Ionophoretic Activity Research Articles

Insights of phase separation in bilayers of ternary mixtures with different sterols

It has been shown that polyene antimycotics are selective according to the sterol present in the membrane, leading to stronger activity in fungal cells. However, it is not clear the role of the membrane structure in ionophoretic activity. In recent years, there has been a debate about if the homogenous model of the membrane is accurate or there exist nanoscopic domains enriched with sphingomyelin and cholesterol. The influence of sterol and its interaction with sphingomyelin can provide information on ordered domain formation.

Morphology and dynamics of domains in ergosterol or cholesterol containing membranes

The effect of cholesterol and ergosterol on supported lipid bilayers composed of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and egg sphingomyelin (eSM) in a 1/1 M ratio was studied using atomic force microscopy. The addition of ergosterol or cholesterol to these membranes considerably modifies both the structure and the dynamics of the domains present in them. The height of the eSM enriched domains increases with concentration of both sterols, but more markedly with ergosterol. The height of the POPC enriched domains increases with concentration in a similar manner for both sterols. This effect is larger for eSM than for POPC when ergosterol, not cholesterol, is present. Domain coverage increases with both sterols at 5 mol% but decreases at 20 mol% and almost disappears at 40 mol%. The size of the eSM enriched domains decreases with sterol concentration, more markedly with cholesterol. Bilayer rupture forces show that overall stiffness increases with the addition of 5 mol% cholesterol, but only for the eSM enriched domains with ergosterol at the same concentration. At larger sterol concentrations the stiffness of both regions becomes reduced. At 40 mol% sterol concentration, both membranes present the same rupture force value. To gain mechanistic insight into these observations we performed Quantum Mechanical calculations and Molecular Dynamics simulations of the sterol molecules. We found that conformational freedom for the sterol molecules is quite different. This difference might be behind the observed phenomena. Finally, the different action of sterols on membrane properties is related to the sterol-dependent ionophoretic activity of polyene antibiotics.

Synthetic hydraphile channels of appropriate length kill Escherichia coli.

Crown ether-based synthetic cation conducting channels called hydraphiles show clear ionophoretic activity in phospholipid vesicles. These compounds are shown to be active against the bacterium E. coli. Disk diffusion assays were performed to assess the toxicity of different hydraphile derivatives. Liquid culture tests were conducted to quantitate the dependence of bacterical activity on channel length. It is proposed that hydraphiles are toxic to bacteria as a result of channel formation in the membrane. The bactericidal activity is found to depend at least on the presence of a functional central relay and proper channel length. It is speculated that hydraphiles insert into the bilayer and disrupt the cell's osmotic balance, leading to cell death.

Synthesis of Sphingadienine-type Glucocerebrosides

Sphinga-4,8-dienine derivatives were synthesized from a vinyl-epoxide 5 via three routes. First, reaction of 5 with 2-dodecenyl cyanocuprate afforded a 1:1 mixture of (4E,8E)- and (4E,8Z)-sphingadienine derivatives in high yield. Second, the (4E,8E)-isomer was selectively synthesized via allylic bromide-allylic sulfone coupling followed by desulfonylation. Third, the (4E,8Z)-isomer was selectively synthesized via SN2′-type addition of di(cyanomethyl)copper-lithium followed by Wittig olefination. These synthetic intermediates were converted to sphingadienine-type glucocerebrosides 1a and 1b which have calcium ionophoretic activity.

Isolation and Structure Determination of Cerebrosides from Garlic, the Bulbs of Allium sativum L.

Five cerebrosides, AS-1-1 (1), AS-1-2 (2), AS-1-3 (3), AS-1-4 (4), and AS-1-5 (5) were obtained from the CHCl3-MeOH extract of Garlic, the bulbs of Allium sativum L. (Liliaceae). On the basis of spectroscopic results, structures of 1-5 have been elucidated. Compounds 1, 2, and 5 were new cerebrosides. Compounds 3 and 4 were identified with the known glucocerebroside soya-cerebroside I and II, respectively, which have been previously obtained from soybean with ionophoretic activity. Positive ion FAB-MS/MS of the (M+Na)+ ion gave important information on the length of the fatty acyl chain of the ceramide moiety.

