 microM verapamil. No significant effect of the drug was found in cells not preincubated with cholesterol. Verapamil induced an increase in the cellular cholesterol content in preloaded cells. Other calcium antagonists such as diltiazem, nifedipine, nitrendipine or amphiphilic drugs such as phenothiazines and propranolol also enhanced HDL3 uptake by Hep G2 cells. These effects of verapamil on HDL3 metabolism could be related to its amphiphilic characteristics, and to its calcium antagonist properties.     

Changes in membrane permeability during semliki forest virus induced cell fusion

The infection of Aedes albopictus cells by Semliki Forest virus (SFV) is a non lytic event. Exposure of infected cells to mildly acidic pH (<6.2) leads to syncytium formation. This polykaryon formation is accompanied by an influex of protons into the cells (Kempfet al. Biosci. Rep. 7, 761–769, 1987). We have further investigated this permeability change using various fluorescent or radiolabeled compounds. A significant, pH dependent increase of the membrane permeability to low molecular weight compounds (Mr<1000) was observed when infected cells were exposed to a pH<6.2. The pH dependence of the peremability change was very similar to the pH dependence of cell-cell fusion. The permeability change was sensitive to divalent cations, protons and anionic antiviral drugs such as trypan blue. The nature of this virus induced, pH dependent permeability change is discussed.     

Influence of hypertension and of antihypertensive drugs on the arterial wall

This paper reviews some of the experimental data regarding the effects of hypertension and antihypertensive drugs on the arterial wall. Hypertension induces major changes in both the arterial media and intima. Experimental studies from our own and other laboratories have demonstrated that medial smooth muscle cells in several forms of hypertension in the rat undergo hypertrophy and nuclear polyploidy which contribute, along with connective tissue alterations, to a large increase in medial mass. Our studies in the deoxycorticosterone/salt-hypertensive rat indicate that such changes may be difficult to regress, despite prolonged control of the hypertension. In the arterial intima, major alterations in the endothelium are induced by hypertension in association with increase in arterial permeability. Marked enhancements of adherence of circulating white blood cells to the endothelium can also be demonstrated along with penetration of blood monocytes and their accumulation in the subendothelial space. Hypertension also appears to stimulate the migration and proliferation of smooth muscle cells in the intima, and evidence is beginning to accumulate that endogenous growth factors within the artery may be involved in this process. Essentially all of the intimal changes which we have observed as a result of arterial hypertension are also present with cholesterol feeding although intimal accumulation of lipid and formation of atherosclerotic plaques do not occur with hypertension alone. On the other hand, in hypercholesterolemic animals, hypertension appears to act as a promoter of atherogenesis. Several antihypertensive drugs may influence the atherosclerotic process. The experimental data regarding the effects of beta blockers and calcium antagonists in the cholesterol-fed rabbit are discussed. Though of considerable interest, the clinical relevance of the findings remains uncertain.     

Structure activity relationships for bradykinin antagonists on the inhibition of cytokine release and the release of histamine

Highly potent bradykinin antagonists were found to inhibit bradykinin-induced release of cytokines but to stimulate histamine release. Both actions show structural requirements completely different from those for bradykinin B1 and B2 receptors, indicating that the release of some cytokines from spleen mononuclear cells and of histamine from rat mast cells is not mediated by these receptors. Most potent bradykinin antagonists release histamine at lower concentrations than does bradykinin itself. Dimers of bradykinin antagonists are the most potent compounds for histamine release. In contrast to enhanced histamine release, potent inhibition of cytokine release enhances the applicability of these compounds as anti-inflammatory drugs. Many of the peptides designed for high B2-receptor antagonism were found to be compared by their concentrations far more potent for inhibition of cytokine release than for smooth muscle contraction. Thus, for some antagonists inhibition of cytokine release was detected at concentrations as low as 10(-15) M. The rational design of peptide and nonpeptide bradykinin antagonists for therapeutic use requires not only knowledge about the potency but also knowledge about the structure-activity relationships of such important side effects as cytokine and histamine release.     

Inhibition of monoamine oxidase B by selective adenosine A2A receptor antagonists

Adenosine receptor antagonists that are selective for the A(2A) receptor subtype (A(2A) antagonists) are under investigation as possible therapeutic agents for the symptomatic treatment of the motor deficits associated with Parkinson's disease (PD). Results of recent studies in the MPTP mouse model of PD suggest that A(2A) antagonists may possess neuroprotective properties. Since monoamine oxidase B (MAO-B) inhibitors also enhance motor function and reduce MPTP neurotoxicity, we have examined the MAO-B inhibiting properties of several A(2A) antagonists and structurally related compounds in an effort to determine if inhibition of MAO-B may contribute to the observed neuroprotection. The results of these studies have established that all of the (E)-8-styrylxanthinyl derived A(2A) antagonists examined display significant MAO-B inhibitory properties in vitro with K(i) values in the low micro M to nM range. Included in this series is (E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine (KW-6002), a potent A(2A) antagonist and neuroprotective agent that is in clinical trials. The results of these studies suggest that MAO-B inhibition may contribute to the neuroprotective potential of A(2A) receptor antagonists such as KW-6002 and open the possibility of designing dual targeting drugs that may have enhanced therapeutic potential in the treatment of PD.     

