PAF effects on transmembrane signaling pathways in rat kupffer cells

Platelet activating factor (PAF) was found to stimulate the metabolism of inositol phospholipids via deacylation and phospholipase C in Kupffer cells, the resident macrophages in liver. PAF-induced phosphoinositide metabolism occurred in two phases. Within seconds after stimulation, in the absence of extracellular Ca⁺⁺, platelet activating factor caused the phosphodiester hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with the release of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate. This was followed by an extracellular Ca⁺⁺-dependent release of glycerophosphoinositol, inositol monophosphates and inositol bisphosphates. Various Ca⁺⁺-mobilizing agonists failed to evoke hydrolysis of phosphoinositides. Platelet activating factor also stimulated the synthesis and release of prostaglandins from these cells. Platelet activating factor-stimulated phosphodiester metabolism of phosphoinositides and prostaglandin synthesis was inhibited by treatment with pertussis toxin and cholera toxin. Pertussis toxin also inhibited platelet activating factor-induced glycerophosphoinositol release. Cholera toxin, in contrast, stimulated platelet activating factor-induced glycerophosphoinositol release and prostaglandin synthesis and synergistically stimulated the effect of platelet activating factor on these processes. The results suggest that platelet activating factor-induced metabolism in the Kupffer cells occurs via specific receptors and may be mediated through the activation of different G-proteins. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Preparation of Poly(lactic acid) Nanocomposite via Catalyst-Modified Montmorillonite

Biopolymer nanocomposite of polylactic acid was synthesized from lactic acid using tin ions exchanged Cloisite Ca⁺⁺. The tin molecules between the Cloisite Ca⁺⁺ layers catalyzed the lactic acid polymerization and also this clay played a role as a nanofiller. Presence of tin molecules increased the molecular weight of the polymer compared with the polymer that was synthesized with the presence of just Cloisite Ca⁺⁺. Both exfoliated and intercalated nanocomposite structures were observed as the results of X-ray diffraction analysis and transmission electron microscopy images. Also, changes of polymer's thermal degradation and other thermal properties such as glass transition temperature, melting temperatures were investigated via thermogravimetric analysis and differential scanning calorimetry.     

Barium chloride crosslinked carboxymethyl guar gum beads for gastrointestinal drug delivery

A mild method for microencapsulation of sensitive drugs, such as proteins, employing a suitably derivatized carboxymethyl guar gum (CMGG) and multivalent metal ions like Ca⁺⁺ and Ba⁺⁺ is reported. Initially, guar gum is derivatized with carboxymethyl groups so that it forms durable, self‐standing microbeads when its solution is dropped into CaCl₂ or BaCl₂ solutions. The swelling data of Ca⁺⁺ and Ba⁺⁺ crosslinked beads suggest that Ba⁺⁺ crosslinks CMGG much more efficiently than Ca⁺⁺. The drug loading efficiency of these Ba⁺⁺/CMGG beads, as a function of concentration of both metal ion as well as drug, was then determined using Bovine Serum Albumin as a model drug. The ability of these beads to protect the drug from the acidic environment of the stomach was investigated. It was found that a very little amount of the drug is released from the beads when they are suspended in NaCl–HCl buffer of pH 1.2 for 6 h. The beads were also shown to release almost the entire encapsulated drug when exposed to TRIS–HCl buffer of pH 7.4. Thus, the results indicate that Ba⁺⁺ crosslinked carboxymethyl guar gum beads can be used for gastrointestinal drug delivery. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 3084–3090, 2001     

The functioning of the lipids and lipoproteins of sarcotubular membranes in calcium transport

In the intact muscle cell, an internal tubular membrane system called the sarcoplasmic reticulum (SR) plays an important role in the contraction-relaxation cycle by controlling the Ca⁺⁺ of the myoplasm; release of Ca⁺⁺ from the SR to myoplasm initiates contractile activity and sequestring Ca⁺⁺ in the SR by means of a transport system causes muscle to relax. Fragments of the SR with a vesicular structure can be isolated from muscle homogenate and these vesicles are able to vigorously transport Ca⁺⁺ from incubation media into the intravesicular space thus enabling study of Ca⁺⁺ transport under precisely defined in vitro conditions. A highly purified fraction of SR vesicles called SF₁ were prepared from rat muscle by means of density gradient centrifugation procedures. The role of SR lipid in Ca⁺⁺ transport was studied. SF₁ was treated in vitro with either phospholipase A or C or D or polyene antibiotics. The effect of essential fatty acid deficiency, induced in vivo, was also investigated. It was concluded that the only structural feature of SF₁-lipid involved in Ca⁺⁺ transport and the associated adenosine triphosphatase is the phosphoryl moiety of the phospholipids. Evidence was obtained which inplicated histidine residues of the SF₁ protein in this transport function. To study the role of SF₁ protein in this process in depth, the membranes were solubilized by a sodium dodecylsulfate system and made free of their lipid components. More than 95% of this protein is soluble in dilute salt solution; of this, more than 90% is composed of a protein fraction which can be isolated by gel filtration (called protein fraction-2). Protein fraction-2 contains large molecular aggregates of small polypeptide subunits of identical or nearly identical molecular weight. They contain solely N-terminal glycine and probably only C-terminal alanine. The significance of such a high percentage of similar polypeptide subunits in SR is discussed. One of five papers to be published from the Symposium “Lipid Transport” presented at the AOCS Meeting, New Orleans, April 1970.     

