Km For ADP Research Articles

Heterologous expression and biochemical characterization of the recombinant nucleoside triphosphate diphosphohydrolase 2 (LbNTPDase2) from Leishmania (Viannia) braziliensis.

Leishmania braziliensis is a pathogenic protozoan parasite that causes American Tegumentary Leishmaniasis (ATL), an important tropical neglected disease. ENTPDases are nucleotidases that hydrolyze intracellular and/or extracellular nucleotides. ENTPDases are known as regulators of purinergic signalling induced by extracellular nucleotides. Leishmania species have two isoforms of ENTPDase, and, particularly, ENTPDase2 seems to be involved in infectivity and virulence. In this study, we conducted the heterologous expression and biochemical characterization of the recombinant ENTPDase2 of L. braziliensis (rLbNTPDase2). Our results show that this enzyme is a canonical ENTPDase with apyrase activity, capable of hydrolysing triphosphate and diphosphate nucleotides, and it is dependent on divalent cations (calcium or magnesium). Substrate specificity was characterized as UDP>GDP>ADP>GTP>ATP=UTP. The enzyme showed optimal activity at a neutral to basic pH and was partially inhibited by suramin and DIDS. Furthermore, the low apparent Km for ADP suggests that the enzyme may play a role in adenosine-mediated signalling. The biochemical characterization of this enzyme can open new avenues for using LbNTPDase2 as a drug target.

Diabetic mitochondria are resistant to palmitoyl CoA inhibition of respiration, which is detrimental during ischemia.

The bioactive lipid intermediate palmitoyl CoA (PCoA) can inhibit mitochondrial ADP/ATP transport, though the physiological relevance of this regulation remains unclear. We questioned whether myocardial ischemia provides a pathological setting in which PCoA regulation of ADP/ATP transport would be beneficial, and secondly, whether the chronically elevated lipid content within the diabetic heart could make mitochondria less sensitive to the effects of PCoA. PCoA acutely decreased ADP‐stimulated state 3 respiration and increased the apparent K m for ADP twofold. The half maximal inhibitory concentration (IC50) of PCoA in control mitochondria was 22 µM. This inhibitory effect of PCoA on respiration was blunted in diabetic mitochondria, with no significant difference in the K m for ADP in the presence of PCoA, and an increase in the IC50 to 32 µM PCoA. The competitive inhibition by PCoA was localised to the phosphorylation apparatus, particularly the ADP/ATP carrier (AAC). During ischemia, the AAC imports ATP into the mitochondria, where it is hydrolysed by reversal of the ATP synthase, regenerating the membrane potential. Addition of PCoA dose‐dependently prevented this wasteful ATP hydrolysis for membrane repolarisation during ischemia, however, this beneficial effect was blunted in diabetic mitochondria. Finally, using 31P‐magnetic resonance spectroscopy we demonstrated that diabetic hearts lose ATP more rapidly during ischemia, with a threefold higher ATP decay rate compared with control hearts. In conclusion, PCoA plays a role in protecting mitochondrial energetics during ischemia, by preventing wasteful ATP hydrolysis. However, this beneficial effect is blunted in diabetes, contributing to the impaired energy metabolism seen during myocardial ischemia in the diabetic heart.

Skeletal muscle mitochondrial protein synthesis and respiration in response to the energetic stress of an ultra-endurance race.