Control of passive permeability of chinese hamster ovary cells by external and intracellular ATP

External ATP causes passive permeability change in several transformed cells, but not in untransformed cells. We studied the effect of external ATP on the passive permeability of CHO-K1 cells, a transformed clone of Chinese hamster ovary cells. Treatment of the cells with external ATP alone did not produce a permeability change, and this was observed only when a mitochondrial inhibitor, such as rotenone or oligomycin, was present together with ATP. These inhibitors reduced the concentration of intracellular ATP and a permeability change by external ATP was observed when intracellular ATP was decreased more than 70%. This requirement for permeability change of CHO-K1 cells was quite unique, since passive permeability change of other transformed cells so far tested was induced by ATP alone. Treatment of CHO-K1 cells with cyclic AMP analogues increased their sensitivity to external ATP about 2-fold. The roles of external and intracellular ATP in controlling passive permeability are discussed.     

Seeing the light: new insights into the molecular pathogenesis of retinal diseases

In the past, most treatments for retinal diseases have been empirical. Steroids and/or laser photocoagulation and/or surgery have been tried for almost every condition with little or no understanding of the underlying disease. Over the past several years vision researchers have uncovered molecular components of processes, such as visual transduction and the visual cycle, that are critical for visual function, and identified other molecules that lead to dysfunction and disease processes such as neovascularization and macular edema. It is becoming clear that dysregulation of certain molecules can have major effects on retinal structure and function. Studies in animal models have suggested that inhibiting or augmenting levels of a single molecule can have major effects in complex disease processes. Although several molecules probably contribute to neovascularization and excessive vascular permeability in the eye, blockade of vascular endothelial growth factor (VEGF) has remarkable beneficial effects in animal models that have now been proven to apply to human diseases in clinical trials. Intraocular injection of VEGF antagonists has revolutionized the treatment of choroidal neovascularization (CNV) and macular edema and serves as a model of targeted ocular pharmacotherapy. Significant progress elucidating the molecular pathogenesis of several disease processes in the eye may soon lead to new treatments following the lead of VEGF antagonists. Initial treatments that provide benefit from frequent intraocular injections are likely to be followed by sustained delivery of drugs and/or prolonged protein delivery by gene transfer. The eye has entered the era of molecular therapy.     

The possible role of calmodulin in the inhibition of prolactin secretion by dopaminergic antagonists

Several previous reports have indicated that a number of dopaminergic antagonists paradoxically inhibit prolactin secretion at micromolar concentrations. It is well known that some of these drugs, including pimozide and the phenothiazines, are inhibitors of calmodulin activity. Here we report that micromolar concentrations of several dopaminergic antagonists inhibit prolactin secretion from isolated rat anterior pituitary cells and calmodulin activity (calmodulin-activated cyclic GMP phosphodiesterase). Inhibition of calmodulin activity may thus, at least partially, explain the inhibitory effect of these drugs on prolactin secretion.     

Calcium Antagonists Prevent Monocyte and Endothelial Cell-Induced Modification of Low Density Lipoproteins

Low density lipoprotein (LDL) incubated in the presence of the calcium antagonists verapamil, nifedipine and flunarizine were more resistant than control LDL to human monocyte- or endothelial cell-induced modification, as assessed by electrophoretic mobility in agarose gel, thiobarbituric acid reactive substance content, and degradation by J774 macrophages. The efficiency of the drugs was: flunarizine > nifedipine > veraparml. Moreover, a 24 h preculture with calcium antagonists significantly impaired the ability of cells to modify LDL in the absence of the drugs. All the studied drugs also inhibited copper-induced autooxidation of LDL. None of the studied calcium antagonists, at concentrations up to 10^-4 M, significantly reacted with free radicals as assessed by the l,1-diphenyl-2-picrylhydrazyl test. It is suggested that such a protective effect of calcium antagonists against LDL peroxidation could play a role in the previously reported antiatherogenic effect of these drugs.     

