Metabolic characteristics of pancreatic beta-cells exposed to calcium-transporting ionophores.

The effects of the ionophores A-23187 and X-537 A on glucose metabolism, ATP content and sucrose permeability in pancreatic islets microdissected from obese-hyperglycemic mice were studied. The formation of 14CO2 from 10 mM D-[U-14C] GLUCOSE WAS INHIBITED BY OMISSION OF Ca2+ from the medium. A-23187 (10 muM) induced a further decrease of 14CO2 formation whereas X-537 A (10 muM) had no effect. At 20 mM glucose both A-23187 (48 muM) and X-537 A (43 muM) decreased the 14CO2 formation in the absence of Ca2+ whereas only X-537 A inhibited in the presence of Ca2+. X-537 A (43 muM) also decreased the formation of 3H2O from 20 mM D-[5-3H] glucose. The islet content of ATP was not changed after incubation in media deficient in either Mg2+ or Ca2+. However, omission of both Mg2+ and Ca2+ resulted in about 50% decrease of the ATP content. A-23187 and X-537 A induced dose-dependent decreases of the islet ATP content. X-537 A was much more potent than A-23187. Both ionophores induced stronger depression of the ATP content when Ca2+ was omitted. X-537 A (43 muM) but not A-23187 (48 muM) increased the beta-cell membrane permeability as indicated by an increased sucrose space in relation to the urea space of islets. Such an effect was not obtained with X-537 A at 1 muM or by omission of Ca2+. It is suggested that the marked metabolic effects of the ionophores reflect an impaired mitochondrial metabolism. These metabolic changes should be considered in interpretations of ionophore action on insulin secretion.     

Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum.

The Ca2+ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca2+ and surface membrane-bound Ca2+ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca2+ in the absence of ATP, 10-12 h are necessary for measurable amount of Ca2+ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca2+ from 'loaded' vesicles only after this period of incubation. A fraction of Ca2+ of 50-60 nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg2+ and K+ in the medium and is readily released by ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca2+ (30-40 nmol/mg protein) is rapidly released X-537A. The results indicate that most of the Ca2+ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca2+ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.     

The kinetics of ionophore X-537A-mediated transport of manganese through dipalmitoylphosphatidylcholine vesicles A 1H-NMR study

We have studied the kinetics of ionophore X-537A-mediated transport of manganese ions into small unilamellar vesicles formed from dipalmitoylphosphatidylcholine. To follow the transport we used the paramagnetic effect of manganese on the ¹H-NMR signal from choline trimethylammonium groups on the inner phospholipid monolayer. The transport of only one manganese ion produces an intravesicular concentration which is high enough (approx. 1 mM) to substantially broaden this signal. The observed signal thus arises predominantly from those vesicles which contain no manganese. Therefore, as manganese is transported into the vesicles the observed signal decreases in intensity, but does not broaden. The initial time-dependence of the intensity of the signal, $\text{S(t)}$, can be approximated by the simple first-order rate law: $\text{S(t) = S}\text{(O)}\text{exp}\text{(?K′t)}$, where $\text{K′}$ is the probability per unit time for the transport of a manganese ion from the external medium to the intravesicular space. From the dependence of $\text{K′}$ on the ionophore X-537A concentration we conclude that manganese is transported into the vesicles via both 1 : 1 and 2 : 1 complexes with ionophore X-537A. At low ratios of ionophore X-537A to vesicles transport via the 1 : 1 complex predominates; at high ratios transport via the 2 : 1 complex predominates. From the dependence of $\text{K′}$ on manganese concentration we determined that under our conditions the equilibration of ionophore X-537A between vesicles is much faster than the transport of manganese through the vesicles. Lastly, from the dependence of $\text{K′}$ on temperature, we conclude that the ionophore X-537A-mediated transport of manganese into the dipalmitoylphosphatidylcholine vesicles is very sensitive to the gel-liquid crystalline phase transition.     

