Poster
# 22
Poster |
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6th Internet World Congress for Biomedical Sciences |
José Manuel Martínez-Martos(1), María Jesús Ramírez-Expósito(2), María Dolores Mayas-Torres(3), Isabel Prieto-Gómez(4), Manuel Ramírez-Sánchez(5)
(1)(3)(4)(5)Unit of Physiology. University of Jaén - Jaén. Spain
(2)Unit of Physiology. University of Jaen - Jaén. Spain
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[Neuroscience] |
[Physiology] |
In 1983, Mosmann used the tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay is based on cleavage of the yellow tetrazolium salt MTT, which forms water-insoluble dark blue formazan crystals. This cleavage takes place in cells by the mitochondrial enzyme succinate-dehydrogenase (Slater, Sawyer & Strauli, 1963). This assay detects living, but not dead cells, and the signal generated is dependent of the degree of activation of the cells. Since this report, MTT reduction is one of the most frequently used methods of measuring cell proliferation and cytotoxicity. Due to the extended use of synaptosomes to study physiological, biochemical and pharmacological aspects of synaptic function, our aim was to study the activity of mitochondrial respiratory chain by MTT assay, under resting and K+- and ATP- stimulated conditions in rat and mouse brain cortex synaptosomes, as an index of the functionality and energetic state of this subcellular fraction.
Frontal cortex synaptosomes from eight male Wistar rats (body weight 265±15 g) and eight male Balb/C mice (body weigh 28.59±0.6 g) were prepared as described previously (Whittaker, Michaelson & Kirkland, 1964) with slight modifications. Briefly, animals were sacrificed by decapitation; their brains were removed and immediately rinsed in cold isotonic saline. The frontal cortex was dissected and maintained on ice until it was homogenized in cold 0.32 M sucrose (approximately 1:20 original tissue weight to volume (g/ml)). All next steps were performed at 4 C. The homogenate was centrifuged at 2000 xg for 12 min, and the resulting supernatant was then centrifuged at 30000 xg for 27 min. The supernatant was discarded and the pellet was resuspended in 0.32 M sucrose. A three layer sucrose gradient was prepared by layering the membrane preparation on top of 1.4 M sucrose plus 0.8 M sucrose for rats, or 1.2 M sucrose plus 0.8 M sucrose for mice. The gradient was centrifugated for 30 min at 30000 xg. The 0.8 M layer pellet was collected and resuspended in an incubation buffer containing 116 mM NaCl, 5.4 mM KCl, 0.9 mM MgCl2, 0.9 mM NaH2PO4, 1.8 mM CaCl2, 25 mM NaHCO3, 10 mM glucose and 1 mM 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) , pH 7.2. Total protein content was adjusted to a final concentration of 1, 2 and 4 mg/ml, measured by the method of Bradford (1976), using BSA as standard. Depolarization of synaptosomes was performed by the addition of increased concentrations of KCl (25, 50 and 100 mM) in the incubation buffer. The increase of K+ was compensated by a proportional reduction of Na+ to maintain osmolarity. ATP stimulation of synaptosomes was carried out by the addition of the corresponding amount of a stock solution of ATP 1 mM in incubation buffer, to get a final concentration of 1, 2, 4, 10 and 20 µM. After that, mitochondrial activity was measured by the colorimetric assay previously described by Mosmann (1983). 50 µl of the suspension of synaptosomes were incubated in triplicate in a 96-well plate for 1 or 2 hours at 37°C. Then, 100 µl of HCl in isopropanol (0.04 M) was added to each well and the mixture was shaked vigorously to dissolve the dark blue crystals. Finally, the samples were measured in a microplate reader using a test wavelength of 550 nm and a reference wavelength of 620 nm. The values were expressed in optical density units as mean±SEM. The one-way analysis of variance (ANOVA) with the Newman-Keul´s post-hoc test, was used for comparisons among the different groups. The linear correlation coefficient test was used for relationships between protein content and MTT formazan production. The null hypothesis was rejected when p<0.05.
MTT reduction in rodent synaptosomes was linearly correlated with the protein content (r=0.999, p=0.000 in both cases) Figure 1. However, the time of incubation did not modify the rate of MTT cleavage. No differences were found on basal MTT formazan production between rat and mouse cortex synaptosomes.
Rat synaptosomes stimulated by K+ 25 mM increased MTT reduction about 100% over control Figure 2. 50 mM and 100 mM K+ increased MTT reduction to 145% of control. Mouse synaptosomes stimulated with 25 mM K+ increased MTT cleavage 60% over control, increasing until 80% with 50 or 100 mM K+.
ATP-stimulation of rat cortex synaptosomes induced an increase in MTT formazan production of about 65% when 1 or 2 µM ATP was used Figure 3. 4 µM ATP increased MTT reduction until 75%, but higher amounts of ATP decreased MTT cleavage until 32% over control (10 µM ATP) and 40% under control with 20 µM ATP. In mouse synaptosomes, the maximum levels of MTT formazan production were observed with ATP 1, 2 or 4 µM (about 40% over control). 10 µM ATP decreased MTT reduction until control levels, whereas 20 µM ATP decreased MTT reduction about 25% under control.
