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The STUDIA UNIVERSITATIS BABEŞ-BOLYAI issue article summary
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Abstract: Male white Wistar rats were maintained in our animal facility for 14-18 weeks and the evolution of their body weight was assessed periodically. One group of the rats, kept on a normal diet, served as control (C), while in another group (A) each rat was supplemented individually with 1.5 ml of 48% ethanol/100 g body weight daily. At the end of the period, the rats were sacrificed and their liver used for the preparation of mitochondria. For membrane potential (ΔΨ), calcium (Ca2+) and swelling measurements, 1 mg of mitochondrial protein/ml was usually incubated directly in the spectrophotometer cuvettes, at room temperature (around 24OC), in different media, usually containing 5 mM Hepes buffer (pH 7.37), 1 mM KPi and various concentrations of mannitol, sucrose, KCl and MgCl2. 8 μM rotenone was always added to the mitochondrial suspension, as well as the appropriate probe for either ΔΨ (2.5 μM diS-C2-(5)) or Ca2+ (30 μM arsenazo III). The respiration and all the associated phenomena monitored by us (ΔΨ, Ca2+ fluxes and swelling) were usually triggered by the addition of succinate (2.5 mM). Different amounts of CaCl2 or other modulating factors were either added gradually or in one pulse, up to the desired final concentration. Recordings of either ΔΨ or calcium fluxes in parallel with the matrix swelling were performed by a diode-array spectrophotometer. In addition, for quantitative measurements of the extension of swelling, special runs were also performed in which mitochondrial protein was used as the triggering factor. From the comparison of the spectrophotometric recordings, one can observe a strong correlation between the concentration of calcium added to the mitochondrial suspension, the moment of the ΔΨ collapse, of the swelling and of the calcium release (massive calcium efflux). As expected, these phenomena occur faster (at shorter times and/or lower calcium concentrations) in ionic media, especially in those that lack magnesium. However, important differences were also noticed between the mitochondria of the control (C) rats and those of the animals treated with ethyl alcohol (A) regarding the degree (amplitude) and the kinetics of the absorbance changes associated with the generation/collapse of ΔΨ, Ca2+ movements and swelling. In general, mitochondria from ethanol-treated rats are more sensitive to calcium. Even though the amplitude of membrane potential does not seem to be affected, it collapses at much lower concentrations of added calcium. Even though, in general, the extension of swelling does not differ statistically significant, this sensitivity is also valid for the matrix swelling and the release of calcium. We have quantified this relationship by considering the number of calcium pulses (of 12.5 μM each) added to the cuvette before massive calcium efflux (release) and ΔΨ collapse occur. As expected, this number is lower in ionic media (with the lowest in the swelling medium), but for the same medium it is also lower for the ethanol-fed rats and the differences are in general statistically very significant (p<0.01) in all media tested. |
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