The 2016 Colorado Trail Race (CTR) was an ultra-endurance mountain bike race in which competitors cycled for up to 24 h/day between altitudes of 1,675 and 4,025 m to complete 800 km and 21,000 m of elevation gain. In one athlete, we had the unique opportunity to characterize skeletal muscle protein synthesis and mitochondrial respiration in response to a normal activity control period (CON) and the CTR. We hypothesized that mitochondrial protein synthesis would be elevated and mitochondrial respiration would be maintained during the extreme stresses of the CTR. Titrated and bolus doses of ADP were provided to determine substrate-specific oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacities in permeabilized muscle fibers via high-resolution respirometry. Protein synthetic rates were determined by daily oral consumption of deuterium oxide (2H2O). The endurance athlete had OXPHOS (226 pmol·s-1·mg tissue-1) and ETS (231 pmol·s-1·mg tissue-1) capacities that rank among the highest published to date in humans. Mitochondrial (3.2-fold), cytoplasmic (2.3-fold), and myofibrillar (1.5-fold) protein synthesis rates were greater during CTR compared with CON. With titrated ADP doses, the apparent Km of ADP, OXPHOS, and ETS increased after the CTR. With provision of ADP boluses after the CTR, the addition of fatty acids (-12 and -14%) mitigated the decline in OXPHOS and ETS capacity during carbohydrate-supported respiration (-26 and -31%). In the face of extreme stresses during the CTR, elevated rates of mitochondrial protein synthesis may contribute to rapid adaptations in mitochondrial bioenergetics. NEW & NOTEWORTHY The mechanisms that maintain skeletal muscle function during extreme stresses remain incompletely understood. In the current study, greater rates of mitochondrial protein synthesis during the energetic demands of ultra-endurance exercise may contribute to rapid adaptations in mitochondrial bioenergetics. The endurance athlete herein achieved mitochondrial respiratory capacities among the highest published for humans. Greater mitochondrial protein synthesis during ultra-endurance exercise may contribute to improved mitochondrial respiration and serve as a mechanism to resist cellular energetic stresses.

A 9-wk docosahexaenoic acid-enriched supplementation improves endurance exercise capacity and skeletal muscle mitochondrial function in adult rats.

Decline in skeletal muscle mass and function starts during adulthood. Among the causes, modifications of the mitochondrial function could be of major importance. Polyunsaturated fatty (ω-3) acids have been shown to play a role in intracellular functions. We hypothesize that docosahexaenoic acid (DHA) supplementation could improve muscle mitochondrial function that could contribute to limit the early consequences of aging on adult muscle. Twelve-month-old male Wistar rats were fed a low-polyunsaturated fat diet and were given DHA (DHA group) or placebo (control group) for 9 wk. Rats from the DHA group showed a higher endurance capacity (+56%, P < 0.05) compared with control animals. Permeabilized myofibers from soleus muscle showed higher O2 consumptions (P < 0.05) in the DHA group compared with the control group, with glutamate-malate as substrates, both in basal conditions (i.e., state 2) and under maximal conditions (i.e., state 3, using ADP), along with a higher apparent Km for ADP (P < 0.05). Calcium retention capacity of isolated mitochondria was lower in DHA group compared with the control group (P < 0.05). Phospho-AMPK/AMPK ratio and PPARδ mRNA content were higher in the DHA group compared with the control group (P < 0.05). Results showed that DHA enhanced endurance capacity in adult animals, a beneficial effect potentially resulting from improvement in mitochondrial function, as suggested by our results on permeabilized fibers. DHA supplementation could be of potential interest for the muscle function in adults and for fighting the decline in exercise tolerance with age that could imply energy-sensing pathway, as suggested by changes in phospho-AMPK/AMPK ratio.

Role of Mitochondria-Cytoskeleton Interactions in Respiration Regulation in Post-Infarct Heart Failure

The aim of this work is to study structural and functional organization of energy metabolism in an ex vivo model of myocardial ischemia-reperfusion, focusing on the role of mitochondria interactions with cytoskeletal proteins in the regulation of mitochondrial respiration and dynamics. Hearts studied were subjected to perfusion under normoxic, ischemia and ischemia-reperfusion conditions using Langendorff system.Hemodynamic measurements showed that reperfusion-dependent left ventricular diastolic dysfunction is related to increased affinity of respiration for ADP (low app. Km for ADP). This loss of ADP microcompartmentalization was associated with decreased functional coupling between oxidative phosphorylation and mitochondrial creatine kinase. Previous studies have shown that in healthy adult cardiomyocytes, characterized by high apparent Km for ADP, cytoskeletal protein β2 tubulin co-localises with mitochondria. Ischemia-reperfusion injury induces the displacement of β2 tubulin from mitochondrial position in cardiac muscle fibers with low apparent Km for ADP.We conclude that ischemia-reperfusion effects on mitochondrial respiration and dynamics could be related to the disruption of mitochondrial outer membrane porin, VDAC, interaction with cytoskeletal proteins such as β2 tubulin.

