Evaluation of lipid peroxidation in red blood cells by monitoring the uptake of sucrose and phenol red.

Red blood cells (RBCs) are prone to lipid peroxidation by virtue of their function as oxygen carriers, and also because of their lipid composition. Malondialdehyde (MDA) content using thiobarbituric reagent is widely used to quantify lipid peroxidation. In this study we compare MDA assay with a newly developed assay that evaluates the uptake of sucrose and phenol red into RBCs under peroxidative stress. Both sucrose and phenol red uptake show significantly higher correlation with incubation time compared with MDA assay. Furthermore, phenol red uptake into RBCs on treatment with H(2)O(2) has a direct linear proportional relationship, whereas it is hyperbolic with MDA. The assay also clearly shows that uptake of sucrose or phenol red is specific for intact cells (RBCs) prior to hemolysis. Assay validation is carried out by using known lipid peroxidation-causing agents, such as ferrous ions, and also by using peroxidation inhibitors such as alpha-tocopherol. This new method can be applied efficiently to evaluate lipid peroxidation in RBCs as well as other cells and tissues.

Structure, function, and control of phosphoinositide-specific phospholipase C.

Phosphoinositide-specific phospholipase C (PLC) subtypes beta, gamma, and delta comprise a related group of multidomain phosphodiesterases that cleave the polar head groups from inositol lipids. Activated by all classes of cell surface receptor, these enzymes generate the ubiquitous second messengers inositol 1,4, 5-trisphosphate and diacylglycerol. The last 5 years have seen remarkable advances in our understanding of the molecular and biological facets of PLCs. New insights into their multidomain arrangement and catalytic mechanism have been gained from crystallographic studies of PLC-delta(1), while new modes of controlling PLC activity have been uncovered in cellular studies. Most notable is the realization that PLC-beta, -gamma, and -delta isoforms act in concert, each contributing to a specific aspect of the cellular response. Clues to their true biological roles were also obtained. Long assumed to function broadly in calcium-regulated processes, genetic studies in yeast, slime molds, plants, flies, and mammals point to specific and conditional roles for each PLC isoform in cell signaling and development. In this review we consider each subtype of PLC in organisms ranging from yeast to mammals and discuss their molecular regulation and biological function.

Dynamics of phosphatidylinositol 4,5-bisphosphate in actin-rich structures.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is known to regulate a wide range of molecular targets and cellular processes, from ion channels to actin polymerization [1] [2] [3] [4] [5] [6]. Recent studies have used the phospholipase C-delta1 (PLC-delta1) pleckstrin-homology (PH) domain fused to green fluorescent protein (GFP) as a detector for PI(4,5)P(2) in vivo [7] [8] [9] [10]. Although these studies demonstrated that PI(4,5)P(2) is concentrated in the plasma membrane, its association with actin-containing structures was not reported. In the present study, fluorescence imaging of living NIH-3T3 fibroblasts expressing the PLC-delta1 PH domain linked to enhanced green fluorescent protein (PH-EGFP) reveals intense, non-uniform fluorescence in distinct structures at the cell periphery. Corresponding fluorescence and phase-contrast imaging over time shows that these fluorescent structures correlate with dynamic, phase-dense features identified as ruffles and with microvillus-like protrusions from the cell's dorsal surface. Imaging of fixed and permeabilized cells shows co-localization of PH-EGFP with F-actin in ruffles, but not with vinculin in focal adhesions. The selective concentration of the PH-EGFP fusion protein in highly dynamic regions of the plasma membrane that are rich in F-actin supports the hypothesis that localized synthesis and lateral segregation of PI(4,5)P(2) spatially restricts actin polymerization and thereby affects cell spreading and retraction.

Prostasomes: current concepts.

BACKGROUND: Prostasomes are membranous vesicles secreted by prostate gland, and they contain large amounts of cholesterol, sphingomyelin, calcium, and several enzymes. Prostasomes are involved in a number of biological functions. At ejaculation, these prostasomes are expelled with prostate secretions and are to be found in the seminal plasma as seminal prostasomes, which facilitate sperm function in various ways. METHODS: In this review, we discuss the structural and functional role of prostasomes, the various enzyme systems associated with these vesicles, and the biological role prostasomes play in male reproduction. RESULTS AND CONCLUSIONS Prostasomes are pluripotent and well-organized organelles secreted by the prostate gland. Prostasomes are ascribed to have many physiologiocal functions, the primary function being enhancement of sperm capacity. The several enzyme systems, small signaling molecules, and neuroendocrine markers associated with prostasomes reveal the complex nature of these vesicles in regulating sperm viability and vitality. The functional significance of these molecules that regulate complex pathways in these small vesicles is still a matter of dogma. Critical evaluation of the biological processes associated with prostasomes might be helpful in modeling new contraceptive agents, improving the techniques of in vitro fertilization, and in furthering our understanding and treatment of male factor infertility.

