diamide and lucifer-yellow

The insecticide lindane (gamma-hexachlorocyclohexane) inhibits gap junction intercellular communication in rat myometrial cells by a mechanism involving oxidative stress. We hypothesized that oxidation of reduced glutathione (GSH) to glutathione disulfide (GSSG) and subsequent S-glutathionylation provide a mechanistic link between lindane-induced oxidative stress and lindane's inhibition of myometrial gap junction communication. Gap junction communication between cultured rat myometrial myocytes was assessed by Lucifer yellow dye transfer after microinjection. A biphasic pattern was confirmed, with dye transfer nearly abolished after 1 h of exposure to 100 microM lindane followed initially by recovery after lindane removal, and then the development 4 h after termination of lindane exposure of a delayed-onset, sustained inhibition that continued for 96 h. As measured by HPLC, cellular GSH varied over a 24-h period in a biphasic fashion that paralleled lindane-induced inhibition of dye transfer, whereas GSSG levels increased in a manner inversely related to GSH. In accordance, GSH/GSSG ratios were depressed at times when GSH and dye transfer were low. Lindane substantially increased S-glutathionylation in a concentration-dependent manner, measured biochemically by GSSG reductase-stimulated release of GSH from precipitated proteins. Furthermore, treatments that promoted accumulation of GSSG (50 microM diamide and 25 microM 1,3-bis(2-chloroethyl)-1-nitrosourea [BCNU]) inhibited Lucifer yellow dye transfer between myometrial cells. Findings that lindane induced GSH oxidation to GSSG with increased S-glutathionylation, together with the diamide and BCNU results, suggest that oxidation of GSH to GSSG is a component of the mechanism by which lindane inhibits myometrial gap junctions.

Topics: Animals; Carmustine; Cell Communication; Cells, Cultured; Diamide; Female; Fluorescent Dyes; Gap Junctions; Glutathione; Glutathione Disulfide; Hexachlorocyclohexane; Insecticides; Isoquinolines; Myocytes, Smooth Muscle; Myometrium; Oxidation-Reduction; Rats; Rats, Sprague-Dawley

The isolated frog lens epithelium can be maintained with its cell shape, cytoskeletal organization and membrane electrophysiological characteristics intact for more than 24 hr. Perifusion with the permeant oxidant diamide (1 mM) led to drastic, but reversible, changes in all the above parameters. After a 20 min exposure to diamide, the regular polygonal arrangement of the epithelial cells become increasingly disrupted as the cells reorganized and a 'rosette' pattern formed. The cells at the edges of the rosette pulled apart from one another while those in the centre maintained a relatively normal appearance. Blebs formed on the apical surface of all of the cells on prolonged exposure and the internal structure was also found to be severely disrupted. The cytoplasm became granular, vacuolated and the nucleus had a banded, non-homogeneous appearance. Phalloidin staining of F-actin microfilaments revealed that there was a general disruption of organization, with actin losing its association with the membrane. The microtubule array, organized around the centrosome, was also severely disrupted although microtubules were still discernible in most cells. During exposure to diamide the membrane potential depolarized and both electrical and dye coupling, which are normally extremely efficient in these cells, were disturbed. If the epithelium was exposed to 1 mM diamide for more than 45 min then all of the above changes were irreversible and cell death followed. If exposure was restricted to less than 30 min, then all of the above changes occurred and, in fact, progressed for over 1 hr; but if the epithelium was perifused for a further 20 hr in control medium, then most of the changes were reversible.

Topics: Actin Cytoskeleton; Animals; Cell Communication; Cytoskeleton; Diamide; Epithelial Cells; Epithelium; Fluorescent Dyes; Isoquinolines; Lens, Crystalline; Membrane Potentials; Microscopy, Electron, Scanning; Microtubules; Rana pipiens