Mitochondrial Calcium Sequestration Research Articles

Evidence that High Frequency Stimulation Influences the Phosphorylation of Pyruvate Dehydrogenase and that the Activity of this Enzyme is Linked to Mitochondrial Calcium Sequestration

Publisher Summary The hippocampal slice contains the major afferent input and the trisynaptic circuitry characteristic of the hippocampus. Approximately 1 hour after preparation of the slice, a bipolar stimulating electrode is placed where it activates the massive Schaffer-commissural input to the CA- 1 pyramidal cells. A recording electrode is placed in the terminal field of these axons where it can record the extracellular representation of the EPSP produced when the Schaffer-commissural axons are stimulated. Single pulses are then delivered until a stable baseline response is obtained and held for 30 min. Then a 1 sec burst of high frequency (100 pulses sec--1) stimulation is delivered for 1 sec and then testing is resumed with stimulation at 1 pulse sec-'. A short-term form of potentiation (STP) can be detected within seconds of the cessation of the high frequency stimulation; and, like the post-tetanic potentiation seen at the neuromuscular junction, this STP decays rapidly. However, the potentiated response does not return to baseline. Rather, within 2–3min of the cessation of stimulation, the potentiated response is stabilized and this potentiated response typically persists for the life of the slice. This chapter describes experimental evidence that have lead to the conclusion that high-frequency stimulation influences the phosphorylation of pyruvate dehydrogenase and that the activity of this enzyme is linked to mitochondrial calcium sequestration.

Mitochondrial matrix granules in soft tissues: I. Elemental composition by X-ray microanalysis

The elemental composition of individual matrix granules in mitochondria of rat brown fat, mouse gall bladder and guinea pig kidney has been examined by X-ray microanalysis. The matrix granules showed a similar elemental composition that was strongly dependent upon the method of sample preparation. Low-temperature oxygen plasma microincineration or wavelength-dispersive X-ray spectrometers were used to demonstrate the presence of phosphorus in matrix granules of osmium tetroxide-fixed specimens. Matrix granule osmiophilia was retained in glutaraldehyde-fixed brown fat only if exposure to polar organic solvents was avoided during subsequent steps, e.g. by cryosectioning. As normally prepared, matrix granules lack detectable calcium but had bound this at detectable levels after fixation in osmium tetroxide, but not glutaraldehyde, supplemented with 5 mM Ca 2+. The results demonstrate that mitochondrial matrix granules of normal soft tissues are not calcium phosphate deposits and contain phospholipids, apparently as a major constituent. Thus they provide evidence against the hypothesis that matrix granules are primarily involved in mitochondrial calcium sequestration and, indirectly, for the hypothesis that the granules may be related to inner membrane assembly.

Energy-dependent calcium sequestration activity in rat liver microsomes.

MgATP-dependent calcium sequestering activity of rat liver microsomes has been characterized. This activity is linear over a 90-min period and specifically requires MgATP. Substitution of CTP, UTP, GTP, or ADP will not support calcium uptake. Oxalate, which serves as a trapping agent in calcium uptake of skeletal muscle microsomes, is required to maintain net accumulation of calcium. The reaction is temperature-dependent and has an apparent Vmax of 11.2 nmol/mg of protein/min. The apparent Km for calcium is 23.2 muM calculated from total calcium concentration, and approximately 4.6 muM based on free calcium concentration, and apparent Km for ATP is 1.8 mM. Calcium uptake activity normally measured in presence of 100 mM KCl is only slightly depressed if 100 mM NaCl is substituted and is considerably depressed when 200 mM sucrose replaces KCl. An appropriate hydrolysis of ATP is associated with the calcium uptake. Separation of smooth and rough endoplasmic reticulum on sucrose gradients indicates a considerably lower specific activity per mg of protein in the fraction enriched with rough endoplasmic reticulum. Azide, at a level which completely inhibits liver mitochondrial calcium sequestration, has no effect on the liver microsomal system. Oligomycin, which inhibits ATP-dependent calcium uptake of liver mitochondria, has a considerably lesser effect on calcium uptake of liver microsomes. p-Chloromercuribenzoate and mersalyl inhibit the liver microsomal calcium pump at levels as low as 10- minus 7 M. Calcium uptake activity is considerably reduced in adult female rats. Weanling rats, both male and female, have calcium uptake activities like that of the adult males. Because of the higher activity in the male rat, the fatty acid composition of the liver microsomal phospholipids was analyzed. The male rat had a higher percentage of linoleic and palmitic acids in the microsomal phospholipids. Endoplasmic reticulum and plasma membrane are postulated to play a role in regulation of the levels of free cytoplasmic calcium in the mammalian liver.