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.     

Time-course of altered islet phospholipids and of calcium binding and ionophoretic properties of islet lipids following glucose stimulation

The time-course of alteration in islet cell phospholipid content following d-glucose exposure in islet cells and in islet cell membranes was related to the ability of lipids extracted from both cultured pancreatic islet cells and from plasma membranes isolated from the islet cells to translocate calcium in two model membrane systems. The first model system (bulk-phase system) detected lipid species with the ability to bind calcium, irrespective of their ability to enhance calcium transport across cell membranes. The second system (multilamellar membrane system) detected lipid species with the ability to both bind calcium and to enhance calcium transport across cell membranes (true ionophores). Pre-exposure to high d-glucose concentration led to a rapid (within 1 min) fall in membrane phosphoinositides. This was partially blocked by mannoheptulose. A concurrent fall in calcium bindig activity of lipids from the plasma membrane was observed. In the whole islet cell fraction, d-glucose induced a marked increase in Ca²⁺ ionophoretic activity. Unlike the fall in membrane polyphosphoinositides and membrane Ca²⁺ binding activity, these changes were dependent on the presence of added extracellular calcium. l-Glucose was without effect on membrane phosphoinositide content. It is concluded that altered membrane and intracellular phospholipids may contribute to the increased availability of intracellular Ca²⁺ following d-glucose stimulation by virtue of theie Ca²⁺ binding and ionophoretic properties.     

The formation constants of the proton and calcium complexes of prostaglandins PGE1 and PGF1b

The formation constants of the proton and calcium complexes of PGE₁ and PGF_1β have been measured in aqueous solution at 20 ± 1°. The proton complexes are shown to be effectively ionized at pH 7, log K_HL values being 5.12 and 5.46, respectively. The calcium complexes, studied radiometrically using ⁴⁵Ca by ion exchange equilibria, have formation constants (K_CaPG) of 37 and 9 (approx.), respectively. “Model” ligands studied in developing the technique were 3-hydroxybutyric acid (K_Ca, 3.1) and 4-hydroxybutyric acid (K_Ca, 5.7).     

Effect of dietary calcium on renal prostaglandins.

The present study was designed to clarify the possible role of renal prostaglandins (PGs) on blood pressure (BP) regulation during calcium (Ca) restriction or supplementation. Twelve normotensive women with a mean age of 21.2 years participated in the study. After 1 week of normal Ca intake (mean +/- SE, 536 +/- 2 mg/day), a low-Ca diet (163 +/- 1 mg/day) was given for a further 1 week. Additional asparagine Ca (3 g as Ca/day) was also given to half of the subjects. BP, heart rate, and serum total and ionized Ca concentrations were measured at the end of each period. Levels of Ca, sodium, PGE2, 6-keto-PGF1 alpha and thromboxane (TX) B2 excreted into urine were also determined. The plasma level of ionized Ca was significantly increased without any change in total Ca in both groups. Low and high Ca intake decreased and increased urinary Ca excretion by 28% and 56%, respectively. BP was not altered after Ca deprivation or loading. However, urinary PGE2 excretion was significantly augmented from 668.9 +/- 68.1 to 959.7 +/- 183.1 ng/day by Ca loading, whereas Ca deprivation decreased PGE2 excretion (695.4 +/- 108.1 to 513.2 +/- 55.2 ng/day). No changes were observed in 6-keto-PGF1 alpha or TXB2 urinary excretion. These results suggest that renal PGE2 synthesis is stimulated or decreased by 1-week Ca loading or deprivation, indicating a possible antihypertensive role of renal PGE2 during high-Ca intake in hypertensives.     