Volatile anesthetics modulate the binding of guanine nucleotides to the alpha subunits of heterotrimeric GTP binding proteins.

The effects of volatile anesthetics on guanine nucleotide binding to the purified alpha subunits of heterotrimeric GTP binding (G) proteins were studied. At sub-anesthetic doses, halothane, isoflurane, enflurane and sevoflurane inhibit exchange of GTPgammaS for GDP bound to Galpha subunits and markedly enhance the dissociation of GTPgammaS, but fail to suppress GDPbetaS release. Nucleotide exchange from non-myristoylated Galpha(i1) is similarly inhibited in the absence of any membrane lipid or detergent. The degrees of inhibition of GDP/GTPgammaS exchange and enhancement of GTPgammaS dissociation are in the same order: alpha(i2)alpha(i1)alpha(i3)alpha(s). By contrast, Galpha(o), which is closely related to Galpha(i), is completely insensitive to anesthetics. We conclude that volatile agents, at clinically relevant doses, have a direct effect on the conformation and stability of the GTP/Mg(2+) bound state of some, but not all Galpha subunits. By destabilizing this state, volatile agents may uncouple metabotropic and other heptahelical receptors from pathways modulating neuronal excitation.

Identification and characterization of a novel protein that regulates RNA-protein interaction.

In a previous study [Nachaliel et al., 1993], we identified an RNA-binding protein (RBP) in FTO-2B rat hepatoma cells whose activity was stimulated upon the dissociation of a protein factor. We report in this article that the RBP is a complex protein of about 400 kDa, composed of RNA-binding subunit(s) (RBS), and regulatory subunit(s) (RS). We purified the RS to near-homogeneity (Mr approximately 25,000) and determined the amino acid sequence of a peptide derived from RS. On the basis of this sequence information, the cDNA for RS was obtained. Recombinant RS protein expressed in Escherichia coli had the capacity to bind RBS and inhibit its RNA-binding activity. The cDNA contains the complete coding sequence because the recombinant protein has the same electrophoretic mobility as that of the native RS in SDS-polyacrylamide gels. Sequence comparison showed that RS is almost identical to DJ-1, a recently discovered protein with an oncogenic potential, and CAP1, a rat sperm protein. However, the protein does not contain any known motifs that can provide a clue as to its exact function. Indirect immunofluorescence analyses showed that in addition to the cytoplasm, where RS is associated with microtubular filaments, the polypeptide is localized to the cell nucleus. The possible role of RS is discussed.

Androgen induction of urokinase gene expression in LNCaP cells is dependent on their interaction with the extracellular matrix.

Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling and cell invasion. In the present study, we examined the expression of uPA in the prostate cancer cell lines LNCaP, DU-145 and PC-3. In contrast to DU-145 and PC-3, the androgen-responsive cell line LNCaP does not express uPA. However, seeding LNCaP cells on fibronectin-coated plates stimulated a low level of uPA expression which was further induced upon exposure of the cells to dihydrotestosterone (DHT). Concomitant with the expression of uPA, an androgen-regulated expression of uPA receptor (uPAR) was induced. These results suggest that the interaction of LNCaP cells with the extracellular matrix plays a dominant role in the androgen control of uPA and uPAR gene expression.

Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity.

