The differential effects of the carcinogen dimethylnitrosamine on isocitrate and 6-phosphogluconate metabolism in single intact cells

A microspectrofluorimetric study is made of the influence of dimethylnitrosamine on NADP reduction, following sequential microinjections into the same L cell, of two substrates: (1) isocitrate, with activity of isocitrate dehydrogenase both in the extramitochondrial and intramitochondrial compartments, (2) 6-phosphogluconate, with activity of the dehydrogenase in the extramitochondrial compartment. In control L cells a two-step reduction of NAD(P) is obtained followed by relatively slow reoxidation. In the minutes which follow addition of carcinogen, e.g., dimethylnitrosamine, to the cell medium the isocitrate and 6-phosphogluconate-induced transient NADP reoxidation is decreased in magnitude compared to control, while the rate constant of NADPH reoxidation is considerably accelerated, possibly due to requirements at the level of the microsomal metabolizing system. Observations within the first hour of carcinogen addition suggest an interesting system for evaluating the immediate actions of carcinogens at extranuclear sites: i.e., a comparative study of NADP reduction-reoxidation rate constants via injection of substrates for extra- vs. intramitochondrial pathways.     

Microspectrofluorometry of fluorescent photoproducts in photosensitized cells. Relationship to cellular quiescence and senescence in culture

Microspectrofluorometry of L and WI-38 cells reveals chemical/structural changes due to quiescence or senescence, i.e., lipid peroxidation, spontaneous or photosensitized by hematoporphyrin. Cells treated with hematoporphyrin and a lysosomal umbelliferone probe show a fast-rising umbelliferone emission, plus a fluorescent photoproduct. Studies in rapidly growing versus quiescent L, early passage/late passage WI-38 cells, suggest accumulation of fluorescence Schiff bases (i.e., their association with granular regions of cells in stationary phase, spectral properties, fast increase in photosensitized cells) and a possible lysosomal membrane permeabilization in quiescent or senescent cells.     

Recent advances in chemiluminescence-based immunoassays for steroid hormones

Several formats of solid-phase separation techniques for the measurement of steroids and urinary steroid conjugates using chemiluminescence as an end point are described. These formats include: (1) immunoadsorption of second antibodies directed against the first antibody on solid support; (2) specific immunoadsorbents consisting of primary antibodies covalently coupled to polymer beads; (3) second antibody coupled to a polymer containing magnetic particles. In these assays a steroid-chemiluminescent marker conjugate serves as the labelled ligand, and highly specific homologous monoclonal antibodies are used to provide optimal specificity and rigorous standardization. These techniques enabled the direct measurement of steroid glucuronides in diluted urine and of steroids in plasma.     

Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl^– uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations50 mM. In contrast, in the absence of Cl^–, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid, 200 M Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl^–/vanadate ratios may result from competition between vanadate and Cl^– for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - FC fusicoccin - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid We thank Drs. R.T. Leonard (University of California, Riverside, USA) and K.A, Rubinson (Yellow Springs, Oh., USA) for helpful comments on the research, Janet Sherwood (Harvard University) for excellent plant care, and Angela Ciamarra, Anne Gershenson, Gustavo Lara (Harvard University) and Orit Tal (Hebrew University) for valuable technical assistance. This research was supported by a grant from the National Science Foundation (DCB-8904041) to S.M.A.     

Ovarian lipoxygenase activity and its regulation by gonadotropin in the rat

In our previous study a dose-dependent blockage of follicular rupture at ovulation by inhibitors of lipoxygenase was demonstrated. Here the presence of 5-lipoxygenase activity in the whole ovary and in the Graafian follicle is estimated by a chemiluminescence assay using unlabeled arachidonic acid as substrate in the presence of luminol and by conversion of 14C-arachidonic acid into lipoxygenase products as separated by HPLC. Both approaches demonstrated lipoxygenase activity in whole ovarian homogenates and in homogenates of preovulatory Graafian follicles. Furthermore, within 6 h after stimulation in vivo with hCG, lipoxygenase activity was increased by 2-fold in the whole ovarian homogenate and by 5-fold in the follicular homogenate. These results confirm the presence of lipoxygenase in rat ovaries, and its stimulation by gonadotropin and thus corroborate the suggested involvement of lipoxygenase products in follicular rupture at ovulation.     