Role of mitochondria–cytoskeleton interactions in respiration regulation and mitochondrial organization in striated muscles

The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers, focusing also on the role of cytoskeletal protein tubulin βII isotype in mitochondrial metabolism and organization. By analyzing accessibility of mitochondrial ADP, using respirometry and pyruvate kinase–phosphoenolpyruvate trapping system for ADP, we show that the apparent affinity of respiration for ADP can be directly linked to the permeability of the mitochondrial outer membrane (MOM). Previous studies have shown that MOM permeability in cardiomyocytes can be regulated by VDAC interaction with cytoskeletal protein, βII tubulin. We found that in oxidative soleus skeletal muscle the high apparent Km for ADP is associated with low MOM permeability and high expression of non-polymerized βII tubulin. Very low expression of non-polymerized form of βII tubulin in glycolytic muscles is associated with high MOM permeability for adenine nucleotides (low apparent Km for ADP).

Regulation of Respiration in Permeabilized Muscle Cells: Apparent KM for ADP Shows the Mitochondrial Outer Membrane Permeability

The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers. Despite certain similarities of mitochondrial organization into Intracellular Energetic Units (ICEUs), different muscle types have distinct metabolic pattern with specific regulatory mechanisms of mitochondrial respiration. In this work we measured ADP fluxes through mitochondrial outer membrane (MOM) in permeabilized fibers oxygraphically using pyruvate kinase - phosphoenolpyruvate system for trapping ADP produced in mitochondrial creatine kinase localized behind MOM. These fluxes were high in permeabilized fibers from glycolytic muscles and very low or absent in soleus muscle fibers and cardiac myocytes. These results indicate that the permeability of voltage-dependent anion channel (VDAC) in MOM correlates with the apparent Km for ADP in regulation of respiration. In cardiac cells, the MOM permeability seems to be regulated by the interaction of VDAC with cytoskeletal protein βII tubulin. To ascertain the role of this protein in skeletal muscles we visualized the localization pattern of βII tubulin together with mitochondrial protein VDAC immunocytochemically. We used the Metabolic Control Analysis to evaluate the Flux Control Coefficients of the respiratory chain complexes (I,III, IV), ANT, ATP synthase, and MtCK in permeabilized soleus muscle fibers under conditions of respiration stimulated by exogenous ADP and by endogenous ADP produced in activated MtCK reaction.

Comparative investigation of bioenergetic properties of human colorectal and breast cancer

Today, there is only very limited information of the regulation of energy metabolism in neuroblastoma (NB) cells, although this is a prerequisite condition for development of more efficient treatment protocols. It is believed that NBs have increased rates of aerobic glycolysis and display a Warburg phenotype. The aim of the present studywas to determine if there are specific alterations of aerobic energy metabolism in NBs, or if there is an overall down regulation of oxidative phosphorylation (OXPHOS) complexes. To clarify the mechanisms of regulation of mitochondrial respiration in NBs, we determined the apparent Km value for exogenously-added ADP. We found that the Km for ADP in non-differentiated and differentiated (normal) Neuro-2a (N2A) cells have very similar values, 20.3±1.4 μM and 19.4±3.2 μM, respectively, and the maximal (in the presence of 2 mM ADP) rate of O2 consumption by differentiated N2A cells exceeds considerably (N2 times) that measured for non-differentiated cells. Our findings suggest that NB cells have, in comparison with normal differentiated neural cells, a decreased activity of OXPHOS. Metabolic control analysis (MCA) performed onN2A cells suggest that in NBs the key sites of the regulation of OXPHOS are Complex-I (Flux control coefficient, FCC=1.11), Complex-II (FCC=0.99) and IV (FCC=0.92), since FCC(s) for other mitochondrial complexes were found to be substantially lower and they have approximately equal values. In the mitochondria of differentiated N2A cells the key sites of respiratory regulation were found to be Complex-II (FCC=1.34) and IV (FCC=0.99).Moreover, our data suggest that in differentiatedNB cells the Complex-II activitymay exceed considerably that inmalignant cells. It ismost interesting that inN2Acells (like for example in breast cancer cells in situ) the sum of FCC(s) for ADP activated respiration exceeds significantly 1 normally observed in oxidative tissues and isolated mitochondria and is close to 4. This indicates the altered structure of the mitochondrial respiratory chain in N2A cells. Indeed, our results suggest that the mitochondrial respiratory chain and OXPHOS system contain large respiratory supercomplexes with direct substrate transfer inside these complexes.