Synaptosomes have been widely used to understand the neurochemical mechanisms which underlie to the brain function, including uptake/release of neurotransmitters (Sheng, Westenbroek & Catteral, 1998; Langley & Grant, 1997; Erecinska, Zaleska, Chiv & Nelson, 1991; Szutowicz, Tomaszewicz & Bielarczyk, 1997), intracellular free calcium homeostasis (Sheng et al. 1998; Huang, Toral-Barza & Gibson, 1991), second messenger or protein studies (Hertz & Peng, 1992) and energy metabolism (Curti, Izzo, Brambilla, Faccheti, Sangiovanni & Brambilla, 1995; Erecinska, Nelson & Silver, 1996). These studies include the variations on the functional status of this subcellular fraction depending of its depolarization by K+ and/or its activation with different substances (including ATP). Furthermore, several reports describe the metabolic status of rodent synaptosomal mitochondria as a key factor of dysfunctions in different brain diseases such as traumatic spinal cord injury (Azbill, Mu, Bruce-Keller, Mattson & Springer, 1997), ischemia (Santos, Moreno & Carvalho, 1996), reactive oxygen species formation and membrane lipid peroxidation (Keller et al. 1997), aging (Gabbita, Butterfield, Hensley, Shaw & Carney, 1997), chronic lead intoxication (Struzynska, Dabrowska-Bouta & Rafalowska, 1997) or drug-induced neurotoxicity (Callahan, Yuan, Strover, Hatzidimitrion & Ricaurte, 1998). Our results describe the physiological changes in the energetic status of rodent cortical brain synaptosomes under three conditions:1) in a resting (basal) state; 2) after depolarization with high K+, where the respiration is accelerated by increased Na+-permeability through the plasma membrane, which stimulates the function of Na+/K+ ATPase, and thus increases the energy demand (Erecinska, Nelson, Deas & Silver, 1996; Raatikainen, Kauppinen, Komulainen, Taipale, Pirttila & Tuomisto, 1991); 3) after stimulation with ATP, acting through its ionotropic and metabotropic receptors (Zimmermann, 1994). Our results showed a similar MTT formazan production in rat and mouse synaptosomes, which is in agreement with previous reports that demonstrated an essentially identical behaviour between synaptosomes prepared from mouse, rat, dog and chicken cerebra in several energy metabolism parameters (Kyriazy & Basford, 1986), other than the use of MTT assay.
Although it is widely assumed that MTT is reduced by active mitochondria in living cells, and MTT assay is near to be exclusively used for measuring cell proliferation and cytotoxicity, we report that MTT assay could be useful as an index of the functional status and energetic behaviour of rodent brain cortex synaptosomes, which could be interesting to relate with several other biochemical and physiological parameters in different types of studies under resting and/or stimulating conditions.
Synaptosomes have been widely used to understand the neurochemical mechanisms which underlie to the brain function, including uptake/release of neurotransmitters (Sheng, Westenbroek & Catteral, 1998; Langley & Grant, 1997; Erecinska, Zaleska, Chiv & Nelson, 1991; Szutowicz, Tomaszewicz & Bielarczyk, 1997), intracellular free calcium homeostasis (Sheng et al. 1998; Huang, Toral-Barza & Gibson, 1991), second messenger or protein studies (Hertz & Peng, 1992) and energy metabolism (Curti, Izzo, Brambilla, Faccheti, Sangiovanni & Brambilla, 1995; Erecinska, Nelson & Silver, 1996). These studies include the variations on the functional status of this subcellular fraction depending of its depolarization by K+ and/or its activation with different substances (including ATP). Furthermore, several reports describe the metabolic status of rodent synaptosomal mitochondria as a key factor of dysfunctions in different brain diseases such as traumatic spinal cord injury (Azbill, Mu, Bruce-Keller, Mattson & Springer, 1997), ischemia (Santos, Moreno & Carvalho, 1996), reactive oxygen species formation and membrane lipid peroxidation (Keller et al. 1997), aging (Gabbita, Butterfield, Hensley, Shaw & Carney, 1997), chronic lead intoxication (Struzynska, Dabrowska-Bouta & Rafalowska, 1997) or drug-induced neurotoxicity (Callahan, Yuan, Strover, Hatzidimitrion & Ricaurte, 1998). Our results describe the physiological changes in the energetic status of rodent cortical brain synaptosomes under three conditions:1) in a resting (basal) state; 2) after depolarization with high K+, where the respiration is accelerated by increased Na+-permeability through the plasma membrane, which stimulates the function of Na+/K+ ATPase, and thus increases the energy demand (Erecinska, Nelson, Deas & Silver, 1996; Raatikainen, Kauppinen, Komulainen, Taipale, Pirttila & Tuomisto, 1991); 3) after stimulation with ATP, acting through its ionotropic and metabotropic receptors (Zimmermann, 1994). Our results showed a similar MTT formazan production in rat and mouse synaptosomes, which is in agreement with previous reports that demonstrated an essentially identical behaviour between synaptosomes prepared from mouse, rat, dog and chicken cerebra in several energy metabolism parameters (Kyriazy & Basford, 1986), other than the use of MTT assay.
Although it is widely assumed that MTT is reduced by active mitochondria in living cells, and MTT assay is near to be exclusively used for measuring cell proliferation and cytotoxicity, we report that MTT assay could be useful as an index of the functional status and energetic behaviour of rodent brain cortex synaptosomes, which could be interesting to relate with several other biochemical and physiological parameters in different types of studies under resting and/or stimulating conditions.
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[Neuroscience] |
[Physiology] |
