Effects of caffeine and other methylxanthines on the development and metabolism of sea urchin eggs. Involvement of NADP and glutathione.

Abstract

Methylxanthines (MX) inhibit cell division in sea urchin and clam eggs, This inhibitory effect is not mediated via cAMP. MX also inhibit respiration in marine eggs, at concentrations which inhibit cleavage. Studies showed that no changes occurred in ATP and ADP levels in the presence of inhibitory concentrations of MX, indicating an extra-mitochondrial site of action for the drug. Subsequent studies revealed decreased levels of NADP § and NADPH, when eggs were incubated with inhibitory concentrations of MX, but no change in levels of NAD § and NADH. MX did not affect the pentose phosphate shunt pathway and did not have any effect on the enzyme NAD+-kinase. Further studies showed a marked inhibitory effect on the glutathione reductase activity of MX-treated eggs. Reduced glutathione (GSH) could reverse the cleavage inhibitory effect of MX. Moreover, diamide, a thiol-oxidizing agent specific for GSH in living cells, caused inhibition of cell division in sea urchin eggs. Diamide added to eggs containing mitotic apparatus (MA) could prevent cleavage by causing a dissolution of the formed MA. Both MX and diamide inhibit a Ca2+-activated ATPase in whole eggs. The enzyme can be reactivated by sulfhydryl reducing agents added in the assay mixture. In addition, diamide causes an inhibition of microtubule polymerization, reversible with dithioerythritol. All experimental evidence so far suggests that inhibition of mitosis in sea urchin eggs by MX is mediated by perturbations of the in vivo thiol-disulfide status of target systems, with a primary effect on glutathione levels.Our earlier studies have shown that neither cAMP nor its butyrated derivatives, at concentrations up to 2 • 10 -~ M, affect early cleavage of sea urchin or clam eggs, at least through the late gastrulation and feeding stages (47). Incorporation experiments with both radioactive cAMP and dibutyryl cAMP demonstrated uptake of exogenously added nucleotides in sea urchin eggs. Identification and quantitation of the intracellular compounds showed that intracellular levels of cAMP or N emonobutyryl cAMP (the converted intracellular product of dibutyryl cAMP) reached at least 10 -8 M by first cleavage (at an external concentration of 10 -8 M), two orders of magnitude higher 440

Role of the Cell Membrane in Mitosis: Effects on HeLa Cell Division of Ouabain, Amphotericin B, Concanavalin A and Cyclic Nucleotides

“…1). Consistent with our present results, there is a report (14) on the effect of cyclic AMP and its butyrated derivatives on sea urchin and clam eggs. It shows that these compounds did not affect the early cleavage of the eggs at concentrations up to 20 mM.…”

“…1). Theophylline might affect cell division by combining with a metal ion that is necessary for enzyme activity or, as Nath and Rebhun (14) proposed for the division of sea urchin eggs, by indirectly inhibiting glutathione reducta se.…”

Microtubule assembly and conanavalin A capping in lymphocytes: reappraisal using normal and abnormal human peripheral blood cells.

“…(We assume, of course, that lymphocytes and PMN are equivalently permeable to colchicine.) In contrast with colchicine, microtubule disassembly caused by diamide is independent of the assembly/disassembly equilibrium (5,11,12) and so occurs with equal rapidity in both lymphocytes and PMN.…”

“…As described (5,8), the majority of PMN were also readily capped after exposure to 100uM diamide for 10 min. This glutathione-oxidizing agent is thought to cause microtubule disassembly and to inhibit microtubule assembly by causing oxidation of critical tubulin-SH residues (11,12).…”

Why the xanthine derivatives are used to study of P-glycoprotein-mediated multidrug resistance in L1210/VCR line cells

“…as: inhibitors of metabolism in sea urchin eggs (Nath and Rebhun 1976), suppressor of cytokineinduced NO production via inhibition of the expression of inducible NO-synthase mRNA in macrophages (Trajkovic et al 1997), hemorheologic agents (Porter et al 1982), improvers of circulatory failure in murine models of endotoxaemia (Wu et al 1999), anti-inflammatory agents (Rao et al 2005), nonspecific inhibitors of cyclic nucleotide phosphodiesterases (Nicholson et al 1991). Moreover, alkyl-xanthine derivatives constitute nonspecific phosphodiesterase inactivators -they increase the cyclic adenosine monophosphate (cAMP) level in the cells thus inhibiting the synthesis not only of TNF-α, but also of IL-1L , IL-6, and IL-8 (Han et al 1990;Semmler et al 1993;Zabel et al 1993;Neuner et al 1994).…”