Striking Differences Between the Kinetics of Regulation of Respiration by ADP in Slow‐Twitch and Fast‐Twitch Muscles In Vivo

Abstract

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...

Metabolic consequences of functional complexes of mitochondria,myofibrils and sarcoplasmic reticulum in muscle cells

“…A trivial explanation of this phenomenon by formation of ADP concentration gradients between the medium and the core of the cells is excluded (Kay et al, 1997), since the Brownian movement of adenine nucleotides in water solution across the diffusion distance of <10·µm (permeabilized cardiomyocytes) is more rapid than the metabolic turnover of ADP and ATP . Besides, this trivial explanation is in conflict with the tissue specificity of the phenomenon mentioned above (Kuznetsov et al, 1996;Veksler et al, 1995). Another important recent observation is that the kinetics of regulation of mitochondrial respiration in ADP is approximately 20·µmol·l -1 ).…”

“…(Duchen, 1999;. These structural differences are paralleled by differences in functional characteristics, in particular in the apparent Km for exogenous ADP (Kuznetsov et al, 1996;Burelle and Hochachka, 2002). Thus, both intracellular arrangement and regulation of mitochondrial respiration are tissue specific.…”

“…It is also known that the high values of apparent Km for exogenous ADP are significantly decreased from 300-350·µmol·l -1 to 40-70·µmol·l -1 by selective proteolysis (Kuznetsov et al, 1996;Saks et al, 2001). Treatment of permeabilized cardiomyocytes for a short time with 1·µmol·l -1 trypsin also results in rapid disorganisation of the regular arrangement of mitochondria in cardiomyocytes and a collapse in the microtubular network (Appaix et al, 2003).…”

“…Determinations were carried out at 25°C, and solubility of oxygen was taken as 215·nmol·ml -1 (Kuznetsov et al, 1996). The method of calculation of free calcium concentration in solution B is given below.…”

“…One of the interesting observations in this area, made in numerous laboratories, is that in permeabilized oxidative muscle cells the apparent Km for exogenous ADP in the control of mitochondrial respiration is very high, in the range of 200-300·µmol·l -1 , in contrast to permeabilized fibers from fast-twitch skeletal muscles and isolated mitochondria in vitro: in both cases, the apparent Km for ADP is 15-20·µmol·l -1 (Anflous et al, 2001;Braun et al, 2001;Burelle and Hochachka, 2002;Dos Santos et al, 2002;Fontaine et al, 1995;Kay et al, 1997;Kummel, 1988;Kuznetsov et al, 1996;Liobikas et al, 2001;Milner et al, 2000;Saks et al, 1991Saks et al, , 1993Saks et al, , 1994Saks et al, , 1995Saks et al, , 1998aSaks et al, , 2001Seppet et al, 2001;Toleikis et al, 2001;Veksler et al, 1995). A trivial explanation of this phenomenon by formation of ADP concentration gradients between the medium and the core of the cells is excluded (Kay et al, 1997), since the Brownian movement of adenine nucleotides in water solution across the diffusion distance of <10·µm (permeabilized cardiomyocytes) is more rapid than the metabolic turnover of ADP and ATP .…”