ATP-citrate lyase (CL) catalyzes the conversion of citrate and CoA to oxaloacetate (OA) and acetyl-CoA. As the coupled malic dehydrogenase (MDH) assay is not able either to study the effect of oxaloacetate (OA) on CL activity or to measure accurately CL activity in biological samples, a new assay was developed. The CL-citrate coupled CAT assay measures the amount of acetyl-CoA formed by transferring radiolabeled acetyl-CoA synthesized from [14C]citrate to chloramphenicol with chloramphenicol acetyltransferase (CAT). Employing this assay, the rate of increase in acetyl-CoA synthesis from citrate is linear with respect to added CL. Kinetic values for ATP, CoA and citrate are similar to those obtained using the MDH assay. The effect of CL phosphorylation on enzyme activity was determined. CL phosphorylated by cAMP-dependent protein kinase or by this kinase and glycogen synthase kinase-3 (GSK-3) decreases the apparent Vmax without changing the apparent Km. The effect of OA, a product of the enzyme reaction, on CL activity was also determined. Computational analysis of the data obtained without added OA and at three concentrations of OA indicate that the apparent Km for the substrate is not altered even though the apparent Vmax is decreased. The effect of OA on the activity of phosphorylated enzyme was also determined. OA decreases the apparent Vmax of the phosphorylated enzyme to the same extent as in control CL. This assay is able to measure CL activity in cytosol from 3T3-L1 adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Lead alters inositol polyphosphate receptor activities: protection by ATP.

Receptor-mediated phosphoinositide signaling pathway which generates a variety of second messengers is regulated by intracellular free Ca2+ concentrations. Since toxic metal cations like Pb2+ are known to alter Ca(2+)-dependent processes, the present study was initiated to study the effects of Pb2+ on inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) receptor binding and InsP3-mediated Ca(2+)-release. Rat cerebellar membrane and microsomal fractions were incubated with various concentrations of Pb2+ (0.01-100 microM). Pb2+ significantly stimulated [3H]-InsP3 and [3H]-InsP4 receptor binding (EC50 22.7 and 13.5 microM respectively) as a function of metal concentrations. However, InsP3-mediated Ca2+ release, determined by measuring the changes in fluorescence intensity of Fura-2, was significantly inhibited by varying concentrations of Pb2+. Re-uptake of Ca2+ into the microsomes was also inhibited by Pb2+. A significant inhibition of microsomal Ca(2+)-pump by micromolar concentration of Pb2+ was also observed. ATP at 5-1000 microM concentration range inhibited [3H]-InsP3 and [3H]-InsP4 binding to the specific receptors. [3H]-InsP4 receptor binding was more sensitive to ATP inhibition as compared to [3H]-InsP3 receptor binding. Furthermore, varying concentrations of ATP also inhibited Pb(2+)-mediated increase in [3H]-InsP3 and [3H]-InsP4 receptor binding. The kinetic analysis of ATP effect on Pb(2+)-stimulated [3H]-InsP4 receptor binding revealed non-competitive type of interaction. The results of the present study suggest that Pb2+ may be increasing the binding of [3H]-InsP3 and [3H]-InsP4 to the specific receptors by modulating the conformation of the receptor sites. ATP may be playing a protective role in Pb2+ induced alteration of the receptor sites.

Effect of carbon tetrachloride on inositol 1,4,5-trisphosphate dependent and independent regulation of rat brain microsomal Ca2+ flux.

Carbon tetrachloride (CCl4) is a highly toxic industrial solvent with pronounced effects on the liver and brain. CCl4 is enzymatically cleaved to produce free radicals which attack membrane components, including proteins. Earlier reports indicated that CCl4 affects Ca(2+)-regulated events in the brain. Hence, the present study was initiated to determine whether CCl4 affects inositol 1,4,5-trisphosphate (IP3) receptor binding, free-Ca2+ movements across the microsomal membrane and protein kinase C (PKC) activity in rat brain, since IP3, Ca2+ and PKC are known to be involved in signal transduction. [3H]IP3 binding, free-Ca2+ movements and 45Ca2+ uptake were determined using rat brain microsomes and PKC activity was determined in the cytosolic fraction. CCl4 in vitro decreased [3H]IP3 binding to microsomes. IP3 mediated Ca2+ release from microsomes was inhibited and also the reuptake of IP3-released Ca2+ into microsomes was decreased in the presence of CCl4. CCl4 at concentrations < 2 microM independently released Ca2+ from microsomes. Uptake of total Ca2+ into microsomes was inhibited by CCl4 as observed with 45Ca(2+)-uptake studies. CCl4 at 1 microM inhibited PKC activity by 50%. Thus, perturbations in the binding of IP3 to its receptor sites, changes in the Ca2+ flux across the microsomal membrane and modulation of PKC activity by CCl4 in vitro suggested that CCl4 may exert neurotoxicity by altering signal transduction pathways.