Measurement of intracellular Na + concentration by a Na +-sensitive fluorescent dye, sodium-binding benzofuran isophthalate, in porcine adrenal chromaffin cells—usage of palytoxin as a Na + ionophore

Palytoxin was found to equilibrate sodium ions (Na +) across the cell membrane much faster than dose gramicidin, which has been frequently used to calibrate the intracellular Na + concentration ([Na +]in), in cells treated with a Na +-sensitive fluorescent dye, sodium-binding benzofuran isophthalate (SBFI). Palytoxin was capable of equilibrating Na + in cells treated with SBFI-acetoxymethyl ester (SBFI-AM) and in voltage-clamped cells loaded with SBFI through a patch pipette. Nicotine caused a dose-dependent increase in [Na +]in in porcine adrenal chromaffin cells treated with SBFI-AM and caused a simultaneous increase in [Na +]in and inward current in the voltage-clamped cells loaded with SBFI. Palytoxin has an advantage of calibrating [Na +]in in a shorter time than dose gramicidin because of its powerful ionophoretic activity.

Enhancement of ricin toxin a chain immunotoxin activity: Synthesis, ionophoretic ability, and in vitro activity of monensin derivatives

Site-selective toxin delivery was achieved by coupling monoclonal antibody to the A chain subunit of ricin (RTA-IT). The cell-killing potency of RTA-IT can be drastically increased in vitro by using ionophores such as monensin. To reduce the intrinsic toxicity of monensin and to enhance its in vitro and in vivo activity, we synthesized 7 derivatives characterized by different lipophilicities. These derivatives were also analyzed for ionophoretic activity on intact cells, toxicity, and RTA-IT-enhancing activity. Two different RTA-IT were assayed on a human leukemia cell line. A correlation between lipophilicity, ionophoretic activity, and RTA-IT enhancement was observed. The compounds with the highest polar charge showed low intrinsic toxicity, revealed moderate ionophoretic activity, and were able to enhance RTA-IT only at high concentrations, whereas more lipophilic compounds (with a C28 tail or a phenyl group) showed significant ionophoretic activity and good enhancing properties.

Insulinotropic action of (2S)-2-benzyl-3-( cis-hexahydro-2-isoindolinyl-carbonyl) propionate II. Ionophoretic and conformational aspects

1. 1. The non-sulphonylurea insulinotropic agent sodium (2S)-2-benzyl-3-( cis-hexahydro-2-isoindolinylcarbonyl) propionate (KAD-1229) was found to display calcium ionophoretic activity in an artificial membrane model. 2. 2. Conformation analysis indicated that a complex between calcium and KAD-1229, with a 1:2 stoichiometry, indeed displays favourable attributes for ionophoretic activity across a hydrophobic environment. 3. 3. It is speculated that the ionophoretic property of KAD-1229 might participate to the remodelling of cationic fluxes evoked by this insulinotropic agent in pancreatic islet cells.

Tri-Calciphor (16,16-dimethyl-15-dehydroprostagalndin B 1 trimer)-mediated mitochondrial Ca 2+ movements: modulation by phosphate

The trimeric derivative of 16,16-dimethyl-15-dehydroprostaglandin B 1 (termed tri-Calciphor), which protects tissues against ischemic damage, induced Ca 2+ efflux and swelling in mitochondria in the absence of phosphate, Mg 2+ and ATP. When glutamate/malate rather than succinate was the substrate, higher tri-Calciphor concentrations were required for the ionophoretic activity. Ca 2+ efflux and mitochondrial swelling induced by tri-Calciphor were completely inhibited by ATP, phopsphate and Mg 2+ added together, and partially inhibited with phosphate plus either ATP or Mg 2+. Between 0 and 7 μM added Ca 2+ and in the presence of phosphate, ATP and Mg 2+, tri-Calciphor stimulated the uptake of Ca 2+ by mitochondria and increased the efficiency of buffering of extramitochondrial Ca 2+. Thus depending on the assay conditions, two different effects involving Ca 2+ movements and mitochondria are observed with tri-Calciphor.