Inhibition of ruthenium red-induced Ca2+ efflux from liver mitochondria by the antibiotic X-537A

It has been reported (Becker, G.L., Fiskum, G. and Lehninger, A.L. (1980) J. Biol. Chem. 255, 9009-9012) that respiring rat liver mitochondria suspended in KC1 medium containing ATP, Mg2+ and phosphate, maintain a steady state extramitochondrial free Ca2+ concentration of about 0.5 microM. The results reported here show that the addition of the antibiotic X-537A, at concentrations far below those required for ionophorous activity, caused a perturbation in this steady state, lowering the extramitochondrial free Ca2+ concentration by about 0.20 microM. This shift in steady state was clarified by a study of X-537A inhibition of the Ca2+ efflux induced by ruthenium red; a half-maximum effect was observed at approximately 25 nM X-537A. No effect on Ca2+ transport through the influx uniporter was observed. The possibility of a generalized stabilizing action of the antibiotic on the mitochondrial membrane seems to be ruled out by its effectiveness at very low concentrations.     

Effects of the ionophores,X-537A and A-23187 on catecholamine release from the in vitro frog adrenal

1. The ionophore X-537A increases the rate of catecholamine release from the $\text{in vitro}$ frog adrenal.2. The ratio of epinephrine/norepinephrine measured during X-537A stimulation was the same as that during spontaneous release.3. Even when Ca⁺⁺ was removed from the Ringer, X-537A stimulated catecholamine release, but depolarization by elevated extra-cellular K⁺ was no longer effective.4. X-537A also increases the release of dopamine β-hydroxylase, suggesting that the ionophore acts, at least in part, by stimulating the exocytosis of the chrommaffin granule contents.5. Therefore, it is questionable whether the release of catecholamines by X-537A is owing to its action as a Ca⁺⁺- ionophore.6. The divalent cation ionophore, A-23187 (50μM), did not affect the rate of catecholamine release.     

Crystal structure of valinomycin-monohydrate cage complexes crystallized from dioxane.

Valinomycin, cyclo-[(L-Val-D-Hyv-D-Val-L-Lac)3-], was crystallized from aqueous dioxane solvent as a monohydrate complex in which water molecules were found within the ion-binding cavity of the ionophore: monoclinic P2(1), a = 14.377 (3), b = 41.554 (14), c = 14.080 (3) A, beta = 118.27 (2) degrees, Z = 4. There are two non-equivalent valinomycin-water complexes and three dioxane molecules in the asymmetric unit. The ionophore molecules adopt two similar but non-identical, octahedral, bracelet, cage conformations that are a consequence of two distinct ways in which the complexed water molecules can deform the normal octahedral coordinate geometry of the metal binding site. In the first complex the water molecule forms hydrogen donor bonds to the carbonyl oxygens of two L-valine residues on one facial side of the cavity, while in the second complex the water molecule is trigonal-planar coordinate and binds to two L-valine residues on one entrant face of the cavity plus a third D-valine residue from the opposite side of the cavity.     

Modifying actions of calcium ionophores on insulin release.

Beta-Cell-rich pancreatic islets were microdissected from noninbred ob/obmice and exposed to the calcium ionophores X-537A and A-23187. X-537A differed from A-23187 in being a potent insulin secretagogue at non-stimulating glucose concentrations. Both ionophores inhibited the stimulation of insulin release obtained after adding 20 mM glucose to the incubation medium. The latter observation is consistent with the idea of a reduced beta-cell function when the Ca-2+ in the functionally important intracellular pool (s) exceeds a certain concentration. The ionophore inhibition of the glucose-stimulated insulin release may at least in part result from decreased formation of cyclic AMP, since X-537A proved to be as effective as L-epinephrine in reducing the islet content of this nucleotide in the presence of a phosphodiesterase inhibitor. The secretagogic action of X-537A at a low glucose concentration persisted when different ions were omitted from the incubation medium and was actually considerably enhanced in the absence of extracellular Ca-2+. The insulin-releasing action of X-537A was neither influenced by 3-O-methyglucose nor by drugs blocking the alpha or beta-adrenergic receptor sites. Exposure of the pancreatic beta-cells to metabolic inhibitors in concentrations which significantly reduced the secretory response to glucose, potentiated stimulation of insulin release by X-537A, suggesting that this effect may in part be accounted for by intracellular dissolution of secretory granules.     

Synthesis of adenosine triphosphate during release of intravesicular and membrane-bound calcium ions from passively loaded sarcoplasmic reticulum.