Comparison of some properties of free and immobilized alpha-amylase by Aspergillus sclerotiorum in calcium alginate gel beads

Some properties of immobilized alpha-amylase by Aspergillus sclerotiorum within calcium alginate gel beads were investigated and compared with soluble enzyme. Optimum pH and temperature were found to be 5.0 and 40 degrees C, respectively, for both soluble and immobilized enzymes. The immobilized enzyme had a better Km value, but kcat/Km values were the same for both enzymes. Entrapment within calcium alginate gel beads improved, remarkably, the thermal and storage stability of alpha-amylase. The half life values of immobilized enzyme and soluble enzyme at 60 degrees C were 164.2, and 26.2 min, respectively. The midpoint of thermal inactivation (Tm) shifted from 56 degrees C (for soluble enzyme) to 65.4 degrees C for immobilized enzyme. The percentages of soluble starch hydrolysis for soluble and immobilized alpha-amylase were determined to be 97.5 and 92.2% for 60 min, respectively.     

Insulin and glucagon secretion from isolated islets of Langerhans. The effects of calcium ionophores.

The role of Ca2+ in the secretion of insulin and glucagon was investigated by studying the effects of Ca2+ ionophores on hormone secretion from isolated perifused islets of Langerhans. Ionophore X537A (100 muM), which binds alkaline earth cations and also complexes some univalent cations, caused a rapid transient increase in insulin and glucagon secretion which was not dependent on the presence of Ca2+ in the perifusion medium. Ionophore A23187 (100 muM), which specifically binds bivalent cations at neutral pH values, similarly increased insulin secretion in complete and Ca2+-free medium, but only stimulated glucagon release in the presence of extracellular Ca2+. Since the stimulatory effects of both ionophores were associated with an increased Ca2+ flux in the islets, these experiments support the hypothesis that Ca2+ may trigger the release of insulin and suggest that it is also involved in the secretion of glucagon. The basal rate of both insulin and glucagon release was significantly increased when Ca2+ was omitted from the perifusion medium, but it is proposed that this finding may be due to adverse effects on cell-membrane function under these conditions.     

Influence of ionophores which bind calcium on the release of norepinephrine from synaptosomes.

A preparation of synaptosomes isolated from rat brain was used as a model of nerve to study affects of drugs on uptake and release of biogenic amines. The influence of ionophores, which bind calcium, on the release of noripinephrine from synaptosomes was examined to determine their effect on the release of the amine. A23187 induced release of norepinephrine mainly as the amine and this action was enhanced by calcium and depressed by magnetism. X-537A however, released norepinephrine mostly as deaminated metabolites but acted independently of calcium or magnetism. A23187, therefore is thought to be associated at least in part, with exocytotic amine release, possibly by enhancing entry of calcium across the plasma membrane. X-537A on the other hand may act as a carrier of the amine across the vesicular membrane and expose the amine to intrasynaptosomal monoamine oxidase.     

Effect of calcium ionophores on early development in fucoid algae

Although much studied, the role of Ca²⁺ in early fucoid development remains unclear. One technique to investigate Ca²⁺ function that has not been fully exploited is the use of ionophores to drastically increase cytosolic Ca²⁺ activity. We have therefore conducted an analysis of the effects of uniform application of the Ca²⁺ ionophores, A23187 and ionomycin, on early development of fucoid algae. Both ionophores had substantially the same effects. Cell adhesive secretion, rhizoid growth and negative phototropism were reduced but not abolished by ionophore treatment, and germination was delayed. One plausible interpretation of these data is that secretion is partially compromised in the treated zygotes. Surprisingly, photopolarization and cytokinesis were unaffected, indicating that Ca²⁺ homeostasis may not be required for these processes.     

Phosphatidate and oxidized fatty acids are calcium ionophores. Studies employing arsenazo III in liposomes