Effects of training at mild exercise intensities on quadriceps muscle energy metabolism in patients with chronic obstructive pulmonary disease

To study the effects of physical training at mild intensities on skeletal muscle energy metabolism in eight patients with chronic obstructive pulmonary disease (COPD) and eight paired healthy sedentary subjects. Energy metabolism of patients and controls vastus lateralis muscle was studied before and after 3 months of cycling training at mild exercises intensities. The total amount of work accomplished was about 4059 ± 336 kJ in patients with COPD and 7531 ± 1693 kJ in control subjects. This work corresponds to a mechanical power set at 65.2 ± 7.5% of the maximum power for patients with COPD and 52 ± 3.3% of the maximum power in control group. Despite this low level of exercise intensities, we observed an improvement in mitochondrial oxidative phosphorylation through the creatine kinase system revealed by the increased apparent K(m) for ADP (from 105.5 ± 16.1 to 176.9 ± 26.5 μm, P < 0.05 in the COPD group and from 126.9 ± 16.8 to 177.7 ± 17.0, P > 0.05 in the control group). Meanwhile, maximal mechanical and metabolic power increased significantly from 83.1 ± 7.1 to 91.3 ± 7.4 Watts (P < 0.05) and from 16 ± 0.8 to 18.7 ± 0.98 mL O(2) kg(-1) min(-1) (P < 0.05) only in the COPD group. This study shows that physical training at mild intensity is able to induce comparable changes in skeletal muscles oxidative energy metabolism in patients with COPD and sedentary healthy subjects, but different changes of maximal mechanical and metabolic power.

The rate of ATP export in the extramitochondrial phase via the adenine nucleotide translocator changes in aging in mitochondria isolated from heart left ventricle of either normotensive or spontaneously hypertensive rats

To find out whether and how deficit of cellular energy supply from mitochondria to cytosol occurs in aging and hypertension, we used mitochondria isolated from 5 to 72 week-old heart left ventricle of either normotensive (WKY) or spontaneous hypertensive (SH) rats as a model system. Measurements were made of the rate of ATP appearance outside mitochondria, due to externally added ADP, as an increase of NADPH absorbance which occurs when ATP is produced in the presence of glucose, hexokinase and glucose-6-phosphate dehydrogenase. Such a rate proved to mirror the function of the adenine nucleotide translocator (ANT) rather than other processes linked to the both oxidative and substrate level phosphorylation. The changes in both Ki for atractyloside and Km for ADP suggest the occurrence of modification of the ANT conformation during aging in which the ANT Vmax was found to decrease in normotensive but to increase under spontaneously hypertension in 24 week-old rats with a subsequent decrease in both cases. ANT function, as investigated in the ADP physiological range (20–60μM), is expected to decrease in normotensive, but to increase in hypertensive rats up to 48 weeks. Later a decrease in the ATP rate of export outside mitochondria should occur in both cases.

Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle.

