Uptake and toxic effects of heavy metal ions: interactions among cadmium, copper and zinc in cultured cells

Absorption of metal ions by KB, HeLa and L-59 cells has been analyzed by atomic absorption spectrophotometry in the course of culture. Ions of the elements of the fourth period in the periodic chart such as Fe(II), Cu(II), Zn(II), Mn(II) and Ni(II) were not taken up, but those of the higher periods, such as Cd(II), Pb(II), Hg(II) and Ag(I) were were taken up easily. The uptake behavior by the cultured cells was in accordance with the characteristic features of metals, that metals in the fourth period are essential elements, and most of the elements of the fifth and the sixth periods are non-essential or toxic elements. The initial rate of Cd(II) uptake and the Cd(II) concentration has a sigmoidal relationship. Cd(II) was absorbed homotropically through cell membranes. The uptake of Cd(II) was specifically inhibited by Cu(II), but was affected little by Zn(II). The toxicity of Cd(II) to KB cells was greatly enhanced in the presence of Cu(II). On the contrary, the toxicity of Cd(II) was reduced by the addition of Zn(II) at several concentrations of Cd(II). The toxicity of Cd(II) did not depend on the amount of Cd(II) absorbed in the cells, but was determined by cofactors such as Cu(II). The interaction between Cd(II) and Cu(II) may be important for Itai-itai disease.     

Uptake and toxic effects of heavy metal ions: Interactions among cadmium,copper and zinc in cultured cells

Summary Absorption of metal ions by KB, HeLa and L-59 cells has been analyzed by atomic absorption spectrophotometry in the course of culture. Ions of the elements of the fourth period in the periodic chart such as Fe(II), Cu(II), Zn(II), Mn(II) and Ni(II) were not taken up, but those of the higher periods, such as Cd(II), Pb(II), Hg(II) and Ag(I) were were taken up easily. The uptake behavior by the cultured cells was in accordance with the characteristic features of metals, that metals in the fourth period are essential elements, and most of the elements of the fifth and the sixth periods are non-essential or toxic elements.The initial rate of Cd(II) uptake and the Cd(II) concentration has a sigmoidal relationship. Cd(II) was absorbed homotropically through cell membranes. The uptake of Cd(II) was specifically inhibited by Cu(II), but was affected little by Zn(II). The toxicity of Cd(II) to KB cells was greatly enhanced in the presence of Cu(II). On the contrary, the toxicity of Cd(II) was reduced by the addition of Zn(II) at several concentrations of Cd(II). The toxicity of Cd(II) did not depend on the amount of Cd(II) absorbed in the cells, but was determined by cofactors such as Cu(II). The interaction between Cd(II) and Cu(II) may be important for Itai-itai disease.     

Hydrolyse enzymatique de la propionylcholine,de l'acétylthiocholine et de la butyrylthiocholine par le rectus de grenouille

Summary The enzymatic hydrolysis of propionylcholine (PrCh), acetylthiocholine (AcThCh), and butyrylthiocholine (BuThCh), by extracts of the muscle rectus abdominis, was determined. Inhibition of this hydrolysis by D.F.P. and 3318 CT [bis(pipéridinométhylcoumaranyl- 5)cétone diméthiodide] — utilized over a range of concentrations covering both specific and non-specific concentrations—showed that PrCh is hydrolyzed by an acetylcholinesterase (70%) and an Xcholinesterase (30%), AcThCh by the AcChE (70%), the XChE (15%) and a thioesterase (15%) and BuThCh by the XChE (70%) and a thioesterase (30%).     

Modification of theophylline-induced lipolysis in human fat cells after trypsination.

Trypsin-treatment of human fat cells results in the potentiation of the lipolytic response and the cAMP accumulation induced by theophylline (5 . 10(-4) M) but not of those induced by theophylline (5 . 10(-3) M). The amount of cAMP formed after exposure to theophylline (5 . 10(-3) M) plus norepinephrine (5 . 10(-6) M) remains, however, 2.6 fold higher in trypsin-treated human fat cells than in the control ones.     

Glioma cell activation by Alzheimer's peptide Abeta1-42, alpha1-antichymotrypsin,and their mixture

We compared the effects ofAlzheimer's peptide (Abeta1-42), a,-antichymotrypsin (ACT) and an ACT/Abeta1-42 mixture on human glioma DK-MG cells. The solution of Abeta (5 microM) formed by 2-h incubation at room temperature induced tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 levels by 55 and 45%, respectively, and increased gelatinase B activity by 67%, while exposure of cells to the ACT/Abeta1-42 mixture (1:10 molar ratio ACT: Abeta1-42) under the same experimental conditions showed no effect on IL-6 levels or gelatinase B activity, but strongly induced TNF-alpha (by 190%), compared to the controls. Stimulation of the cells with Abeta1-42 alone, but not with ACT, increased by about 20% low-density lipoprotein (LDL) uptake and mRNA levels for LDL receptor and HMG-CoA reductase, while the ACT/Abeta1-42 mixture significantly increased LDL uptake (by 50%), up-regulated mRNA levels for LDL receptor and HMG-CoA reductase by 48 and 63%, respectively, and increased lipid accumulation by about 20-fold. These data suggest a possible new role for Abeta in Alzheimer's disease through its interaction with the inflammatory reactant, ACT.     