Transition Metals Potentiate Paraquat Toxicity

The involvement of transition metal ions in paraquat toxicity was studied in bacterial model system. We show that the addition of micromolar, or lower, concentrations of copper dramatically enhanced the rate of bacterial inactivation. In contrast, the addition of chelating agents totally eliminated the killing of E. coli. No inactivation was observed under anaerobic exposure to paraquat, both in the absence and presence of copper. However, in the presence of copper, the anaerobic addition of hydrogen peroxide resulted in complete restoration of inactivation as under aerobiosis.

Paraquat either produces superoxide ions or directly reduces bound copper ions in a catalytic mode. The reduced cuprous complexes react with hydrogen peroxide to locally form hydroxyl radicals (OH) which are probably responsible for the deleterious effects.

This study indicates the involvement of a site-specific metal-mediated Haber-Weiss mechanism in paraquat toxicity. It is in agreement with earlier observations that copper unusually enhance biological damage induced by either superoxide or ascorbate.     

Hydrocortisone inhibits antigen-induced rise in intracellular free calcium concentration and abolishes leukotriene C4 production in leukemic basophils

Antigenic stimulation of rat basophilic leukemia cells (RBL-3H3) elevates intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and induces production of leukotriene C₄ (LTC₄). This model was used to examine the role of Ca²⁺ in LTC₄ formation, and inhibition by hydrocortisone (HC). HC, at a physiological concentration (2×10⁻⁷M), selectively prevented the stimulatory effect of the antigen on LTC₄ production whereas the response to calcium inophore (A23187) remained unimpaired. The inhibition by HC was time-dependent: half maximal response was reached at 2 hour and maximal response at 3 hours. Addition of arachidonic acid (3 μg/ml) did not overcome the inhibitory action of HC. An elevated [Ca²⁺]_i is known to be essential for the activation ob both 5-lipoxygenase and phospholipase A₂. The stimulatory effect of the antigen on LTC₄ production was abolished when the cells were incubated in Ca²⁺-deficient medium. Likewise, calcium ionophore stimulation shows dependence on extracellular Ca²⁺. Half maximal stimulation by the antigen and calcium ionophore was observed at external Ca²⁺ concentration of 150 μM and 40 μM respectively. Treatment with HC largely prevented the antigen-induced rise in [Ca²⁺]_i, measured by Quin 2. In addition, HC reduced by 70% the accumulation of ⁴⁵Ca²⁺ induced by the antigen. Collectively, these results demonstrate for the first time that HC reduces antigen-induced elevation of [Ca²⁺]_i, and this may be associated with the inhibitory action of HC on LTC₄ formation. This property could be partly responsible for the antiallergic and antiinflammatory activities of HC.     

Antibodies to spiroperidol and their anti-idiotypes as probes for studying dopamine receptors

Spiroperidol was covalently conjugated to bovine serum albumin (BSA). Conjugated spiroperidol was almost as efficient as free spiroperidol in its binding capacity to dopamine receptor. Antibodies to spiroperidol were produced in rabbits following repeated immunizations with the conjugate of spiroperidol and BSA. The obtained antibodies have an apparent K_D of 0.02 nM for [³H]-spiroperidol. These antibodies bind also to other butyrophenones with IC₅₀ values three to four orders of magnitude higher than the IC₅₀ obtained with unlabeled spiroperidol. Antibodies were purified from anti-spiroperidol sera by affinity chromatography. Anti-idiotypic antibodies were raised in rabbits by immunization with the purified anti-spiroperidol antibodies. Some rabbits produced anti-idiotypic antibodies which bind to rat and calf striatum.     

The development of non-separation time-resolved fluoroimmunoassays for the measurement of urinary metabolites

We describe the principles of a new generation of sequential or simultaneous time-resolved fluoroimmunoassays, namely, simple, rapid, liquid-phase non-separation procedures which may be applied to the measurement of urinary steroid and drug metabolites. As an example, a method for the measurement of estrone-3-glucuronide in undiluted urine is reported. This method has a similar sensitivity, specificity and accuracy to a conventional separation fluoroimmunoassay or radioimmunoassay but in terms of speed, convenience, precision, reliability and clinical utility the new method has many advantages. The labelled antigen is a novel fluorescent europium chelate covalently linked to estrone-3-glucuronide. The antibody-binding reaction involves the incubation of the labelled antigen (2ng) with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-BSA and an aliquot of standard or sample (undiluted urine; 10 μl) in microtitre wells. After a 10 min incubation, the fluorescence which emanates from the antibody-free label is measured in a time-resolved fluorometer and is proportional to the concentration of estrone-3-glucuronide in the standard or sample. The method may be applied for the monitoring of ovarian function in women.