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Despite extensive research, the regulation of mitochondrial function is still not understood completely. Ample evidence shows that cytosolic Ca2+ has a strategic task in co-ordinating the cellular work load and the regeneration of ATP by mitochondria. Currently, the paradigmatic view is that Cacyt2+ taken up by the Ca2+ uniporter activates the matrix enzymes pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and isocitrate dehydrogenase. However, we have recently found that Ca2+ regulates the glutamate-dependent state 3 respiration by the supply of glutamate to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier. Since this activation is not affected by ruthenium red, glutamate transport into mitochondria is controlled exclusively by extramitochondrial Ca2+. Therefore, this discovery shows that besides intramitochondrial also extramitochondrial Ca2+ regulates oxidative phosphorylation. This new mechanism acts as a mitochondrial "gas pedal", supplying the OXPHOS with substrate on demand. These results are in line with recent findings of Satrustegui and Palmieri showing that aralar as part of the malate-aspartate shuttle is involved in the Ca2+-dependent transport of reducing hydrogen equivalents (from NADH) into mitochondria. This review summarises results and evidence as well as hypothetical interpretations of data supporting the view that at the surface of mitochondria different regulatory Ca2+-binding sites exist and can contribute to cellular energy homeostasis. Moreover, on the basis of our own data, we propose that these surface Ca2+-binding sites may act as targets for neurotoxic proteins such as mutated huntingtin and others. The binding of these proteins to Ca2+-binding sites can impair the regulation by Ca2+, causing energetic depression and neurodegeneration., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The effect of phorbol 12-myristate 13-acetate (PMA) on the expression and shedding of intercellular adhesion molecule-1 (ICAM-1) was investigated on the hematopoietic cell lines K 562 and U 937 using flow cytometry, fluorescence microscopy and ELISA technique. At low concentration of 1 nM, PMA stimulated the expression of ICAM-1 on the cell surface about 4-fold within 24 h, whereas a short-term treatment with 100 nM PMA led to the shedding of 35% of ICAM-1 from the surface of K 562 cells. The release of surface ICAM-1 was found on single cells by fluorescence microscopy to be a uniform process proceeding within 15 min. The shedding of ICAM-1 correlated with elevated levels of sICAM-1 in the supernatants of cultured cells. Also on K 562 cells stimulated by TNF-alpha, a PMA-induced release of ICAM-1 was observed in addition to the known spontaneous shedding. In contrast to the results with K 562 cells, no PMA-induced shedding of ICAM-1 was found on U 937 cells. This indicates a cell-specific process for K 562 cells. The PMA-mediated release of ICAM-1 from K 562 cells suggests that the shedding process does not only occur in parallel to the surface expression of ICAM-1, but may be controlled by particular mechanisms of down-regulation.

The conditions of treatment of human skeletal muscle fibers from M. vastus lateralis with saponin were optimized to achieve complete permeabilization of cell membrane at intact mitochondrial oxidative phosphorylation. After 30 min of incubation with saponin all lactate dehydrogenase, 50% of creatine kinase, 30% of adenylate kinase and less than 20% of citrate synthase was released into the permeabilization medium. These skinned fibers behave similar to isolated mitochondria from human skeletal muscle: (i) the respiration with mitochondrial substrates can be stimulated by ADP, (ii) inhibited by carboxyatractyloside and (iii) it is possible to detect fluorescence changes of mitochondrial NAD(P)H on additions of substrates, uncoupler and cyanide. From a comparison of rates of respiration per cytochrome aa3 content of isolated human skeletal muscle mitochondria and saponin-skinned muscle fibers it was possible to calculate that almost 85% of mitochondria in those fibers are accessible for the investigation of oxidative phosphorylation. As shown by the investigation of biopsy samples of two patients with undefined myopathies these fibers are a suitable object for the replacement of isolated mitochondria in the diagnosis of mitochondrial myopathies and encephalomyopathies.