Osmotic behaviour and permeability properties of liposomes

In this overview liposomes are described as bilayer-bounded vesicles which under defined conditions act as ideal osmometers according to the Boyle van't Hoff law. Investigations on osmotic volume changes, directly or indirectly by taking advantage of changes in light scattering, are considered and applications in permeability measurements are discussed. Solute-solvent interactions occuring in isotonic swelling experiments are analysed in view of an irreversible thermodynamic description. In a second part liquid crystalline lipid bilayers are characterized as highly selective permeability bilayers and the physical principles underlying this selectivity are considered. Attention is given to special physical and chemical conditions that may cause structural defects in the bilayer organization and can affect the selective permeability properties of the bilayer or completely deteriorate its barrier function. Finally an evaluation is given of intrinsic ionophoretic activity in lipid bilayers containing negatively charged lipids.

Non-ionophoretic elevation of intracellular Ca 2+ by Lonidamine

Lonidamine is an antispermatogenic and anticancer drug that is believed to act by inhibition of energy metabolism. In this study, the effects of Lonidamine on the concentration of intracellular free Ca 2+ of several tumor cell lines were assessed because of the important role that cytosolic Ca 2+ plays in cell viability and proliferation. The presence of 300 μM Lonidamine resulted in large elevations of cytosolic Ca 2+ (> 100nM) in AS-30D rat ascites hepatoma cells and in cultured EMT6 murine mammary adenocarcinoma cells but had little effect on cultured NCI-H345 human small cell lung cancer cells. The apparent EC 50 for Lonidamine was approximately 175 μM. The source of elevated cytosolic Ca 2+ was primarily intracellular stores, and the effects of Lonidamine on Ca 2+ efflux from these stores did not appear to be due to an ionophoretic action of this compound or to a decline in the level of cellular ATP. These results indicate that the Ca 2+ homeostasis of certain lines of tumor cells is specifically altered by Lonidamine at concentrations known to affect cell proliferation.

Application of in vivo bioluminescence to the study of ionophoretic action.

Ionophores (carbonyl cyanide m-chlorophenylhydrazone; valinomycin; and the hop-derived compounds colupulone, trans-isohumulone and trans-humulinic acid) reduced the rate of dodecanal-dependent light emission from IuxA/B-transformed cells of Lactococcus lactis subsp. diacetylactis F712 when the cells were suspended in a buffered medium (pH 6.4) containing glucose. This allowed an assay for ionophores to be devised and permitted measurement of the effects of such compounds on the test organism by the use of a non-destructive technique in real time.

Sphingolipids and Glycerolipids. IV. Syntheses and Ionophoretic Activities of Several Analogues of Soya-cerebroside II, a Calcium Ionophoretic Sphingoglycolipid Isolated from Soybean.

For examination of the structure-activity relationship five analogues [(2'R)-2'-hydroxypalmitoyl (3), palmitoyl (4), (2'S)-2'-hydroxypalmitoyl (5), β-D-galactosyl (6), and 8, 9-dihydro (7) relevancies] of soya-cerebroside II (2), which is a calcium ionophoretic sphingoglycolipid isolated from soybean, have been synthesized by using our previously reported synthetic method for sphingoglycolipids. Examinations by using a W-08 (liquid-membrane type) apparatus and by means of the human erythrocyte membrane method, have shown that the (2'R)-2'-hydroxypalmitoyl analogue (3) exhibits higher ion-binding and ion-permeation activities for calcium ion than soya-cerebroside II (2), which contains 3 as the major component. It has also been found that the other analogues(5, 6, 7, 8) do not show those ionophoretic activities.An enantiomer (8) of the (2'R)-2'-hydroxypalmitoyl analogue (3) has been synthesized and its calcium ionophoretic activity examined. Compound 8 exhibits calcium ion-binding activity equal to that of 3, but 8 lacks the ability to support calcium ion-permeation through human erythrocyte membrane. Thus, human erythrocyte membrane precisely distinguishes the absolute configurations of 3 and 8 as regards calcium ionophoretic activity.