Liposomes which have entrapped the metallochromic dye, arsenazo III, constitute a sensitive assay system for ionophoresis of divalent cations. By this means we have compared known calcium ionophores (A23187, ionomycin) with membrane phospholipids, fatty acids, prostanoids, and retinoids. Added at micromolar concentrations to preformed multilamellar liposomes (phosphatidylcholine 7:dicetyl phosphate 2: cholesterol 1) both A23187 and ionomycin, as well as phosphatidic acid and products derived from linoleic acid, linolenic acid, and two eicosatrienoic acids provoked Ca influx (e.g. phosphatidic acid: 0.13 mol of Ca2+/mol of membrane lipid/5 min). A variety of other phospholipids (e.g. phosphatidylinositol), fatty acids (e.g. arachidonic acid), prostanoids (e.g. PGE1) retinoids (e.g. retinoic acid), and glyceryl ether phosphorylcholines ("platelet-activating factors") were without effect. Phosphatidic acid and oxidized fatty acids translocated divalent cations selectively, demonstrating the same rank order as A23187 or ionomycin: Mn greater than Ca greater than Sr much greater than Mg. Membrane lysis did not contribute to the perceived translocation; the liposomes remained impermeable to EDTA, EGTA, arsenazo III, or Mg. Liposomes with phosphatidic acid or oxidized trienoic acids preincorporated at 1-5 mole % of total lipids also permitted translocation of Ca but not Mg. Reduction of ionophoretic fatty acids or ionomycin with stannous chloride abolished their ionophoretic activity. Release of Ca from liposomes which had entrapped arsenazo III-Ca complexes into a medium rich in EGTA permitted calculation of efflux induced by ionophores, whether these were added to the outside of liposomes or preincorporated. Data suggest that phosphatidic acid and oxidized di- and trienoic fatty acids, which act as calcium ionophores in model bilayers, could serve as "endogenous ionophores" in cells.     

IL-1 secretion by macrophages. Enhancement of IL-1 secretion and processing by calcium ionophores

In the present study we have demonstrated that the murine IL-1 alpha precursor lacks a cleavable signal sequence and does not undergo cotranslational translocation across microsomal membranes in vitro. Culture supernatants of the murine macrophage cell line, P388D, or from normal peritoneal macrophages collected within 0.5 to 3 h after stimulation contained the 33,000 m.w. precursor as the predominant form of IL-1 alpha. Over an 18-h period, the level of low m.w. IL-1 alpha increased as the secreted precursor was processed by extracellular and/or cell surface-associated proteolytic enzymes. The calcium ionophores A23187 and ionomycin were found to dramatically enhance the release and processing of murine and human IL-1. The rapid release of IL-1 in response to a change in the intracellular level of calcium does not appear to be caused by release of a membrane-bound form of the protein, nor is there evidence that IL-1 is packaged and released from cytoskeletal associated secretory granules. In marked contrast, calcium ionophores do not induce secretion of IL-1 from a nonmacrophage cell line that synthesizes but does not normally secrete IL-1. Our results suggest that activated macrophages possess a novel processing independent, possibly calcium-dependent, mechanism that allows for the release of the precursor forms of IL-1 alpha and IL-1 beta.     

Mobilization of intracellular calcium by extracellular ATP and by calcium ionophores in the Ehrlich ascites-tumour cell

We have studied the changes of the intracellular free calcium concentration ([Ca2+]i) effected by external ATP, which induces formation of inositol trisphosphate, and by the divalent cation ionophores ionomycin and A23187. Both, ATP (40 microM) and ionophores (1-80 mumol/l cells ionomycin; 20-400 mumol/l cells A23187), produced a transient rise of [Ca2+]i which reached its maximum within 15-30 s and declined near resting values (about 200 nM) within 1-3 min. When the [Ca2+]i peak surpassed 500 nM a transient cell shrinkage due to simultaneous activation of Ca2+-dependent K+ and Cl- channels was also observed. The cell response was similar in medium containing 1 mM Ca2+ and in Ca2+-free medium, suggesting that the Ca mobilized to the cytosol comes preferently from the intracellular stores. Treatment with low doses of ionophore (1 mumol/l cells for ionomycin; 20 mumol/l cells for A23187) depressed the response to a subsequent treatment, either with ionophore or with ATP. Treatment with ATP did also inhibit the subsequent response to ionophore, but in this case the inhibition was dependent on time, the stronger the shorter the interval between both treatments. This result suggests that the permeabilization of Ca stores by ATP is transient and that Ca can be taken up again by the intracellular stores. Refill was most efficient when Ca2+ was present in the incubation medium. Addition of either ATP or ionomycin (1-25 mumol/l cells) to cells incubated in medium containing 1 mM Ca2+ decreased drastically the total cell Ca content during the following 3 min of incubation. In the case of ATP the total cell levels of Ca returned to the initial values after 7-15 min, whereas in the case of the ionophore they remained decreased during the whole incubation period. These results indicate that Ca released from the intracellular stores by either ATP or ionophores is quickly extruded by active mechanisms located at the plasma membrane. They also suggest that, under the conditions studied here, with 1 mM Ca2+ outside, the Ca-mobilizing effect of ionophores is stronger in endomembranes than in the plasma membrane.     