Pharmacologic circumvention of multidrug resistance

The ability of malignant cells to develop resistance to chemotherapeutic drugs is a major obstacle to the successful treatment of clinical tumors. The phenomenon multidrug resistance (MDR) in cancer cells results in cross-resistance to a broad range of structurally diverse antineoplastic agents, due to outward efflux of cytotoxic substrates by themdr1 gene product, P-glycoprotein (P-gp). Numerous pharmacologic agents have been identified which inhibit the efflux pump and modulate MDR. The biochemical, cellular and clinical pharmacology of agents used to circumvent MDR is analyzed in terms of their mechanism of action and potential clinical utility. MDR antagonists, termed chemosensitizers, may be grouped into several classes, and include calcium channel blockers, calmodulin antagonists, anthracycline andVinca alkaloid analogs, cyclosporines, dipyridamole, and other hydrophobic, cationic compounds. Structural features important for chemosensitizer activity have been identified, and a model for the interaction of these drugs with P-gp is proposed. Other possible cellular targets for the reversal of MDR are also discussed, such as protein kinase C. Strategies for the clinical modulation of MDR and trials combining chemosensitizers with chemotherapeutic drugs in humans are reviewed. Several novel approaches for the modulation of MDR are examined.Abbreviations ALL acute lymphocytic leukemia - AML acute myelogenous leukemia - CaM calmodulin - CsA cyclosporin A - MDR multidrug resistance - P-gp P-glycoprotein - PMA phorbol 12-myristate 13-acetate - PKC protein kinase C     

Characterization of the proteins of intracisternal type A and extracellular oncornavirus-like particles produced by MOPC-460 myeloma cells.

A number of translation inhibitors were tested for their effects on both control and encephalomyocarditis virus-infected mouse 3T6 cells. The virus-infected cells were specifically inhibited by gougerotin, edeine, and blasticidin S, whereas these drugs failed to penetrate into uninfected cells. Inhibition of infected cells by gougerotin became apparent when the synthesis of viral proteins commenced, suggesting that the latter process is accompanied by a permeability change in the cells that allows uptake of the drug. This permeability change was not observed in cells treated with cycloheximide soon after viral infection, although treatment with actinomycin D did not prevent inhibition of gougerotin. It is possible, therefore, that a specific viral protein is involved in the permeability change of the plasma membrane. Moreover, gougerotin was unable to inhibit protein synthesis in the presence of zinc ions, thus preventing gougerotin from entering into the infected cell. Membrane leakiness was not restricted to the encephalomyocarditis virus-3T6 system; it was also observed in mengovirus-infected 3T6 cells, Semliki Forest virus-infected BHK cells, and simian virus 40-infected CVI1 cells at the time in which the synthesis of late proteins is maximal.     

Effects of nonsteroidal anti-inflammatory drugs on the uptake of various cations by lymphoid cells.

Several acidic nonsteroidal anti-inflammatory drugs (NSAID) as well as their corresponding alcohol molecules which are known to induce swelling of isolated lymphocytes by changing cell membrane permeability to water, are demonstrated also to induce changes of membrane permeability of lymphoid cells to one divalent cation, calcium, and to three monovalent cations, rubidium, cesium and sodium. According to the cells ionic environment, they increase or decrease the cellular uptake of cation which is itself also closely dependent on the ionic composition of the incubation medium. This drug-effect is very rapid, directly related to the medium NSAID concentration and almost totally reversible except to the most potent drugs such as flufenamic acid. Changes in intracellular ionic balance could have important catalytic effects on the metabolism of normal as well as of pathological cells. This fact could explain side-effects of these drugs as well as some of their therapeutic effects.     

Functional consequences of agonist-mediated state transitions in the cholinergic receptor. Studies in cultured muscle cells.

Parameters associated with activation and desensitization of the nicotinic receptor in the BC3H-1 muscle cell line have been compared with the state transitions that result upon combination with agonist. 125I-labeled cobra alpha-toxin is found to bind to an apparent single class of surface nicotinic receptors on the cells in situ with a rate constant of 1.15 x 10(5) M-1 s-1. The competition between cholinergic ligands and alpha-toxin reveals that agonists, but not classical antagonists, will promote a slow conversion to a receptor state where the affinity for agonists is enhanced. Moreover, agonists such as carbamylcholine elicit a permeability increase to 22Na+ ions that slowly decrements at a rate and to an extent closely paralleled by the conversion of the receptor to the high affinity state. Upon removal of the agonist, both the affinity increase and the diminished permeability change are completely reversible and again exhibit similar kinetics for their return to the original state. A comparison of the capacity of full agonists to compete with alpha-toxin binding and elicit a permeability change suggests that in the absence of agonist, receptor predominates in a low affinity activatable state. Binding of agonists to the low affinity state exhibits little if any cooperativity (n = 0.97 to 1.31), while the corresponding permeability change appears more cooperative (n = 1.31 to 1.52). By contrast, when receptors have been previously equilibrated with agonists, occupation of the receptor occurs over a 3- to 5-fold lower concentration range. Binding following equilibration closely correlates with a concomitant decrease in activatable receptor resulting from equivalent exposure to agonist. Furthermore, under equilibrium conditions, the binding of full agonists is typified by a moderate degree of homotropic cooperativity (1.25 to 1.44), enabling the receptor to desensitize over a narrow range of agonist concentration. Simultaneous measurement of occupation and activation parameters has enabled us to compare a state function for desensitization which is generated from binding parameters with the reduction in permeability seen in the desensitization process. A scheme describing the association of agonist with two functionally distinct receptor states is developed to account for the cooperative relationship between agonist binding and desensitization of the receptor.