Carnosine prevents necrotic and apoptotic death of rat thymocytes via ouabain-sensitive Na/K-ATPase

Abstract

It is known that ouabain, a selective inhibitor of Na/K-ATPase, not only can cause the activation of signal cascades, which regulate the cell viability, but also can cause the accumulation of free radicals, which can evoke the oxidative stress. We have shown that the nanomolar concentrations of ouabain result in the temporary increase in the level of intracellular free radicals, but the millimolar concentration of ouabain induces a stable intracellular accumulation of free radicals in rat thymocytes. The increasing level of free radicals resulting from both low and high concentrations of ouabain can be attenuated by the antioxidant, carnosine. Moreover, the long-term incubation with ouabain leads to the cell death by necrosis and apoptosis. Ouabain-mediated apoptosis and necrosis were also abolished by carnosine.

Figure 1

Effect of ouabain on DCF fluorescence levels in rat thymocytes. DCF-preloaded cells were incubated with different concentrations of ouabain (1 nM, 100 nM or 1 mM as noted) for 30 min at 37°C before measurement.

Figure 2

Effect of Ca-free media on DCF fluorescence level in ouabain-treated thymocytes. DCF-loaded cells were pre-incubated in calcium-free Tyrode’s solution containing 1mM EGTA for 30 min at 37°C and then treated with ouabain (15 min at 37°C). Sign * corresponds to significance vs control, # – to significance vs samples containing Ca, n = 3, p < 0,05.

Figure 3

Effect of an inhibitor of NO-synthase on accumulation of free radicals in ouabain-treated thymocytes. DCF-loaded cells were pre-treated with N-nitroarginine (NNA, 30 μM) for 30 min at 37°C followed by incubation with ouabain (for 15 min at 37°C). Sign * corresponds to significance vs control, # – to significance vs samples containing no NNA, n = 3, p < 0,05.

Figure 4

Profile of distribution the thymocytes along the DCF fluorescence axis. DCF fluorescence value corresponds to free radicals levels in cells. Thymocytes were incubated with carnosine (1 mM for 40min) and then were treated with low (100 nM, A) or high (1 mM, B) concentrations of ouabain for 15 min at 37°C.

Figure 5

Effect of carnosine on ouabain-mediated free radical accumulation. DCF-loaded cells were pretreated with 1 mM carnosine for 40 min and then were incubated with different concentrations of ouabain (100 nM and 1 mM) for 5, 15 and 30 min at 37°C. Sign * corresponds to significance vs control, # – to significance vs samples containing no carnosine, n = 3, p < 0,05.

Figure 6

Effect of ouabain on the numbers of apoptotic (A) and necrotic (B) cells under different conditions. Cells were treated with 1 mM carnosine and then incubated with ouabain (100nM, 1mM) and 100 nM dexametasone during 5 h. Sign * corresponds to significance vs control, # – to significance vs samples containing no carnosine, n = 3, p < 0,05.