ATP citrate-lyase and glycogen synthase kinase-3 beta in 3T3-L1 cells during differentiation into adipocytes.

ATP citrate-lyase (CL), acetyl-CoA carboxylase (ACC) and glycogen synthase kinase-3 beta (GSK-3 beta) levels were measured in cytosol from 3T3-L1 cells during differentiation from fibroblasts into fat-cells. Protein levels were estimated from immunoblots using specific antisera. Cytosol from confluent cells contain significant amounts of GSK-3 beta, which fell during differentiation of these cells into adipocytes. CL from confluent cells was found to be mostly in the form of a single protein band of apparent mass 110 kDa. Levels of CL and ACC increased during cell differentiation into adipocytes. During the first 3 days of differentiation, CL migration changed, and it was expressed as a complex of protein bands of apparent mass 110 kDa, 113 kDa and 115 kDa. At later stages of differentiation, when these cells had assumed the phenotype of fat-cells, they expressed CL mainly as protein bands of 110 and 113 kDa. When samples containing these bands were treated with alkaline phosphatase, the 113 kDa protein band collapsed into the 110 kDa species. This suggests that the slower-migrating species of CL is a higher-order phosphorylation state of the same protein. Furthermore, when purified CL, mostly expressed as the 110 kDa species, was phosphorylated with cyclic AMP-dependent protein kinase alone or together with GSK-3 and resolved by SDS/PAGE, the phosphorylated CL now migrated more slowly as the 113 kDa and 115 kDa forms. CL phosphorylation was hormone-regulated, since, in samples from fat-cells that had the complex two-band pattern, when cultured in medium without serum or hormones, CL migration reverted to a single band of 110 kDa, similar to confluent cells. Treatment of these 'down-regulated' cells with insulin rapidly induced substantial amounts of the 113 kDa species, with a concomitant decrease in the 110 kDa species.

Permeability changes in the blood-brain barrier of neonate and adult rats after thiobencarb exposure.

Thiobencarb, an organocarbamate herbicide, induces toxic neuropathies in neonates and adults. Permeability changes in the blood brain barrier under thiobencarb stress in neonate and adult rats were assessed using the dye, trypan blue. Acute and subacute doses of thiobencarb increased the dye concentrations in the brains of both neonates and adults in a time dependent manner. Concentrations of the dye were found to be greater in neonate rat brains when compared to adult brains.

Comparative study on the changes in AChE and ATPase activities in neonate and adult rat brains under thiobencarb stress.

Thiobencarb (S-(4-chlorobenzyl)-N,N-diethyl thiol carbamate), a dithiocarbamate herbicide, was found to cause neuronal dysfunction in adult and neonate albino rats. In general, organocarbamates exert their action by inhibiting acetylcholinesterase (AChE) activity. Thiobencarb inhibited both acetylcholinesterase and adenosine triphosphatase (ATPase) activities in rat brain. Withdrawal of thiobencarb treatment resulted in the recovery of AChE activity to a normal level, whereas there was no recovery of Na(+)-K(+)-ATPase activity in either neonate or adult rat brains. The results suggest that neuronal dysfunction caused by thiobencarb is mainly due to the inhibition of ATPase activity rather than to the inhibition of AChE activity.

Amiodarone and desethylamiodarone increase intrasynaptosomal free calcium through receptor mediated channel.

Long term amiodarone (AM) therapy has been associated with several side effects including neurotoxicity. Since AM alters Ca2+ regulated events, we have studied its effects on the compartmentation of free Ca2+ in the synaptosomes as an attempt to understand the mechanism of AM and its metabolite, desethylamiodarone (DEA)-induced neurotoxicity. Intact brain synaptosomes were prepared from male Sprague-Dawley rats. Both AM and DEA produced a concentration dependent increase in intrasynaptosomal free Ca2+ concentration ([Ca2]i) to micromolar levels. The increase in [Ca2]i was not transient and a steady rise was observed with time. Omission of Ca2+ from the external medium prevented the AM- and DEA-induced rise in [Ca2+]i suggesting that AM and DEA increased the intracellular [Ca2+]i due to increased influx of Ca2+ from external medium. AM- and DEA-induced increase in intrasynaptosomal [Ca2+]i was neither inhibited by a calcium channel blocker, verapamil, nor with a Na+ channel blocker, tetrodotoxin. However, the blockade of [Ca2+]i rise by AM and DEA was observed with MK-801, a receptor antagonist indicating that AM and DEA induced rise in [Ca2+]i is through receptor mediated channel. Both AM and DEA also inhibited N-methyl-D-aspartic acid (NMDA)-receptor binding in synaptic membranes in a concentration dependent manner, DEA being more effective, indicating that AM and DEA compete for the same site as that of NMDA and confirm the observation that these drugs increase intrasynaptosomal [Ca2+]i through receptor mediated channel. 45Ca accumulation into brain microsomes and mitochondria was significantly inhibited by AM and DEA, but without any effect on the Ca2+ release from these intracellular organelles.(ABSTRACT TRUNCATED AT 250 WORDS)