Sarcoplasmic reticulum isolated from rabbit skeletal muscle and incubated in a medium containing Ca2+ in the absence of ATP retains intravesicular and/or membrane-bound Ca2+. The synthesis of ATP coupled with the release of intravesicular Ca2+ is totally inhibited by the ionophore X-537A. Release of the membrane-bound Ca2+, retained after short periods of incubation (10min) or after release of the intravesicular Ca2+ by ionophore X-537A, still supports some synthesis of ATP. The ratios of Ca2+ released to ATP synthesized are 2.5-3.2, when bound and intravesicular Ca2+ are released simultaneously, and 3.1-4.0, when only bound Ca2+ is released. The results show that the synthesis of ATP by sarcoplasmic reticulum during release of passively accumulated Ca2+ by EGTA [ethanedioxybis(ethylamine)tetra-acetic acid] is accompanied by a loss of membrane-bound Ca2+.     

Some effects of the ionophore X-537A on the isolated rat tail artery

The effects of micromolar concentrations of the ionophore X-537A (RO 2-2985) were studied using isolated preparations of the rat tail artery. The ionophore causes complete release of catecholamines from adrenergic nerves, which is the sole cause of the transient contractile response. The amines are released by a nonexocytotic process which seems to be related to the ability of X-537A to act as an efficient transmembrane carrier of Na+, k+, and H+. The ionophore also causes an almost complete and irreversible loss of the cocaine-sensitive component of metaraminol uptake by the tissue. X-537A dissipates the transmembrane concentration gradients of Na and K in the smooth muscle component of the preparation. This effect is unrelated to the release of endogenous catecholamines, and it can also be observed after the Na pump has been inhibited with ouabain. It is fully reversible, though not readily, and it can be induced repeatedly. In catecholamine-depleted strips, X-537A dissipates the transmembrane Na+ and K+ gradients without causing any change in tension. Stimulation of the rate of O2 consumption by X-537A in catecholamine-depleted tissue is reversible, and it is unaffected by ouabain and (or) removal of external Ca2+.     

The stoichiometry of A23187- and X537A-mediated calcium ion transport across lipid bilayers

Initial rates of ionophore-mediated Ca2+ transport across egg phosphatidylcholine bilayers of large unilamellar vesicles were measured using the absorbance change of arsenazo III at 650 nm as an indicator of Ca2+ translocation. A23187 induced the movement of Ca2+ in a 2:1 ionophore: Ca2+ complex, whereas its methyl ester (CH3A23187) and X537A mediated Ca2+ movement in a 1:1 ionophore: Ca2+ complex. The relative potencies of these ionophores in transporting Ca2+ across lipid membranes were A23187 much greater than X537A greater than CH3A23187.     

The circular polarization of fluorescence of the ionophore lasalocid A (X-537A) and some of its metal complexes

The conformation of the ionophore lasalocid A (X-537A) and its complexes with metal ions was probed by the circular polarization of their luminescence (CPL). The CPL of each complex in methanol was found to be different than when in n-hexane. Furthermore, the different metal ion complexes investigated had a different CPL spectrum in each solvent. These findings indicate wide variability in the conformation of the complexes depending on the metal ion and the solvent. From the spectral behaviour of the CPL it was concluded that at least some of the complexes exist in more than one form in solution. A comparison between the CPL and CD spectra indicates a change in the conformation of the ionophore in the vicinity of the salicylate chromophore upon electronic excitation.     

Particulate guanylate cyclase of skeletal muscle: effects of Ca2+ and other divalent cations on enzyme activity.

The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.     

The effect of holotoxin A1 on transport of calcium ions across the lipid models of biological membranes

Planar bilayer lipid membranes formed from trepang phospholipids possess an intrinsic Ca2(+)-permeability. These phospholipids dissolved in a non-polar solvent can extract 45Ca2+ from the aqueous to the organic phase. The triterpenic glycoside holotoxin A isolated from the trepang Stichopus japonicus inhibits the Ca2+ flux of lipid bilayers from trepang phospholipids as well as the Ca2+ flux induced in phosphatidylcholine bilayers by the calcium ionophore X-537A. Toxin inhibits the Ca2+ ionophore A23187 induced Ca2+ efflux from phosphatidylcholine liposomes and 45Ca2+ transition from the aqueous to the organic phase. Holotoxin A does not inhibit the 45Ca2+ transfer to the non-polar phase induced by holoturia phospholipids and does not affect the phosphatidylcholine hydroperoxide-induced Ca2+ flux of lipid bilayers. Using the fluorescent probe pyrene, it was demonstrated that toxin increases the microviscosity of liposomal membranes and trepang oocyte "ghosts".     