Effect of water hardness ions on the solution properties of an anionic surfactant

The solution characteristics of the system linear alkylbenzene sulfonate (LAS)/Ca⁺⁺/builder were studied using a dynamic surface tension technique. The results showed that the rate of CA⁺⁺/LAS interaction is slower than the CA⁺⁺ binding rate of zeolite A. Consequently, zeolite A is effective in preventing precipitation of LAS by calcium ions. The data obtained from the study of Ca⁺⁺ binding detergent builders on solubilization of Ca(LAS)₂ showed that zeolite A was effective but the rate of solubilization was much slower than that for STPP. Incorporation of a small amount of phosphate with zeolite A in a detergent significantly increases the rate of solubilizing of Ca(LAS)₂.     

Lipid Production by Lipomyces lipoferus

Lipomyces lipoferus was studied for its lipid production by investigating the inhibitory and stimulatory effects of different metals on lipid synthesis. The nature of metallic ions present in the medium exercise a considerable influence on the lipid storage. Chlorides of Ca⁺⁺ and Mg⁺⁺ ' show a marked inhibition on the formation of lipid materials while the acetates of the same metallic ions show improving effect on the amount of lipid produced. It is further noted that Na⁺ and K⁺ ions show better results for lipid production as compared to Ca⁺⁺ and Mg⁺⁺ when used as their acetates. The utilisation of sugar is also less effective with Ca⁺⁺ than with Na⁺⁺. The fatty acid composition of lipids extracted from cells of L. lipoferus shows that palmitic acid and oleic acid are the major components. However, the percentage of these acids increases with the age of mycelium by 11 % and 6% respectively. TLC results reveal that triglycerides are the major components of the neutral lipids (90 % ).     

Lysolecithin acyltransferase in hamster intestinal mucosa

1-Acyl-lysolecithin acyltransferase has been demonstrated in the microsomal fraction of hamster intestinal mucosa. The characteristics of the enzyme, with respect to substrate concentration, time of incubation and protein concentration, were studied. Ca⁺⁺ was found to severely inhibit enzymatic activity. More modest inhibitors were found to be Mg⁺⁺ and F⁻; EDTA and albumin had no effect. Enzyme activity was reduced when palmityl CoA was substituted for oleoyl CoA as substrate. The specific activity of intestinal microsomes was modestly greater than liver microsomes.     

Calcium activation of peanut lipoxygenase

Peanut lipoxygenase isozyme 1 (pH optimum, 8.3) was strongly activated by 0.5–1.0 mM Ca⁺⁺, and the rate of activation was maximum when the ratio of substrate to Ca⁺⁺ was ca. 2∶1. Peanut lipoxygenase isozymes 2 and 3 (pH optima, 6.2) were activated by calcium but did not have an optimum level of activity. Calcium differentially activated peanut lipoxygenase causing the rate of pentane production to increase much more rapidly than the rate of oxygen consumed by the enzyme reaction. At pH 6.2, in the absence of calcium, the percentages of the hydroperoxide isomers produced by peanut lipoxygenase were 74.9% 13-hydroperoxycis-9,trans-11-octadecadienoic acid (13 LOOHcis-trans), 2.6% 13-hydroperoxytrans-9,trans-11-octadecadienoic acid (13 LOOHtrans-trans) and 22.5% 9-hydroperoxy 10, 12-octadecadienoic acid (9 LOOH). The presence of 1 mM Ca⁺⁺ at pH 6.2 did not significantly affect the percentage distribution of the hydroperoxides produced. However, at pH 8.3, the percentage distribution of hydroperoxides produced was 45.2% 13 LOOHcis-trans, 10.9% 13 LOOHtrans-trans and 43.9% 9 LOOH in the absence of Ca⁺⁺ and 57.0% 13 LOOHcis-trans, 8.0% 13 LOOHtrans-trans and 35.0% 9 LOOH in the presence of 1 mM Ca⁺⁺. Paper No. 5110 of the Journal of the North Carolina Agricultural Experiment Station, Raleight, NC 27607.     