Assessment of mitochondrial ADP-stimulated respiratory kinetics in PmFBs (permeabilized fibre bundles) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (~20-300 μM) and tend to overestimate respiration at rest. Noting that PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. BLEB (blebbistatin), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >~250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the Km for ADP decreased ~2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (Km<100 μM), whereas blocking contraction (+BLEB) and including a phosphocreatine/creatine ratio of 2:1 to mimic the resting energetic state yielded a Km for ADP of ~1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate that the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.

Impairment of maximal aerobic power with moderate hypoxia in endurance athletes: do skeletal muscle mitochondria play a role?

This study investigates the role of central vs. peripheral factors in the limitation of maximal oxygen uptake (Vo(2max)) with moderate hypoxia [inspired fraction (Fi(O(2))) =14.5%]. Fifteen endurance-trained athletes performed maximal cycle incremental tests to assess Vo(2max), maximal cardiac output (Q(max)), and maximal arteriovenous oxygen (a-vO(2)) difference in normoxia and hypoxia. Muscle biopsies of vastus lateralis were taken 1 wk before the cycling tests to evaluate maximal muscle oxidative capacity (V(max)) and sensitivity of mitochondrial respiration to ADP (K(m)) on permeabilized muscle fibers in situ. Those athletes exhibiting the largest reduction of Vo(2max) in moderate hypoxia (Severe Loss group: -18 +/- 2%) suffered from significant reductions in Q(max) (-4 +/- 1%) and maximal a-vO(2) difference (-14 +/- 2%). Athletes who well tolerated hypoxia, as attested by a significantly smaller drop of Vo(2max) with hypoxia (Moderate Loss group: -7 +/- 1%), also display a blunted Q(max) (-9 +/- 2%) but, conversely, were able to maintain maximal a-vO(2) difference (+1 +/- 2%). Though V(max) was similar in the two experimental groups, the smallest reduction of Vo(2max) with moderate hypoxia was observed in those athletes presenting the lowest apparent K(m) for ADP in the presence of creatine (K(m+Cr)). In already-trained athletes with high muscular oxidative capacities, the qualitative, rather than quantitative, aspects of the mitochondrial function may constitute a limiting factor to aerobic ATP turnover when exercising at low Fi(O(2)), presumably through the functional coupling between the mitochondrial creatine kinase and ATP production. This study suggests a potential role for peripheral factors, including the alteration of cellular homeostasis in active muscles, in determining the tolerance to hypoxia in maximally exercising endurance-trained athletes.

Impaired ATP turnover and ADP supply depress cardiac mitochondrial respiration and elevate superoxide in nonfailing spontaneously hypertensive rat hearts

Although most attention has been focused on mitochondrial ATP production and transfer in failing hearts, less has been focused on the nonfailing hypertensive heart. Here, energetic complications are less obvious, yet they may provide insight into disease ontogeny. We studied hearts from 12-mo-old spontaneously hypertensive rats (SHR) relative to normotensive Wistar-Kyoto (WKY) rats. The ex vivo working-heart model of SHR showed reduced compliance and impaired responses to increasing preloads. High-resolution respirometry showed higher state 3 (with excess ADP) respiration in SHR left ventricle fibers with complex I substrates and maximal uncoupled respiration with complex I + complex II substrates. Respiration with ATP was depressed 15% in SHR fibers relative to WKY fibers, suggesting impaired ATP hydrolysis. This finding was consistent with a 50% depression of actomyosin ATPase activities. Superoxide production from SHR fibers was similar to that from WKY fibers respiring with ADP; however, it was increased by 15% with ATP. In addition, the apparent K(m) for ADP was 54% higher for SHR fibers, and assays conducted after ex vivo work showed a 28% depression of complex I in SHR, but not WKY, fibers. Transmission electron microscopy showed similar mitochondrial volumes but a decrease in the number of cristae in SHR mitochondria. Tissue lipid peroxidation was also 15% greater in SHR left ventricle. Overall, these data suggest that although cardiac mitochondria from nonfailing SHR hearts function marginally better than those from WKY hearts, they show dysfunction after intense work. Impaired ATP turnover in hard-working SHR hearts may starve cardiac mitochondria of ADP and elevate superoxide.