Movement of calcium through artificial lipid membranes and the effects of ionophores

The calcium efflux from multi-layered vesicles (liposomes) of different lipid composition has been studied. Liposomes composed of lipids extracted from cattle retinas are compared with liposomes which consist of phosphatidylcholine or a 1 : 1 phosphatidylcholine/phosphatidylserine mixture. The percentages of ⁴⁵Ca capture by these three types of liposomes are 10, 1 and 4% respectively.The efflux rates are 2.5 · 10^?6, 2 · 10^?6 and 4 · 10^?5 s^?1 respectively. The semilogarithmic efflux curves for phosphatidylcholine and phosphatidylcholine/phosphatidylserine liposomes are linear with time, but those for the retinal lipid liposomes are discontinuous. The activation energy for the calcium efflux from the latter liposomes is about 10.5 kcal/mol, both before and after the discontinuity.The ionophores X537A and A23187 enhance the calcium leakage from retinal lipid liposomes, the latter ionophore being much more effective than the former. At high concentrations both ionophores seem to transport calcium as a 1 : 2Ca · ionophore complex. At low ionophore concentrations, however, X537A appears to transport calcium as a 1 : 1 complex, but A23187 as a 2 : 1 complex.     

Induction of acrosomal reaction and calcium uptake in ram spermatozoa by ionophores

Ram spermatozoa incubated in the presence of Ca2+ and the Ca2+-ionophore A23187 undergo a process which is known as the acrosome reaction. This reaction is characterized by fusion of the outer acrosomal membrane and the overlying plasma membrane to form mixed vesicles which can be seen in the electron microscope. As a result, the trypsin-like acrosin is released from the cells to the medium. The occurrence of the acrosome reaction was determined by following acrosin activity in the medium. After 2 h of incubation of the cells in the presence of ionophore and Ca2+, the released acrosin activity is related to the ionophores according to the sequence: A23187 greater than monensin greater than valinomycin greater than FCCP = without ionophore. The study of Ca2+ uptake by the cells revealed that Ca2+ enters the cell prior to the release of acrosin. Monensin can induce Ca2+ uptake and acrosin release only when Na+ is present in the incubation medium. There is no increase in Ca2+ uptake with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). We suggest that the Na+/H+ exchange induced by monensin causes an increase in intracellular Na which is the driving force for the Ca2+ entry via a Ca2+/Na+ antiporter. Since monensin can induce an increase in Ca2+ uptake only in the presence of Na+, FCCP enhances Ca2+ uptake in the presence of valinomycin, and A23187 is a Ca2+/2H+ exchanger, we suggest that alkalization of the intracellular space is involved in the acrosome reaction. Calcium uptake in the presence of monensin is not affected by the uncoupler FCCP, a result which indicates that Ca2+ is not accumulated in the mitochondria. Incubation of cells for 3 h in the absence of Ca2+ or ionophore caused a 3-fold increase in the rate of acrosin release when monensin and Ca2+ were added together. There was no change in this rate when A23187 was used. We suggest that during the preincubation time (known as capacitation) the permeability of the plasma membrane to Ca2+ is enhanced. This study shows that acrosin release and Ca2+ uptake can be used as a quantitative asay for the determination of the acrosome reaction.     