Effects of dietary n−3 polyunsaturated fatty acids on phospholipid composition and calcium transport in mouse cardiac sarcoplasmic reticulum

The effects of dietary n−3 and n−6 polyunsaturated fatty acids on the fatty acid composition of phospholipid, Ca⁺⁺· Mg⁺⁺ ATPase and Ca⁺⁺ transport activities of mouse sarcoplasmic reticulum were investigated. Mice were fed a 2 weight percent fat diet containing either 0.5 weight percent ethyl esters of 18∶3n−3, 20∶5n−3 or 22∶6n−3 as a source of n−3 polyusaturated fatty acid or 0.5 weight percent safflower oil as a cource of n−6 polyunsaturated fatty acid for 10 days. Olive oil (2 weight percent) was used as a control diet. Although feeding n−6 polyunsaturated fatty acid induced very little modifications of the phospholipid sarcoplasmic reticulum fatty acid composition, feeding n−3 polyunsaturated fatty acid altered it markedly. Inclusion of 18∶−3, 20∶5n−3 or 22∶6n−3 in the diet caused an accumulation of 22∶6n−3, which replaced 20∶4n−6 and 18∶2n−6 in phospholipid sarcoplasmic reticulum. The saturated fatty acids were significantly increased with a concurrent reduction of 18∶1n−9. These changes in the fatty acid composition resulted in a decrease in the values of the n−6/n−3 polyunsaturated fatty acid ratio and a decrease in the ratio of 20 carbon to 22 carbon fatty acids esterified in the phospholipid sarcoplasmic reticulum. This was associated with a decrease in Ca⁺⁺ uptake by n−3 polyunsaturated fatty acid enriched sarcoplasmic reticulum vesicles as compared with n−6 fatty acid and control diet sarcoplasmic reticulum vesicles. However, neither the affinity for Ca⁺⁺ nor the maximal velocity of ATP hydrolysis activity of Ca⁺⁺·MG⁺⁺ ATPase were altered by the different diets. The data suggest that the incorporation of 22∶6n−3 and/or the decrease of 20∶4n−6 plus 18∶2n−6 in the phospholipid sarcoplasmic reticulum may affect the membrane lipid bilayer structure and make it more permeable to Ca⁺⁺.     

Lipid Metabolism in Rats is Modified by Nitric Oxide Availability Through a Ca<Superscript>++</Superscript>-Dependent Mechanism

We studied lipid metabolism and the antioxidant defense system in plasma and liver of rats fed diets supplemented with l ^ω-nitro-l-arginine methyl ester (l-NAME), isosorbide dinitrate (DIS), l-arginine (Arg), or the associations of these drugs. Liver hydroperoxide and thiobarbituric-acid-reactive substance (TBARS) levels were decreased by Arg and increased by l-NAME or DIS treatments. Oxidized glutathione and conjugated dienes were increased by DIS. Nitrate + nitrite levels and serum calcium ([Ca⁺⁺]) were incremented by Arg or DIS and reduced by l-NAME. Superoxide dismutase and catalase activities decreased under Arg treatment, while l-NAME or DIS caused stimulation. Liver high-density lipoprotein (HDL) cholesterol was increased by DIS or NAME (alone or associated with Arg). Free fatty acids and neutral and polar lipids were increased by Arg, l-NAME, and DIS. However, predominating phospholipid synthesis increased the neutral/polar ratio. Decreased levels of nitric oxide (NO) (low [Ca⁺⁺]) was directly associated with increased fatty acid synthetase, decreased phospholipase A₂, carnitine-palmitoyl transferase, and fatty acid desaturase activities. Raised NO (high [Ca⁺⁺]) inversely correlated with increased phospholipase-A₂ and acyl-coenzyme A (CoA) synthetase and decreased fatty acid synthetase and β-oxidation rate. Arg or DIS produced changes that were partially reverted by association with l-NAME. Based on these observations, prolonged therapeutical approaches using drugs that modify NO availability should be carefully considered. Julio Nella author in memoriam.     