Selective Regulation of Mitochondrial Outer Membrane VDAC Permeability in situ in Permeabilized Cardiomyocytes

The aim of this study was to investigate localized restrictions of diffusion of phosphocreatine (PCr) and adenine nucleotides across mitochondrial outer membrane (MOM). Complete kinetic analysis of mitochondrial creatine kinase (MtCK) - activated respiration in situ and in vitro showed that apparent dissociation constants of MgATP from complexes with MtCK were increased by several orders of magnitude in situ system in comparison with values in vitro. No difference of apparent dissociation constant for PCr was observed. To study the selective permeability of the VDAC in MOM in situ, we measured the rates of PCr synthesis and channelling from mitochondria into medium in permeabilized cardiomyocytes. An external PEP-PK system was used to trap extramitochondrial ADP and prevent PCr utilization after activation of the MtCK system by Cr. The concentrations of the ATP stayed constant as the concentration of the PCr showed linear increase in time. The rate of mitochondrial synthesis of PCr and its diffusion into medium at 5 mM ATP was equal to 0,55 μmol/min/mg giving PCr/O ratio equal to 5,5. Thus, in permeabilized CM the permeability of VDAC for PCr was high; at the same time apparent Km for ADP and for ATP rise tenfold and 100-fold respectively from in vitro to in situ. These results show that diffusion restriction through the VDAC is selective due to the interaction of the anion channels with some of the components of cytoskeletal network. We found that beta-tubulin cDNA (beta-tubulin gene M-beta-4) is present in mouse myocardium and oxidative m. soleus (high apparent Km(ADP)) but is absent in m. extensor digitorum longus (low apparent Km(ADP)). This tubulin isoform may participate in the organization of the intracellular energetic units as the regulator of VDAC permeability in oxidative muscle cells.

Governing Respiration: Tubulin's C-Terminus Interaction with VDAC

Mitochondria have long been known to localize within and move along the tubulin-microtubule network. It was shown that tubulin binds to isolated mitochondria with high-affinity and specifically associates with the mitochondrial voltage-dependent anion channel (VDAC). We found that nanomolar concentrations of dimeric tubulin vastly increase VDAC sensitivity to voltage allowing for VDAC blockage at low transmembrane potentials. Tubulin interaction with VDAC requires the presence of anionic C-terminal tails (CTT) on the intact protein. Tubulin with proteolytically cleaved CTTs does not block the channel. Actin, also an acidic protein but lacking CTTs, does not induce VDAC blockage. Two synthetic peptides with the sequences of mammalian α and β brain tubulin CTT do not induce detectable channel closure up to micromolar concentrations. These results suggest a completely new role for dimeric tubulin and its charged CTT. We propose a model for tubulin-VDAC channel interaction in which the tubulin CTT penetrates into the channel lumen, potentially reaching through the channel and interacting with a positively charged domain of VDAC. We found that tubulin/VDAC interaction is greatly dependent on the state of VDAC phosphorylation. Remarkably, phosphorylated VDAC is more than an order of magnitude more sensitive to tubulin-induced closure than dephosphorylated VDAC.Tubulin addition to isolated mitochondria increases Km for ADP and lowers oxygen consumption, most likely by restricting flux of ADP through VDAC. Thus interaction of tubulin CTT with VDAC blocks nucleotide passage into and out of mitochondria, thereby regulating oxidative phosphorylation. Examination of the evolution of CTT sequences in mitochondria-containing cells reveals high conservation of charge and length of the tails. Considering the known conservation of VDAC folding pattern throughout mitochondria-containing eukaryotes, we propose that this interaction is widespread and ancient.

Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase

The role of ubiquitous mitochondrial creatine kinase (uMtCK) reaction in regulation of mitochondrial respiration was studied in purified preparations of rat brain synaptosomes and mitochondria. In permeabilized synaptosomes, apparent Km for exogenous ADP, Km (ADP), in regulation of respiration in situ was rather high (110 +/- 11 microM) in comparison with isolated brain mitochondria (9 +/- 1 microM). This apparent Km for ADP observed in isolated mitochondria in vitro dramatically increased to 169 +/- 52 microM after their incubation with 1 muM of dimeric tubulin showing that in rat brain, particularly in synaptosomes, mitochondrial outer membrane permeability for ADP, and ATP may be restricted by tubulin binding to voltage dependent anion channel (VDAC). On the other hand, in synaptosomes apparent Km (ADP) decreased to 25 +/- 1 microM in the presence of 20 mM creatine. To fully understand this effect of creatine on kinetics of respiration regulation, complete kinetic analysis of uMtCK reaction in isolated brain mitochondria was carried out. This showed that oxidative phosphorylation specifically altered only the dissociation constants for MgATP, by decreasing that from ternary complex MtCK.Cr.MgATP (K (a)) from 0.13 +/- 0.02 to 0.018 +/- 0.007 mM and that from binary complex MtCK.MgATP (K (ia)) from 1.1 +/- 0.29 mM to 0.17 +/- 0.07 mM. Apparent decrease of dissociation constants for MgATP reflects effective cycling of ATP and ADP between uMtCK and adenine nucleotide translocase (ANT). These results emphasize important role and various pathophysiological implications of the phosphocreatine-creatine kinase system in energy transfer in brain cells, including synaptosomes.

Different kinetics of the regulation of respiration in permeabilized cardiomyocytes and in HL-1 cardiac cells: Importance of cell structure/organization for respiration regulation

The aim of this study was to investigate the mechanism of cellular regulation of mitochondrial respiration in permeabilized cardiac cells with clearly different structural organization: (i) in isolated rat cardiomyocytes with very regular mitochondrial arrangement, (ii) in HL-1 cells from mouse heart, and (iii) in non-beating (NB HL-1 cells) without sarcomeres with irregular and dynamic filamentous mitochondrial network. We found striking differences in the kinetics of respiration regulation by exogenous ADP between these cells: the apparent Km for exogenous ADP was by more than order of magnitude (14 times) lower in the permeabilized non-beating NB HL-1 cells without sarcomeres (25 ± 4 μM) and seven times lower in normally cultured HL-1 cells (47 ± 15 μM) than in permeabilized primary cardiomyocytes (360 ± 51 μM). In the latter cells, treatment with trypsin resulted in dramatic changes in intracellular structure that were associated with 3-fold decrease in apparent Km for ADP in regulation of respiration. In contrast to permeabilized cardiomyocytes, in NB HL-1 cells creatine kinase activity was low and the endogenous ADP fluxes from MgATPases recorded spectrophotometrically by the coupled enzyme assay were not reduced after activation of mitochondrial oxidative phosphorylation by the addition of mitochondrial substrates, showing the absence of ADP channelling in the NB HL-1 cells. While in the permeabilized cardiomyocytes creatine strongly activated mitochondrial respiration even in the presence of powerful competing pyruvate kinase–phosphoenolpyruvate system, in the NB HL-1 cells the stimulatory effect of creatine was not significant. The results of this study show that in normal adult cardiomyocytes and HL-1 cells intracellular local restrictions of diffusion of adenine nucleotides and metabolic feedback regulation of respiration via phosphotransfer networks are different, most probably related to differences in structural organization of these cells.

Altered energy transfer from mitochondria to sarcoplasmic reticulum after cytoarchitectural perturbations in mice hearts