Triorganotin inhibition of rat cardiac adenosine triphosphatases and catecholamine binding.

Triorganotins have been reported to affect heme metabolism as well as the cardiovascular system. Our recent studies indicated that these organotins inhibit cardiac sarcoplasmic reticulum Ca(2+)-transport and cAMP-stimulated phosphorylation of specific proteins involved in Ca2+ transport, suggesting their interference with cardiac adrenergic function. The present study determines the effect of three organotins--tributyltin bromide (TBT), triethyltin bromide (TET) and trimethyltin chloride (TMT)--on rat cardiac ATPases and catecholamine binding, since these phenomena are involved in cardiac function. Cardiac membrane fraction was prepared from heart ventricles of male Sprague-Dawley rats. All three organotins inhibited cardiac Na+,K(+)-ATPase, [3H]ouabain binding, K(+)-activated p-nitrophenyl phosphatase (K(+)-PNPPase) and oligomycin-sensitive (OS) and oligomycin-insensitive (OI) Mg(2+)-ATPase in a concentration-dependent manner. K(+)-PNPPase was less sensitive to these triorganotins when compared to Na+K(+)-ATPase, suggesting that triorganotins affect the Na(+)-pump activity by acting on the Na(+)-dependent phosphorylation process. OS Mg(2+)-ATPase was more sensitive to these organotins when compared to OI Mg(2+)-ATPase, confirming their potent effect on the enzymes of oxidative phosphorylation. The order of potency is TBT greater than TET greater than TMT. TET and TMT, but not TBT, inhibited [3H]norepinephrine and [3H]dopamine binding to cardiac membranes in a concentration-dependent manner, the effect being more with TET. These results suggest that triorganotins inhibit sodium pump activity as well as ATP synthesis. Since Na+,K(+)-ATPase is involved in the active transport of catecholamines, triorganotins not only inhibited the catecholamine transport but also to some extent affected catecholamine binding, thus interfering with cardiac function.

Combined effects of carbon tetrachloride and chlordecone on calmodulin activity in gerbil brain.

The potentiation of carbon tetrachloride (CCl4) toxicity by chlordecone (CD) pretreatment in different animal models is well established. However, these studies have only dealt with hepatotoxicity. The present study was initiated to determine whether CD preexposure potentiates CCl4 neurotoxicity in gerbils. Gerbils were chosen for the reason that the metabolism of CD in gerbil is similar to that of humans. Gerbils (50-80 g), fed on diet without or with CD (10 ppm) for 15 d, were challenged with a single dose of CCl4 (15 microliters, ip). Ca(2+)-ATPase and calmodulin (CaM) activities were determined in gerbil brain P2 fraction and cytosol, respectively, at intervals of 0.5, 2, 6, 12, and 24 h after CCl4 administration. Ca(2+)-ATPase and CaM activities were decreased at 0.5 and 2 h in both CD-preexposed and CCl4-treated gerbils. However, CaM activity returned to normal levels after 6 h and Ca(2+)-ATPase activity showed 80% recovery after 2 h. In vitro experiments showed that CCl4 alone at 5 microM concentration inhibited Ca(2+)-ATPase activity up to 50%. Combination of CD (0.5 microM) and CCl4 (1 and 5 microM) on Ca(2+)-ATPase activity showed no additive effect in vitro. Interaction between CCl4 and CaM was studied in the presence and absence of CD by monitoring NPN fluorescence. The decrease in NPN fluorescence observed with CCl4 was not potentiated by CD preincubation. These data suggest that CD does not enhance CCl4-induced alterations of Ca(2+)-ATPase and CaM activities in gerbil brain.