P2X4 Receptors Mediate Ca2+ Release from Lysosomes in Response to Stimulation of P2X7 and H1 Histamine Receptors

The P2X4 purinergic receptor is targeted to endolysosomes, where it mediates an inward current dependent on luminal ATP and pH. Activation of P2X4 receptors was previously shown to trigger lysosome fusion, but the regulation of P2X4 receptors and their role in lysosomal Ca²⁺ signaling are poorly understood. We show that lysosomal P2X4 receptors are activated downstream of plasma membrane P2X7 and H₁ histamine receptor stimulation. When P2X4 receptors are expressed, the increase in near-lysosome cytosolic [Ca²⁺] is exaggerated, as detected with a low-affinity targeted Ca²⁺ sensor. P2X4-dependent changes in lysosome properties were triggered downstream of P2X7 receptor activation, including an enlargement of lysosomes indicative of homotypic fusion and a redistribution of lysosomes towards the periphery of the cell. Lysosomal P2X4 receptors, therefore, have a role in regulating lysosomal Ca²⁺ release and the regulation of lysosomal membrane trafficking.     

Über den Kationenaustausch bei anionischen Polyurethan-Dispersionen mit Natriumsulfonat-gruppen

In aqueous dispersions of anionic polyurethanes with SO groups and Na⁺ as counter-ion, Na⁺ was ion exchanged for other cations (H⁺, Ag⁺, Ca⁺⁺, Ba⁺⁺, Ni⁺⁺, Cu⁺⁺, Zn⁺⁺, Pb⁺⁺ and AI⁺⁺⁺). With the exception of AI⁺⁺⁺ the ion exchange took place nearly quantitatively, indicating that site binding of counter-ions did not occur at the particle surface. In addition, the influence of the counter-ion was studied with respect to stability of the dispersion and the mechanical properties of the films.     

Effect of soluble ash components on the viscosity of lignite-water mixtures

The removal of multivalent cations from lignite-water mixtures (LWM) was shown to lower mixture apparent viscosity by a factor of about 40, for example, at 100 s^?1 by reducing both the yield stress and plastic viscosity. The cations Ca⁺⁺, Mg⁺⁺, Al⁺⁺⁺, K⁺, and Na⁺, among others, were found to be present in the aqueous phase of the LWM in concentrations ranging from 546 ppm to 8 ppm. The primary anions present were CI^? and SO₄⁼. The degree to which the cations affected viscosity were of order trivalent > divalent > monovalent. The nature of the anion was found to be relatively unimportant. A process utilizing ion-exchange resins for removing multivalent cations to produce low viscosity lignite-water or coal-water mixtures is described.     

Reducing Myosin II and ATP-Dependent Mechanical Activity Increases Order and Stability of Intracellular Organelles

Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca⁺⁺ probe as a small and mobile tracer in live T cells. Ca⁺⁺, highlighted by Fluo-4, is localized in intracellular organelles. Inhibiting intracellular mechanical work by myosin II through blebbistatin treatment increased cellular dis-homogeneity of Ca⁺⁺-rich features in length scale < 1.1 μm. We detected a similar effect in cells imaged by label-free bright-field (BF) microscopy, in mitochondria-highlighted cells and in ATP-depleted cells. Blebbistatin treatment also reduced the dynamics of the Ca⁺⁺-rich features and generated prominent negative temporal correlations in their signals. Following Guggenberger et al. and numerical simulations, we suggest that diffusion in the viscoelastic and confined medium of intracellular organelles may promote spatial dis-homogeneity and stability of their content. This may be revealed only after inhibiting intracellular mechanical work and related cell vibrations. Our described mechanisms may allow the cell to control its organization via balancing its viscoelasticity and mechanical activity, with implications to cell physiology in health and disease.     

Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1

In pancreatic β-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca²⁺-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca²⁺ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca²⁺ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 β-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca²⁺ entry were investigated in Fura-2 Ca²⁺-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca²⁺ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S₁₁₇₂A mutant displayed enhanced Ca²⁺ entry. The TRPM3-mediated Ca²⁺ entry in INS-1 β-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 β-cells.     

Studies on phospholipid biosynthesis in hepatocytes from alcoholic rats by using radiolabeled exogenous precursors

We have studied the synthesis of phospholipids in hepatocytes isolated from chronically ethanol-treated rats by using isotopically labelled serine, ethanolamine, and choline as exogenous precursors. Our results demonstrate that ethanol induces specific effects on the biosynthesis of phosphatidyl-ethanolamine and phosphatidylcholinevia CDP-derivatives and also on the synthesis of phosphatidylserinevia the Ca⁺⁺-dependent base-exchange reaction. Thus, the synthesis of phosphatidylethanolamine from [³-H]ethanolamine and the incorporation of [³H]serine into phosphatidylserine were clearly higher in hepatocytes from ethanol-treated rats compared to controls. The synthesis of phosphatidylcholine from [methyl-¹⁴C] choline, on the other hand, decreased markedly, suggesting a specific inhibition of cholinephosphotransferase activity. We have also demonstrated that the phosphatidylcholine levels are markedly decreased in hepatocytes isolated from chronically ethanol-treated rats as a consequence of the lower phosphatidylcholine biosynthesis. The decrease in the incorporation of radioactivity from choline to betaine, which we also found, is interpreted as being the result of a higher use of betaine as methyl donor instead of methionine to maintain the hepaticS-adenosylmethionine levels in chronic alcoholism.     