Phosphoramidon enhances allatostatin-mediated inhibition of juvenile hormone biosynthesis in the corpora allata of the cockroach, Diploptera punctata.

Use of enkephalinase inhibitor phosphoramidon in the in vitro radiochemical assay for juvenile hormone biosynthesis enhanced allatostatin-mediated inhibition of hormone production by corpora allata of the cockroach, Diploptera punctata. Significant increases in inhibition in day 2 virgin female CA by AST 1 (at 10(-7) M) and AST 4 (10(-8)-10(-7) M) were observed in the presence of phosphoramidon (10(-5) M or greater). No significant increase in inhibition were seen in CA from day 6 mated females with AST 4 (10(-9)-10(-7) M) and phosphoramidon combined. Phosphoramidon alone had no effect on JH biosynthesis. Analysis of allatostatin content of the CA, as determined by ELISA, revealed that addition of phosphoramidon to the medium increased the endogenous allatostatin content in CA of virgin and mated females. The similarity in primary structure between allatostatins and enkephalin-like peptides and their similar distribution makes it probable that phosphoramidon acts by preventing breakdown of allatostatins within the CA.     

Comparison of intradermal pigeon crop-sac bioassay and double antibody radioimmunoassay for rat prolactin

Summary On an absolute basis, the intradermal pigeon crop-sac bioassay (PCA) gave results that were 20.5% higher than the radioimmunoassay (RIA) in rat anterior pituitary (AP) preparations. A highly significant correlation (r=0.87) was obtained between RIA (in g) and PCA (in Reece-Turner units) when 58 medium samples obtained by culturing rat APs in vitro were assayed for prolactin (PRL) content.Supported by the Charles and Johanna Busch Memorial Fund and by Hatch Amended Funds, Rutgers University-The State University of New Jersey, New Brunswick, NJ 08903, USA.The authors acknowledge the generous supply of rat PRL reference preparation and iodination material for the RIA by NIAMDD, NIH.     

Heterogeneous responses of canine basilar and middle cerebral arteries to serotonin at normal and high CO2 tension.

The responses of basilar arteries (BAs) to serotonin were attenuated by high PCO2 (86 +/- 1 mm Hg) and the pH matched acidotic solution (PCO2 37 +/- 1 mm Hg), whereas the responses of middle cerebral arteries (MCAs) were not. High PCO2 decreased the basal tone of both arteries, and the changes in basal tone due to high PCO2 were not influenced by 3 x 10(-7) M imipramine, 10(-5) M pargyline or 10(-4) M aspirin. The responses of BAs to serotonin were attenuated by high PCO2 in the presence of imipramine, pargyline and aspirin. The responses of MCAs to serotonin were not influenced by high PCO2 in the presence of pargyline and aspirin, but attenuated by high PCO2 in the presence of imipramine.     

The influence of 6-OHDA on hypothermia produced by intrahypothalamically-injected carbachol in the pigeon

Summary The involvement of the noradrenergic system in hypothermia induced by intrahypothalamically-injected carbachol (CCh) was studied by depleting hypothalamic noradrenaline (NA) with the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) and repeating the CCh injections after 6-OHDA treatment. The results suggest that noradrenergic neurons may be involved in hypothermia produced by CCh in the pigeon.     

Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle

The contraction induced by a Ca2+-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (Fo), immediate elastic recoil (SE), unloaded shortening velocity (Vus), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca2+ -induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. Fo developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. Vus in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3 +/- 8.9% less than Vus induced by Ca2+ (1.6 X 10(-6) M) in the control fibers. Addition of Ca2+ (1.6 X 10(-6) M) to a contraction induced by MLCK-resulted in small increases in both Fo and Vus. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca2+. We conclude that with respect to actin-myosin interaction, MLCK-and Ca2+ -induced contractions are similar.     

Stimulation of sodium transport by antidiuretic hormone in bullfrog intestine

Summary Antidiuretic hormone (ADH) increased the potential difference (PD) and shortcircuit current (SCC) across the small intestine of the bullfrog. This effect was independent of those produced by amiloride or high calcium but was masked by a theophylline-induced effect. Net active sodium (Na⁺) absorption accounted for the observed electrical changes.This work was supported by Whitehall Foundation, grant No. 78 156 ck-1 DSR No.     

Stimulation of sodium transport by antidiuretic hormone in bullfrog intestine.

Antidiuretic hormone (ADH) increased the potential difference (PD) and shortcircuit current (SCC) across the small intestine of the bullfrog. This effect was independent of those produced by amiloride or high calcium but was masked by a theophylline-induced effect. Net active sodium (Na+) absorption accounted for the observed electrical changes.     

Der Einfluss verschiedener Psychopharmaka auf den Bluthistamingehalt von Ratten

Summary The concentration of histamine in plasma of rats is changed by injection of psychopharmacons as follows: decreased by narcotics (ether, chloroform) and phantastics (LSD, mescaline), increased by tranquilizers (reserpine, chlorpromazine) a hypnotic (numal) and psychotomimetics (iproniacide, amphetamine).     