Conformational studies of A23187 with mono-, di- and trivalent metal ions by circular dichroism spectroscopy

CD studies carried out on A23187 indicate a solvent-dependent conformation for the free acid. Alkali metal ions were found to bind to the ionophore weakly. Divalent metal ions such as Mg2+, Ca2+, Sr2+, Ba2+ and Co2+ and trivalent lanthanide metal ions like La3+ were found to form predominantly 2:1 (ionophore-metal ion) complexes at low concentrations of metal ions, but both 2:1 and 1:1 complexes were formed with increasing salt concentration. Mg2+ and Co2+ exhibit similar CD behaviour that differs from that observed for the other divalent and lanthanide metal ions. The structure of 2:1 complexes involves two ligand molecules coordinated to the metal ion through the carboxylate oxygen, benzoxazole nitrogen and keto-pyrrole oxygen from each ligand molecule along with one or more solvent molecules. Values of the binding constant were determined for 2:1 complexes of the ionophore with divalent and lanthanide metal ions.     

Solution conformation of various uridine diphosphoglucose salts as probed by NMR spectroscopy

The solution conformations of uridine diphosphoglucose (UDP-Glc) under a variety of conditions (solvent, ionic strength, various mono- and divalent cations) have been studied by NMR spectroscopy (1H, 13C, 31P, and 25Mg). In the case of divalent cations (Ca2+, Mg2+, Mn2+) the phosphate oxygens are the preferred coordination sites and analysis of the 25Mg linewidths of solutions with various [Mg2+]/[UDP-Glc] ratios, indicates that the 1:1 Mg2+ UDP-Glc complex is the major species. From 13C relaxation data and hydrodynamic theory, it has been demonstrated that under all conditions UDP-Glc adopts a fairly extended overall shape and that magnesium ions lead to a significant increase in the average length of the UDP-Glc molecule as compared to monovalent cations. Thus, one of the roles of the metal ion in enzymic reactions involving nucleotide sugars may be to preorganize the nucleotide sugar.     

Structure of the lipid A-inner core region and biological activity of Plesiomonas shigelloides O54 (strain CNCTC 113/92) lipopolysaccharide

Plesiomonas shigelloides is a Gram-negative rod associated with episodes of intestinal infections and outbreaks of diarrhea in humans. The extraintestinal infections caused by this bacterium, for example, endopthalmitis, meningitidis, bacteremia, and septicemia, usually have gastrointestinal origin and serious course. The lipopolysaccharide (LPS, endotoxin) as virulence factor is important in enteropathogenicity of this bacterium. LPSs of P. shigelloides and especially their lipid A part, that is, the immunomodulatory center of LPS, have not been extensively investigated. The structure of P. shigelloides O54 lipid A was determined by chemical analysis combined with MALDI-TOF mass spectrometry, and the intact Kdo-containing core region was investigated by NMR spectroscopy on deacylated LPS. Products from alkaline deacylation of LPS, containing 4-substituted uronic acids, are usually very complex and difficult to separate. Since Kdo residues, like sialic acids, form complexes with serotonin, we used immobilized serotonin for one-step isolation of oligosaccharide containing the intact Kdo region from the reaction mixture by affinity chromatography. The major form of lipid A was built of beta-d-GlcpN4PPEtn-(1-->6)-alpha-d-GlcpN1P disaccharide substituted with 14:0(3-OH), 12:0(3-OH), 14:0(3-O-14:0), and 12:0(3-O-12:0) acyl groups at N-2, O-3, N-2', and O-3', respectively. This is a novel structure among known lipid A molecules. Analysis of intact Kdo-lipid A region, lipid A and its linkage with the core oligosaccharide completes the structural investigation of P. shigelloides O54 LPS, resolving the entire molecule. Biological activities and observed discrepancy between in vitro and in vivo activity of P. shigelloides and Escherichia coli LPS are discussed.     

