Methylene blue-mediated hexose monophosphate shunt stimulation in human red blood cells in vitro: Independence from intracellular oxidative injury

Abstract

Abstract-I. The red blood cellhexose monophosphate shunt (HMS) and proteolytic responses to several concentrations of Methylene Blue or sodium nitrite were measured.2. The results suggested two distinct mechanisms for activation of the HMS: (I) nitrite treatment increased HMS activity in response to oxidative challenge to red cellprotein; (2) Methylene Bluetreatment activated HMS without injurious oxidative challenge. Nitrite-treated cells actively degraded protein, whereas Methylene Blue-treated red cells did not activate proteolytic systems that degrade oxidized red cell protein.3. These observations are relevant to proposed in vitro systems for evaluation of drug hemolytic toxicity potential on the basis of HMS stimulation capacity.

Meiotic Induction in Cumulus Cell-Enclosed Mouse Oocytes: Involvement of the Pentose Phosphate Pathway1

“…In the first series of experiments, cumulus cell-enclosed oocytes, maintained in meiotic arrest for 17-18 h with hypoxanthine, were treated with increasing concentrations of methylene blue, P5C, or PES, agents known to generate NADP from NADPH and consequently stimulate the PPP [25][26][27][28][29][30][31][32][33]. With all three agents, a dose-dependent increase in the incidence of GVB was observed ( Fig.…”

“…The PPP is regulated by the intracellular NADPH/NADP ratio [50]. Methylene blue, PES, and P5C are electron acceptors that can lower the NADPH/NADP ratio and activate the PPP by promoting oxidation of NADPH to NADP [25][26][27][28][29][30][31][32][33]; by their action, the oxidized dinucleotide is made available to the two dehydrogenase enzymes in the oxidative arm of the PPP that depend on this coenzyme for their activity (see Fig. 1).…”

An in vitro micro-volume procedure for rapid measurement of erythrocytic hexose monophosphate shunt activity

“…In this laboratory the micro-volume procedure has been employed to examine the relationship between HMS activation and the recently described proteolytic system in erythrocytes which is activated by oxidative stress (Goldberg and Boches, 1982). It has been found that sodium nitriteinduced proteolysis is sensitive to the level of HMS activity, whereas Methylene Blue may stimulate the HMS independently of proteolytic activity (Baird, 1984). These observations may be related to the fact that nitrite-induced activation of the HMS was distinctly sigmoidal (Fig.…”

“…These observations may be related to the fact that nitrite-induced activation of the HMS was distinctly sigmoidal (Fig. 2) and that induced by Methylene Blue was strongly hyperbolic (Kirkman et al, 1980;Baird, 1984). The micro-volume procedure is well-suited to detecting such differences in HMS response.…”

“…However, comparison of relative HMS activities reveals published values in good agreement with those obtained using the microvolume procedure. For example, Methylene Blueinduced elevation of HMS activity has been reported as 51-fold (Gaetani et aI., 1974), 48-fold (Metz et al, 1977), and 52-fold (Kirkman et al, 1980), whereas the micro-volume procedure measured a 45-fold elevation of HMS above baseline activity (Baird, 1984). Further agreement is evident among estimates of the Methylene Blue-induced glucose carbon recycling contribution to total HMS activity; 34% (Brin andYonemoto, 1958), 27% (Harvey andKaneko, 1977), 39% (Kirkman et al, 1980), and 28% using the micro-volume procedure (data not given).…”