Taurine-conjugated bile acids act as calcium ionophores

The ionophoretic properties of several taurine-conjugated bile acids have been investigated in two experimental systems: in a two-phase bulk partitioning system and in proteoliposomes. In the former, a bile acid/Ca2+ complex was extracted into the bulk organic phase and had an experimental stoichiometry of 1.75. Extraction was specific for Ca2+ over Mg2+; Na+ and K+ did not compete with the extraction of Ca2+. In the second system, bile acids at concentrations as low as 5-100 molecules/vesicle lowered the steady-state Ca2+ gradient maintained by a reconstituted sarcoplasmic reticulum Ca(2+)-ATPase. The effect was not due to nonspecific membrane perturbation. In addition to releasing intravesicular Ca2+ in a transmembraneous process, bile acids caused partition of Ca2+/bile acid complexes into the hydrophobic core of the bilayer. In both experimental systems, the Ca2+ ionophoretic activity correlated well with the concentration and the hydrophobicity of the bile acid. Taurolithocholate was most active, with a significant effect measurable at 10 microM in either system. Since bile acid concentrations equal to those used in our experiments can occur in the blood in certain liver diseases, the results support the notion that bile acids can increase the intracellular Ca2+ concentration bypassing the regulatory systems that maintain cellular Ca2+ homeostasis.

Counteraction by nicardipine of the ionophoretic effect of gliclazide: A mitochondrial study

Using mitochondria, we demonstrate that gliclazide exhibits a calcium ionophoretic activity. Indeed, gliclazide induces a decrease in the respiratory control of mitochondria; this effect is increased by addition of Ca 2+ and corrected by ruthenium red, all characteristics of a calcium ionophoretic compound. Our results, on a biological membrane, confirm previous findings in vitro. Moreover, we show that nicardipine counteracts the action of gliclazide. Besides the effect on ATP-stimulated K + channels, the ionophoretic effect of gliclazide may play a role in its hypoglycaemic effect, thus this counteracting action of nicardipine might induce drug interaction during the concomitant clinical administration of gliclazide and nicardipine.

Effects of apical vs. basolateral palytoxin on LLC-PK1 renal epithelia

Research on palytoxin focuses on its action as a tumor promoter and its ionophoretic action in cell membranes. The first property is unusual because palytoxin is not a protein kinase C activator. The second property is remarkable in that it may require interaction with the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Our studies here with palytoxin exposure to the LLC-PK1 renal epithelial cells have yielded the following results: 1) unlike protein kinase C-activating tumor promoters (tetradecanoylphorbol 12,13-acetate or teleocidin), palytoxin does not produce a specific effect on the tight junctions between epithelia; 2) palytoxin instead produces an irreversible cytotoxic effect characterized by a pronounced cell swelling associated with sharply elevated levels of intracellular Na+ and decreased levels of intracellular K+; 3) these fluctuations in intracellular Na+ and K+ levels are explained by marked elevations in the membrane flux of 22Na+ and 86Rb+; 4) the electrophysiological reflection of these altered ion fluxes is a pronounced depolarization of the cell sheet if palytoxin is presented to the basolateral cell surface and a pronounced hyperpolarization (due to sharply elevated apical Na+ flux and transepithelial short-circuit current) if palytoxin is administered apically; 5) the apical effect of palytoxin can be blocked by apical ouabain; and 6) this apical effect of palytoxin decreases as a function of the age of the cell sheet. This first report of palytoxin action in a polar epithelial cell system provides additional evidence for palytoxin effects being mediated by contact with the Na(+)-K(+)-ATPase. It also adds to a growing literature suggesting the existence of Na(+)-K(+)-ATPase in the apical cell surface of epithelia under certain conditions.

Sphingolipids and glycerolipids. I. Chemical structures and ionophoretic activities of soya-cerebrosides I and II from soybean.

Two glycosphingolipids named soya-cerebrosides I and II were isolated from soybean, the seeds of Glycine max Merrill (Leguminosae), and their chemical structures have been elucidated on the basis of physicochemical evidence and several chemical degradation reactions. By using a newly constructed liquid membrane-type apparatus (W-08) for measurement of ion-transport and ion-binding activities and by employing a method using human erythrocyte membranes for measurement of ion-permeability, it has been found that soya-cerebroside II exhibits ionophoretic activity for Ca2+ ion.

