Chapter 20 The Use of T ween-8 0 -Permeabiliyed Mammalian Cells in Studies of Nucleic Acid Metabolism

Billen, Daniel; Olson, Ann C.

doi:10.1016/s0091-679x(08)62024-5

Cited by 7 publications

(2 citation statements)

References 12 publications

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“…The technique of permeabilization of cells to enhance entry of various molecules into the intracellular compartment and attain access to intracellular enzymes has been used successfully in vascular and cardiac muscle as well as in isolated hepatocytes and in ovarian cells in culture. [12][13][14][15] Under these conditions, the cells remain viable and retain normal histological features. In three experiments we performed electron microscopic studies after the application of SDS as described above.…”

mentioning

confidence: 99%

Hydroxyl radical-dependent inactivation of guanylate cyclase in cerebral arterioles by methylene blue and by LY83583.

Kontos

Wei

1993

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118

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Background and Purpose: Methylene blue and 6-anilino,5,8-quinolinedione (LY83583) are used extensively to block activation of guanylate cyclase. Both agents generate oxygen radicals. Therefore, it appeared profitable to investigate whether the generation of oxygen radicals by these agents is responsible for the blockade of responses to nitrodilators that act via activation of guanylate cyclase to relax vascular smooth muscle and cause vasodilation.Methods: We tested in anesthetized cats equipped with cranial windows responses to topical application of nitroglycerin, nitroprusside, and adenosine before and during topical application of methylene blue (5 ,M). Responses to the vasoactive agents were tested during application of methylene blue after permeabilization of the cell membrane with a detergent to allow methylene blue to enter vascular smooth muscle. Responses were also tested in the presence of superoxide dismutase, catalase, deferoxamine, or dimethyl sulfoxide to scavenge reactive products of oxygen metabolism or to eliminate catalytic iron. In additional experiments we tested the effects of topical application of nitroprusside or adenosine before and after application of LY83583. The responses to the vasoactive agents were also tested in the presence of superoxide dismutase, catalase, or dimethyl sulfoxide in addition to LY83583. We also tested responses to calcitonin gene-related peptide before and in the presence of LY83583 with or without superoxide dismutase.Results: Methylene blue eliminated the arteriolar dilation in response to nitroprusside and nitroglycerin after permeabilization of the cell membrane with a detergent but not before. The responses to adenosine were unaffected. The blockade induced by methylene blue was reversed by superoxide dismutase, catalase, or dimethyl sulfoxide but not by deferoxamine. LY83583 blocked responses to nitroprusside but not to

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mentioning

confidence: 99%

Hydroxyl radical-dependent inactivation of guanylate cyclase in cerebral arterioles by methylene blue and by LY83583.

Kontos

Wei

1993

Stroke

118

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“…The technique of permeabilization to facilitate the entry of various agents into the interior of cells where the agents gain access to intracellular enzymes has been used successfully in isolated hepatocytes and in cultured ovarian cells as well as very extensively in various types of muscle including vascular smooth muscle and cardiac muscle. 15 " 18 Subsequently, we tested the responses to 10" 7 M acetylcholine superfusion before and after a 10-minute application of 20 JAM L-NMMA as well as after topical application of 50,100,200, and 1,000 ;u,M L-arginine. Each dose of L-arginine was left for 10 minutes before testing the responses to acetylcholine.…”

Section: Methodsmentioning

confidence: 99%

Effects in cats of inhibition of nitric oxide synthesis on cerebral vasodilation and endothelium-derived relaxing factor from acetylcholine.

Wei

Kukreja

Kontos

1992

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Background and Purpose:We investigated the chemical identity of the endothelium-derived relaxing factor generated by acetylcholine in cerebral microvessels by studying the effects and mechanism of action of inhibitors of nitric oxide synthesis from arginine on the vasodilation and endothelium-derived relaxing factor production induced by topical application of acetylcholine in cerebral arterioles.Methods: We determined cerebral arteriolar dilation and endothelium-derived relaxing factor production by bioassay in anesthetized cats equipped with cranial windows during supervision of 10~7 M acetylcholine before and after administration of either iV

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Ribonucleotide reduction in intact human diploid fibroblasts

Dick

Wright

1980

Journal Cellular Physiology

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There have been very few studies on ribonucleotide reductase activity in human tissue. In this report we describe a rapid and convenient procedure for determining purine and pyrimidine ribonucleotide reduction in normal human diploid fibroblasts and use the method to examine some general properties of the activity in these cells. ADP and CDP reductase was characterized for its response to the positive effectors, ATP and dGTP, the negative effector dATP, and the reducing agent dithiothreitol. Apparent Km values for ADP and CDP were determined to be 0.1 mM and 0.04 mM respectively. THe antitumor agent hydroxyurea inhibited both purine and pyrimidine reductase in a noncompetitive fashion, giving Ki value of 0.40 mM and 0.41 mM for ADP and CDP respectively. These Ki estimates are about four to five times higher than those reported for some permanent cell lines. An examination of the cytotoxic effects of hydroxyurea indicated a close correlation between the concentration of drug which inhibited enzyme activity and decreased colony-forming ability. Clearly the ability to investigate ribonucleotide reduction in low numbers of normal human diploid cells will be useful for genetic and biochemical studies.

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Chapter 20 The Use of T ween-8 0 -Permeabiliyed Mammalian Cells in Studies of Nucleic Acid Metabolism

Cited by 7 publications

References 12 publications

Hydroxyl radical-dependent inactivation of guanylate cyclase in cerebral arterioles by methylene blue and by LY83583.

Hydroxyl radical-dependent inactivation of guanylate cyclase in cerebral arterioles by methylene blue and by LY83583.

Effects in cats of inhibition of nitric oxide synthesis on cerebral vasodilation and endothelium-derived relaxing factor from acetylcholine.

Ribonucleotide reduction in intact human diploid fibroblasts

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