Effect of calcium on absorption of fatty acid by rat jejunum in vitro

The effect of Ca⁺⁺ on jejunal osmiophilic particles was studied in a recirculating system which was not contaminated with plasma lipoproteins. An isolated, infused segment of rat jejunum was suspended in a bath of liquid paraffin. Transudate, containing osmiophilic particles, appeared like beads of sweat on the serosal surface, and fell to the bottom of the bath. In the range of 25–38 C, 30 C proved to be optimal for histological preservation of villous architecture. Production of transudate, 20 mg/min/g of jejunum, and transport of [¹⁴C] oleate proceeded nearly linearly after the first 30 min. Necrosis of mid-villus and crypt cells became obvious by light microscopy after one hour. Therefore, transudate was collected between the period of 30–60 min. Shadow casting of transudates, produced when saline was infused, revealed that 86±9 (SD) % of osmiophilic particles was <800 Å in diameter; 13±8% was 800–1000 Å; 0.4±0.5% was 1000–2000 Å. Corresponding values were 58±10, 25±5, and 16±5% when 5 mM [¹⁴C] oleate+2.5 mM monoolein was infused; 75% of the transported [¹⁴C] appeared in triglyceride. Adding 2 mM Ca⁺⁺ to the infusion doubled the transport of [¹⁴C] triglyceride without increasing particle size further. We conclude that luminal Ca⁺⁺ increases the absorption of luminal fatty acid by rat jejunum in vitro.     

Metabolic studies in isolated rat liver cells: I. Lipid synthesis

Rat liver cells, isolated by chelate perfusion and extrusion through a tissue press, incorporated labeled acetate into cellular lipids and into lipids released into the suspending medium. Optimal rates of incorporation required supplementation of tris-KCl medium with Mg⁺⁺, Mn⁺⁺, succinate, citrate, nicotinamide, Coenzyme A, NADP and glucose-6-phosphate. The rate of acetate incorporation was markedly altered by changes in incubation media; tris-KCl was the most effective buffer. All the major classes of cellular lipids were labeled. ATP, BSA, inorganic phosphate, Ca⁺⁺, 2,4-dinitrophenol and sodium clofibrate were potent inhibitors of acetate incorporation. When added to the incubation mixture, several hormones altered the rate of acetate incorporation into lipids.     

Structural Insights into Retinal Guanylate Cyclase Activator Proteins (GCAPs)

Retinal guanylate cyclases (RetGCs) promote the Ca²⁺-dependent synthesis of cGMP that coordinates the recovery phase of visual phototransduction in retinal rods and cones. The Ca²⁺-sensitive activation of RetGCs is controlled by a family of photoreceptor Ca²⁺ binding proteins known as guanylate cyclase activator proteins (GCAPs). The Mg²⁺-bound/Ca²⁺-free GCAPs bind to RetGCs and activate cGMP synthesis (cyclase activity) at low cytosolic Ca²⁺ levels in light-activated photoreceptors. By contrast, Ca²⁺-bound GCAPs bind to RetGCs and inactivate cyclase activity at high cytosolic Ca²⁺ levels found in dark-adapted photoreceptors. Mutations in both RetGCs and GCAPs that disrupt the Ca²⁺-dependent cyclase activity are genetically linked to various retinal diseases known as cone-rod dystrophies. In this review, I will provide an overview of the known atomic-level structures of various GCAP proteins to understand how protein dimerization and Ca²⁺-dependent conformational changes in GCAPs control the cyclase activity of RetGCs. This review will also summarize recent structural studies on a GCAP homolog from zebrafish (GCAP5) that binds to Fe²⁺ and may serve as a Fe²⁺ sensor in photoreceptors. The GCAP structures reveal an exposed hydrophobic surface that controls both GCAP1 dimerization and RetGC binding. This exposed site could be targeted by therapeutics designed to inhibit the GCAP1 disease mutants, which may serve to mitigate the onset of retinal cone-rod dystrophies.