Studies of the cation binding properties of an oligomeric derivative of prostaglandin B 1

The ionophoretic activity of PGB x, an oligomeric mixture synthesized from 15-dehydro PGB 1, with different cations was measured using arsenazo III-entrapped liposomes. The order of ionophoretic activity was Zn 2+ > Co 2+ > Mn 2+ > Cu 2+ > Ca 2+ > Ba 2+ > Sr 2+ > Mg 2+. The intrinsic fluorescence of PGB x was quenched by the binding of divalent cations as well as by La 3+ and H +. Quenching by K + and Na + was minimal. The order of quenching strength of divalent cations was Zn 2+ > Co 2+ > Cu 2+ = Mn 2+ > Ca 2+ > Ba 2+ > Sr 2+ > Mg 2+. Binding affinities of these cations determined by a murexide indicator method were in good agreement with that determined by the fluorescence quenching reaction. The cation binding affinity of PGB x in aqueous solutions correlates with the ionophoretic activity in liposomes. The binding affinity for K + was estimated from the inhibition by K + of Ca 2+ binding by PGB x. Although PGB x has a lower selectivity for divalent cation binding than the ionophore A23187, the characteristics of the binding affinity of these two compounds for various ions were similar. The pK of PGB x as determined by fluorescence quenching was 6.7. The molecular weight of the divalent cation binding unit was estimated to be about 680, with each PGB x molecule having three such binding sites. The binding of Ca 2+ to such a site is one-to-one.

Diabetes alters postischemic response to a prostacyclin mimetic

Eicosanoid metabolism is altered by diabetes. However, postischemic responses of diabetic hearts (DH) to eicosanoids such as prostacyclin are unknown. a prostacyclin analogue iloprost (Ilo) was given to isovolumically beating rat hearts during 20 min of total global ischemia and 30 min of reperfusion. Acute DH (48 h) but not chronic DH (2 mo) had pronounced postischemic dysfunction (developed pressure = 22 +/- 11%), which was completely reversed by 3 X 10(-8) M Ilo (developed pressure = 113 +/- 15%). Ilo also stimulated endogenous prostacyclin release in postischemic control hearts (CH) but not acute or chronic DH. Ilo significantly decreased postischemic recovery in CH (from 67 +/- 11 to 15 +/- 4%), which was partially blocked by the coadministration of the calcium-entry blocker diltiazem or almost completely reversed by the free radical scavengers superoxide dismutase plus catalase (100 U/ml). These data suggest that Ilo may promote functional recovery in DH at concentrations that produce dysfunction in CH. Furthermore, Ilo may induce dysfunction in CH by a calcium ionophoretic action, which appears depressed in diabetes, and by concomitant free radical production (presumably via prostaglandin hydroperoxidases) during Ilo-stimulated endogenous prostacyclin synthesis.

Ion transport mediated by inverted micellar aggregates across a phosphatidylcholine-octanol liquid membrane

The ionophoretic activity of dipalmitoyl-DL-α-phosphatidylcholine (DPPC) was investigated on the system in which two aqueous NaCl solutions are separated by a porous support membrane impregnated with a 1-octanol solution of DPPC. Transmembrane potential and conductance measurements were carried out with changing temperature to estimate the membrane permeability to ions. To clarify the mechanism of ion transport through an immobilized DPPC-1-ctanol membrane, differential scanning calorimetry and photon correlation spectroscopy were employed to investigate the molecular properties of DPPC in a water-saturated 1-octanol system. An endothermic peak was detected at around 18°C in the water-saturated 1-octanol system, but not in the pure 1-ctanol system. Laser light scattering study indicated that DPPC molecules form aggregates, ca. 0.1 μm in diameter, in water-saturated 1-octanol, but not in pure 1-octanol. Temperature dependences of the relaxation time estimated by the light scattering study changed above and below 18°C. The critical temperature, 18°C, correlates well with that at which modes of the temperature dependences of membrane permeability are altered. Results obtained by these three types of experiments indicate that inverted micellar aggregates of DPPC function as an ionophore.