The effect of amine structure on complexation with lasalocid in model membrane systems: I. Identification of charged complexes in lipid bilayer membranes

The electrical properties of X-537A (lasalocid) doped lipid bilayer membranes were studied in the presence of a series of nine biogenic amines which contain β-phenylethylamine as the basic structural unit. The ionophore antibiotic was found to form charged complexes within the membrane during the transport of some of the amines. The dependence of membrane conductance on the concentration of ionophore and amine was studied. The amines are divided into three classes according to the nature of the complexes formed: (1) charged complex involving two ionophores (phenylephrine, metanephrine, and amphetamine); (2) charged complex containing three ionophores (dopamine, norepinephrine and epinephrine); and (3) no charged species formed (p- and m-tyramine and β-phenylethylamine).     

Crystal structure of the inclusion complex of the antibacterial agent triclosan with cyclomaltoheptaose and NMR study of its molecular encapsulation in positively and negatively charged cyclomaltoheptaose derivatives

The inclusion complexes of triclosan with native cyclomaltoheptaose (beta-cyclodextrin, betaCD) as well as with negatively and positively charged derivatives are studied. The structure of the inclusion complex betaCD/triclosan in the crystalline state [P1, a=15.189(5), b=15.230(6), c=16.293(6), alpha=91.07(4), beta=91.05(3) gamma=100.71(3)] comprises two crystallographically independent host macrocycles A and B. The packing results in betaCD dimers that align head-to-head and form infinite channels along the c-axis. Only one guest molecule statistically disordered over two positions, (the dichlorophenyl ring in the cavities of either A or B) corresponds to each dimer (a 2:1 host/guest complex). The enclosed dichlorophenyl ring enters the dimer through the primary side, whereas the hydrophilic chlorophenol ring extends in the space between dimers. Water molecules in five positions are also enclosed in the intradimer region, arranged on a plane perpendicular to the sevenfold axis of betaCD. The NMR spectroscopic studies in aqueous solution show the presence of both 1:1 and 2:1 betaCD/triclosan complexes. In the first case, two different 1:1 complexes are simultaneously present, each with either ring entering the narrow primary side of one betaCD molecule. In the 2:1 complex both rings of triclosan are included in two independent betaCD hosts, a precursor to the supramolecular arrangement found in the crystalline form. In the case of the negatively charged sodium heptakis[6-deoxy-6-(3-thiopropionate)]-betaCD, the NMR studies at pH 7.9 show a complete inclusion of triclosan inside the host in two orientations, one for the non-ionized (phenol) and reverse for the ionized (phenolate) form. Finally, for the positively charged heptakis(6-aminoethylamino-6-deoxy)-betaCD, inclusion of triclosan is possible only when the pH is raised to 10 and it is concluded that both aromatic rings are alternatively inside the cavity. However in that case also, inclusion of the entire guest in the elongated cavity is suggested.     

Catecholamine secretion by isolated adrenal cells.

Isolated adrenal cells were prepared by collagenase digestion of guinea pig adrenal glands. Acetylcholine stimulates the secretion of catecholamines by these isolated adrenal cells. Acetylcholine-stimulated catecholamine secretion is inhibited by cholinergic blocking agents (atropine and hexamethonium) and by local anaesthetics (tetracaine), and is dependent upon the concentration of Ca2+ in the incubation medium. In the presence of Ca2+, catecholamine secretion is also stimulated by two divalent cation ionophores, A23187 and X-537A. Cyclic nucleotides and 5'-nucleotides cause a small, non-specific stimulation of catecholamine secretion. These results indicate that isolated adrenal cells are a useful system in which to study catecholamine secretion, and support the hypothesis that increased Ca2+ entry into chromaffin cells is a sufficient stimulus for catecholamine secretion.     

Oxalate transfer across the membranes of sarcoplasmic reticulum during the uptake of Ca++

The kinetic profile of Ca++ uptake in the presence of oxalate is biphasic. An initial phase independent on oxalate is followed by an oxalate-dependent phase delayed in time. The ionophore X-537A only abolishes the net Ca++ uptake if added before the onset of the oxalate phase. However, during this phase, X-537A suddenly releases an amount of Ca++ similar in quantity to that released in the initial phase. The delay of the oxalate-dependent phase is a function of pH. At pH of about 5.5, the oxalate phase and simultaneous calcium oxalate precipitation would theoretically start at the beginning, with no delay. Ejection of protons during Ca++ uptake is strongly depressed by oxalate, but not by other organic anions which do not trap Ca++. It is suggested that oxalate is transferred to the inside of the vesicles as a monoprotonated species at expense of protons ejected by the Ca++-pump during the uptake of Ca++.