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The kinetics of in vivo regulation of mitochondrial respiration by ADP was studied in rat heart, slowtwitch skeletal muscle (soleus) and fast-twitch skeletal muscle (gastrocnemius, plantaris, quadriceps and tibialis anterior) by means of saponin-skinned fibres. Mitochondria1 respiratory parameters were determined in the absence and presence of creatine (20 mM), and the effect of proteolytic enzymes (trypsin, chymotrypsin or elastase) on these parameters was investigated in detail. Chem. 270, 19921 -199291, who studied muscle fibres from normal and transgenic mice, that the kinetics of respiration regulation in muscle cells is tissue specific. We found that in rat cardiac and soleus muscle fibres the apparent K,,, for respiration regulation was 300-400 pM and decreased to 50-80 pM in the presence of creatine. In contrast, in skinned fibres from gastrocnemius, plantaris, tibialis anterior and quadriceps muscles, this value was initially very low, 10-20 pM, i.e. the same as that is in isolated muscle mitochondria, and the effect of creatine was not observable under these experimental conditions. Treatment of the fibres with trypsin, chymotrypsin or elastase (0.125 pg/ml) for 15 min decreased the apparent K,,, for ADP in cardiac and soleus muscle fibres to 40-98 pM without significant alteration of V,,,, or the intactness of outer mitochondrial membrane, as assessed by the cytochrome c test. In fibres from gastrocnemius, trypsin increased the apparent K,,, for ADP transiently. The effects of trypsin and chymotrypsin were studied in detail and found to be concentration dependent and time dependent. The effects were characterised by saturation phenomenon with respect to the proteolytic enzyme concentration, saturation being observed above 1 pM enzyme. These results are taken to show that in cardiac and slow-twitch skeletal muscle, the permeability of the outer mitochondrial membrane to adenine nucleotides is low and controlled by a cytoplasmic protein that is sensitive to trypsin and chymotrypsin. This protein may participate in feedback signal transduction by a mechanism of vectorial-ligand conduction. This protein factor is not expressed in fasttwitch skeletal muscle, in which cellular mechanism of regulation of respiration is probably very different from that of slow-twitch muscles.Keywords: heart; skeletal muscle; respiration ; regulation; adenine nucleotide.The cellular mechanism of regulation of respiration in vivo is still unknown. The conventional explanation that the rate of respiration of mitochondria in intact cells is governed by the cytoplasmic ADP concentration according to a simple Michaelis-Menten type relationship is in disagreement with many experimental data [l-51. First, it has been shown in numerous experiments on isolated hearts that the increases in workload and oxygen consumption are usually observed at stable, low and sometimes even decreasing steady-state ADP levels [I -51. Second, Kushmerick et al. have found that in slow-twitch skeletal muscle, an increase of frequency of stimulation does...

The kinetics of regulation of mitochondrial respiration by endogenous and exogenous ADP in muscle cells in situ was studied in skinned cardiac and skeletal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration in the medium were dependent on the calcium concentration, and 70-80% of maximal rate of respiration was achieved at ADP concentration below 20 microM in the medium. In contrast, when exogenous ADP was added, maximal respiration rate was observed only at millimolar concentrations. An exogenous ADP-consuming system consisting of pyruvate kinase (PK; 20-40 units/ml) and phosphoenolpyruvate (PEP; 5 mM), totally suppressed respiration activated by exogenous ADP, but the respiration maintained by endogenous ADP was not suppressed by more than 20-40%. Creatine (20 mM) further activated respiration in the presence of ATP and PK+PEP. Short treatment with trypsin (50-500 nM for 5 min) decreased the apparent K(m) for exogenous ADP from 300-350 microM to 50-60 microM, increased inhibition of respiration by PK+PEP system up to 70-80%, with no changes in MgATPase activity and maximal respiration rates. Electron-microscopic observations showed detachment of mitochondria and disordering of the regular structure of the sarcomere after trypsin treatment. Two-dimensional electrophoresis revealed a group of at least seven low-molecular-mass proteins in cardiac skinned fibres which were very sensitive to trypsin and not present in glycolytic fibres, which have low apparent K(m) for exogenous ADP. It is concluded that, in oxidative muscle cells, mitochondria are incorporated into functional complexes ('intracellular energetic units') with adjacent ADP-producing systems in myofibrils and in sarcoplasmic reticulum, probably due to specific interaction with cytoskeletal elements responsible for mitochondrial distribution in the cell. It is suggested that these complexes represent the basic pattern of organization of muscle-cell energy metabolism.

In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50-100 microg/ml) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (1) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon--tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given.