Roseann L. Vorce

Pyrethroid Insecticides as Phosphatase Inhibitors

In this study we tested the hypothesis that pyrethroid insecticides inhibit calcineurin directly and that inhibition is unaffected by the immunophilin cofactors necessary for calcineurin inhibition by cyclosporin A and FK506. The type II pyrethroid insecticides cis-cypermethrin (c-Cyp), trans-cypermethrin, deltamethrin (Delt), and fenvalerate A alpha (Fen), as well as the type I pyrethroid insecticides cis- and trans-permethrin and S-bioallethrin, were unable to inhibit the phosphatase activity of purified calcineurin under conditions of maximal activation by Ca2+ and calmodulin. Furthermore, c-Cyp, Delt, and Fen did not affect the Ca2+ dependence of calcineurin at 0.1 microM of calmodulin, indicating that Ca2+ binding to calmodulin was not affected by these agents. c-Cyp, Delt, and Fen also failed to inhibit calcineurin phosphatase activity in rat brain supernatant and cultured IMR-32 cells, although potent inhibition was displayed by both cyclosporin A and FK506 in each of these systems. Neither the Ca2+-dependent nor the okadaic acid-inhibitable phosphatase activity toward a 24-amino acid 32P-phospho-peptide substrate was affected by any of the pyrethroid insecticides, indicating that neither type-1 or type-2A phosphatase nor calcineurin is inhibited by pyrethroids. To determine if these results were dependent upon experimental conditions, experiments were repeated using polyethylene glycol-treated glass tubes in place of the standard polypropylene tubes. Regardless of the type of tube, no inhibition of calcineurin by any of the pyrethroid insecticides was observed. These data indicate that the pyrethroid insecticides are not effective inhibitors of calcineurin or other phosphatases.

Folic acid protects SWV/Fnn embryo fibroblasts against arsenic toxicity

It has been proposed that arsenic exerts its toxic effects, in part, by perturbing cellular methyl metabolism. Based on the hypothesis that folic acid treatment will attenuate the cytotoxic and growth inhibitory effects of arsenic, SWV/Fnn embryo fibroblasts were cultured in media supplemented with various concentrations of folic acid during treatment with sodium arsenite or dimethylarsinic acid (DMA). It was found that folic acid protects SWV/Fnn embryo fibroblasts from sodium arsenite and DMA cytotoxicity in a dose-dependent manner. In contrast, folic acid supplementation has no effect on toxicity resulting from treatment with ethanol or staurosporine, suggesting that folic acid is not generally protective against necrosis and apoptosis. Although folic acid protects against acute arsenic toxicity, this agent shows a modest and delayed ability to attenuate the growth inhibitory effect of arsenic on these cells. These results support a model in which perturbations of methyl metabolism contribute to the acute cytotoxicity of arsenic.

Identification of an NPY-Y1 receptor subtype in bovine chromaffin cells

Bovine chromaffin cells have been used in a variety of studies designed to reveal different aspects of neuropeptide Y (NPY) action. Pharmacological data have defined five NPY receptor subtypes, only one of which (Y3) has not been cloned. Some studies with bovine chromaffin cells have concluded that the effects of NPY on this cell type are mediated by the Y3 subtype. Previous work from our laboratory demonstrates that a Y1 subtype mediates the effect of NPY in this tissue. In the current studies we provide further evidence for the existence of the Y1 subtype in bovine chromaffin cells. BIBP3226, the selective Y1 antagonist, potently displaces [125I]NPY from its binding site IC50 = 1.91 x 10(-9) M. Moreover, [125I]BIBP3226 binds to bovine chromaffin cell membranes with high affinity (IC50 = 5.9 x 10(-8) M). Examination of BIBP3226 antagonism of NPY inhibition of forskolin stimulated cyclic AMP accumulation reveals that it is a competitive antagonist with a K(B) similar to the IC50 for [125I]BIBP3226 binding. Northern blot analysis using a porcine cDNA clone for the Y1 subtype demonstrates a 3.5-kb mRNA species in chromaffin cells. These data identify the bovine chromaffin cell NPY receptor as a Y1 subtype.

Cyclosporin a has low potency as a calcineurin inhibitor in cells expressing high levels of P-glycoprotein

Cyclosporin A (CsA) is a widely-used immunosuppressant drug whose therapeutic and toxic actions are mediated through inhibition of calcineurin (CN), a calcium- and calmodulin-dependent phosphatase. Inhibition of CN by CsA requires drug binding to its protein cofactor in the inhibition, cyclophilin. Because cyclophilin is a high affinity target for CsA it is expected that this protein can act as a reservoir for the drug in the cell and may be able to inhibit cellular efflux of CsA. P-glycoprotein (P-gp) is known to increase the rate of CsA efflux from CsA loaded cells but it is not clear if the P-gp drug efflux pump can compete effectively with cyclophilin at therapeutically relevant concentrations of CsA. To test the hypothesis that increased expression of P-gp confers protection against CsA-dependent inhibition of CN phosphatase activity, KB-V cells expressing varying levels of P-gp were analyzed to determine the potency of CsA as a CN inhibitor. When intact cells were treated with CsA, a positive correlation was observed between P-gp expression and resistance to CsA-dependent inhibition of CN: the IC50 is approximately 20-fold higher in the multidrug resistant epidermal carcinoma cell line, KB-V, which expresses P-gp at a high level than in the parental, KB, cell line expressing very low levels of P-gp. The resistance displayed by KB-V cells is abrogated by co-administration of the P-gp inhibitor verapamil, whereas verapamil has no effect on CsA potency in control KB cells. In cell lysates from KB-V cells with different amounts of P-gp CsA exhibits equivalent potency, indicating that the difference in sensitivity to CsA among the cell types requires maintenance of cell integrity. These observations support the view that resistance to CN inhibition by CsA occurs in cells with moderately elevated P-gp activity. Therefore, P-gp activity appears to be an important determinant of CsA cellular specificity for both therapeutic and toxic effects.

Differential effects of arsenic on folate binding protein 2 (Folbp2) null and wild type fibroblasts.

Exposure to arsenic results in a wide variety of adverse effects. It has been postulated that one mechanism of arsenic toxicity is disruption of cellular methyl biochemistry. Because dietary folate is required to generate the methyl donor S-adenosyl methionine, we hypothesized that loss of folate binding protein 2 (Folbp2) results in increased susceptibility to arsenic-induced cytotoxicity. Using Folbp2 +/+ and -/- fibroblasts, we determined that Folbp2 null cells display increased sensitivity to arsenic exposure. Folic acid supplementation partially rescues wild type cells from arsenic toxicity, but Folbp2 null cells are not protected. Arsenic inhibits folic acid uptake in Folbp2 null fibroblasts, but not wild type cells; baseline uptake is similar in both cell types. These results support the possibility that arsenic toxicity occurs, in part, by perturbing cellular methyl biochemistry. Furthermore, identification of Folbp2 as a protective protein presents an opportunity to identify populations at increased risk for serious effects of arsenic exposure.

Compensatory modulation of GAP activity in response to oncogenic stimulation.

GAP is a key negative regulator of the receptor tyrosine kinase (RTK) signal transduction pathway. The purpose of this study was to determine if expression or activity of GAP is modulated by hyperstimulation of the RTK pathway. It was found that cells forced to express wild-type Ha-ras, viral Ha-ras, or v-src exhibit increased GAP activity as compared to control cells. In addition, a novel GAP isoform appears in all ras-expressing NIH3T3 cell clones. These data indicate that there is compensatory regulation of GAP in response to an increase in RTK pathway activity.