Demonstration of "hydroxylase" and "epoxide hydrase" activities in preparations of nucleoli isolated from rat liver]

Aryl hydrocarbon hydroxylase (AHH) activity and epoxyde hydrase (EH) activity have been found in Rat liver nucleoli obtained from untreated (C) and methylcholanthrene (MC) pretreated Rats. Electron microscopic observations of nucleolar preparations did not reveal significant contamination either by intact nuclei or by nuclear membranes. Very low but detectable activity of NADPH cytochrome C reductase was found in the nucleoli. Nucleolar preparations revealed little AHH activity (12-18 pmoles/min/mg). AHH was inducible by MC in nuclei but not in nucleoli. The presence of EH in nucleoli was demonstrated with phenanthrene 9,10-oxide (550-620 pmoles/min/mg) and benzopyrene 4,5-oxide (92-116 pmoles/min/mg). These values were lower than those obtained using intact nuclei. The addition of TCPO (10(-4) M) inhibited EH activity.     

Oyster mushroom (Pleurotus ostreatus) reduces the activity of 3-hydroxy-3-methylglutaryl CoA reductase in rat liver microsomes

The effect of dried oyster mushroom (Pleurotus ostreatus) on cholesterol (C) content in serum, in lipoproteins and in liver, and on the activity of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase in liver microsomes, was studied in male rats (strain Wistar, initial body weight 75 g) fed on low-cholesterol (9 mg/100 g) and high-cholesterol (0.3%) diets. Addition of 5% oyster mushroom to both diets reduced significantly the C-content in serum (by 30%), in very-low- and low-density lipoproteins (in a 11 ratio to the decrease of total serum C) and in liver (by 50%), as well as the activity of HMG-CoA reductase (by more than 30%).     

Identification of in vitro release products of corpora allata in female and male loreyi leafworms,Leucania loreyi

The in vitro release of juvenile hormones (JH) by female, and of JH acids (JHA) by male corpora allata (CA) ofLeucania loreyi was identified by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Separation and quantification were accomplished by HPLC and GC, respectively. JH II and JH III were the major components released by CA of females. Four JHA analogues were identified as the release products of male CA, i.e. JHA III, Iso-JHA II, JHA II and JHA I. JHA III and Iso-JHA II were reported for the first time as the major release products of CA of adult male Lepidoptera. Iso-JHA II is a new member of the insect juvenile hormone analogue family.     

ACTH response induced by interleukin-1 is mediated by CRF secretion stimulated by hypothalamic PGE

We investigated whether hypothalamic prostaglandin E2 (PGE2) and corticotropin releasing factor (CRF) are responsible for the development of the adrenocorticotropic hormone (ACTH) response induced by interleukin-1 alpha (IL-1 alpha). The present results show that ACTH responses induced by intravenous injection of IL-1 alpha were suppressed by systemic pretreatment with indomethacin and that intrahypothalamic injection of PGE2 stimulates the secretion of ACTH. Furthermore, systemic pretreatment with anti-CRF antibody significantly suppressed the ACTH response induced by intrahypothalamic injection of PGE2. These data suggest that the ACTH response induced by IL-1 is mediated by CRF secretion stimulated by hypothalamic PGE2.     

Podocalyxin enhances the adherence of cells to platelets

Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Δ) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin αVβ3/αVβ5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid. Received 14 August 2007; received after revision 12 September 2007; accepted 13 September 2007     

Effect of hyperphenylalaninemia on polyphosphoinositides content of rat brain

Summary Hyperphnylalaninemia (experimental PKU) induced in developing rats by treatment with p-chlorophenylalanine (PCPA) plus phenylalanine (PHE) causes a significant reduction in the triphosphoinositide (TPI) and diphosphoinositide (DPI) content of brain. Since TPI and DPI play an important role in excitable nervous membranes, the functional abnormality in experimental and perhaps in genetic PKU may be related to the reduction in TPI and DPI content.This work was supported by research grant NS14938 from National Institutes of Health.     

Phosphatidylinositol-3,5-bisphosphate lipid-binding-induced activation of the human two-pore channel 2

Mammalian two-pore channels (TPCs) are activated by the low-abundance membrane lipid phosphatidyl-(3,5)-bisphosphate (PI(3,5)P₂) present in the endo-lysosomal system. Malfunction of human TPC1 or TPC2 (hTPC) results in severe organellar storage diseases and membrane trafficking defects. Here, we compared the lipid-binding characteristics of hTPC2 and of the PI(3,5)P₂-insensitive TPC1 from the model plant Arabidopsis thaliana. Combination of simulations with functional analysis of channel mutants revealed the presence of an hTPC2-specific lipid-binding pocket mutually formed by two channel regions exposed to the cytosolic side of the membrane. We showed that PI(3,5)P₂ is simultaneously stabilized by positively charged amino acids (K203, K204, and K207) in the linker between transmembrane helices S4 and S5 and by S322 in the cytosolic extension of S6. We suggest that PI(3,5)P₂ cross links two parts of the channel, enabling their coordinated movement during channel gating.