Ricinoleate and deoxycholate are calcium ionophores in jejunal brush border vesicles

Summary The intestinal secretagogues ricinoleate and deoxycholate have been tested for a capacity to form complexes with Ca²⁺ ions and to affect the passive equilibration of Ca²⁺ ions across the jejunal brush border membrane. Both of these agents formed butanol-soluble Ca²⁺ complexes in a model phase distribution system. They also promote the passive uptake and efflux of Ca²⁺ across brush border vesicles in a concentrationdependent manner. The levels of ricinoleate and deoxycholate that increase the rate of transvesicular Ca²⁺ movement are in the 100 to 300 m range. Concentrations as high as 1.0mm had no significant detergent effects in vesicles as measured by release of entrapped sorbitol. The kinetics of Ca²⁺ uptake and efflux are similar in brush border vesicles treated with A23187, ricinoleate, or deoxycholate. The influx rates observed in this study were high enough to cause the collapse of a Ca²⁺ gradient, which had been generated by Ca-Mg ATPase enzyme activity in the brush border membrane. Ricinoleate did not affect Ca-Mg ATPase activity at concentrations used in this study, but deoxycholate was inhibitory, indicating two potential modes for elevation of intracellular Ca²⁺ content by deoxycholate. When compared with the effects of the Ca²⁺ ionophore, A23187, it appears that both ricinoleate and deoxycholate could have significant intestinal secretory activity due to this Ca²⁺ ionophore property. It is also noteworthy that, at least in this model system, potential secretory effects are expressed at concentrations significantly below levels that have been associated with detergent effects or altered epithelial morphology.     

Determination of association and dissociation rate constants for bilirubin--bovine serum albumin.

The association rate constant for the binding of bilirubin to bovine serum albumin has been determined in a continuous-flow experiment. The value obtained is 0.9 x 10⁶m^?1S^?1. Furthermore the dissociation rate constant is determined from the rate of the peroxidase-catalyzed oxidation of bilirubin in a bilirubin-albumin solution. This figure is 3.1 × 10^?2s ¹. Calculation of the apparent binding equilibrium constant from the two rate constants gives 2.9 x 10⁷m^?1. The above mentioned peroxidase oxidation has also been used for a direct estimation of the binding equilibrium constant giving 2.7 × 10⁷m^?1. All experiments are carried out at 36 °C and pH 7.4.     

Priming effect of calcium ionophores on phorbol ester-induced respiratory burst in mouse peritoneal neutrophils.

The abilities of three calcium ionophores (A23187, 4-bromo-A23187, and ionomycin) to modulate the respiratory burst of neutrophils induced by phorbol ester and to increase the concentration of free intracellular Ca2+ ([Ca2+]i) were compared. The production of reactive oxygen species (ROS) was determined by luminol-dependent chemiluminescence and [Ca2+]i was determined with the Fura-2 fluorescent probe. A23187 (0.05-2 microM) and ionomycin (0.001-0.5 microM) but not 4-bromo-A23187 amplified 3-4-fold the respiratory burst induced by phorbol ester. The integral response (total production of ROS over 6 min) had a bell-shaped dependence on the concentration of ionomycin and A23187 with increase and decrease at low and high concentrations of the ionophores, respectively. The maximal effect was found at 0.5 microM ionomycin and 2 microM A23187, these concentrations resulting in transient increases in [Ca2+]i to 1776 +/- 197 and 955 +/- 27 nM, respectively. The ionophores had no effect in calcium-free media, though they increased [Ca2+]i to approximately 400 nM through the mobilization of intracellular Ca2+. In cells with exhausted stores of Ca2+, the addition of 1.5 mM Ca2+ combined with phorbol ester amplified twofold the production of ROS. The inhibition of phospholipase A2 with 4-bromophenacyl bromide significantly decreased the production of ROS. Thus, the entrance of Ca2+ and generation of arachidonic acid under the influence of phospholipase A2 are necessary for the ionophore-induced priming of production of ROS during cell activation with phorbol esters.