Sarcoplasmic reticulum (SR) calcium pump function requires a high local ATP/ADP ratio, which can be maintained by direct nucleotide channelling from mitochondria, and by SR-bound creatine kinase (CK)-catalysed phosphate-transfer from phosphocreatine. We hypothesized that SR calcium uptake supported by mitochondrial direct nucleotide channelling, but not bound CK, depends on the juxtaposition of these organelles. To test this, we studied a well-described model of cytoarchitectural disorganization, the muscle LIM protein (MLP)-null mouse heart. Subcellular organization was characterized using electron microscopy, and mitochondrial, SR and myofibrillar function were assessed in saponin-permeabilized fibres by measuring respiration rates and caffeine-induced tension transients. MLP-null hearts had fewer, less-tightly packed intermyofibrillar mitochondria, and more subsarcolemmal mitochondria. The apparent mitochondrial Km for ADP was significantly lower in the MLP-null heart than in control (175 +/- 15 and 270 +/- 33 microM, respectively), indicating greater ADP accessibility, although maximal respiration rate, mitochondrial content and total CK activity were unaltered. Active tension in the myofibres of MLP-null mice was 54% lower than in controls (39 +/- 3 and 18 +/- 1 mN mm(-2), respectively), consistent with cytoarchitectural disorganization. SR calcium loading in the myofibres of MLP-null mice was similar to that in control myofibres when energy support was provided via Bound CK, but approximately 36% lower than controls when energy support was provided by mitochondrial (P < 0.05). Mitochondrial support for SR calcium uptake was also specifically decreased in the desmin-null heart, which is another model of cytoarchitectural perturbation. Thus, despite normal oxidative capacity, direct nucleotide channelling to the SR was impaired in MLP deficiency, concomitant with looser mitochondrial packing and increased nucleotide accessibility to this organelle. Changes in cytoarchitecture may therefore impair subcellular energy transfer and contribute to energetic and contractile dysfunction.

Oxidative phosphorylation and its coupling to mitochondrial creatine and adenylate kinases in human gastric mucosa

Energy metabolism in gastrobiopsy specimens of the antral and corpus mucosa, treated with saponin to permeabilize the cells, was studied in patients with gastric diseases. The results show twice lower oxidative capacity in the antral mucosa than in the corpus mucosa and the relative deficiency of antral mitochondria in complex I. The mucosal cells expressed mitochondrial and cytosolic isoforms of creatine kinase and adenylate kinase (AK). Creatine (20 mM) and AMP (2 mM) markedly stimulated mitochondrial respiration in the presence of submaximal ADP or ATP concentrations, and creatine reduced apparent Km for ADP in stimulation of respiration, which indicates the functional coupling of mitochondrial kinases to oxidative phosphorylation. Addition of exogenous cytochrome c increased ADP-dependent respiration, and the large-scale cytochrome c effect (>or=20%) was associated with suppressed stimulation of respiration by creatine and AMP in the mucosal preparations. These results point to the impaired mitochondrial outer membrane, probably attributed to the pathogenic effects of Helicobacter pylori. Compared with the corpus mucosa, the antral mucosa exhibited greater sensitivity to such type of injury as the prevalence of the large-scale cytochrome c effect was twice higher among the latter specimens. Active chronic gastritis was associated with decreased respiratory capacity of the corpus mucosa but with its increase in the antral mucosa. In conclusion, human gastric mucosal cells express the mitochondrial and cytosolic isoforms of CK and AK participating in intracellular energy transfer systems. Gastric mucosa disease is associated with the altered functions of these systems and oxidative phosphorylation.

Rats selectively bred for endurance capacity exhibit enhanced mitochondrial sensitivity to creatine

Qualitative and quantitative measures of mitochondrial function, assessed in chemically permeabilized fiber bundles, were performed in rats selectively bred (generation 15) for high endurance running capacity (HCR; n=8) or low endurance running capacity (LCR; n=8). HCR rats ran 2374.6 (±79.5) m prior to exhaustion, compared to 237.8 (±12.0) m for LCR rats, during an incremental exercise treadmill test. Fiber bundles were obtained from the soleus and chemically permeabilized (50 μg/ml saponin). Respiration was measured in the absence of ADP, in the presence of a submaximally stimulating concentration of ADP (0.1 mM ADP, with and without 20 mM creatine), and in the presence of a maximally stimulating concentration of ADP (2 mM). Non-ADP stimulated and maximally ADP stimulated rates of respiration were 13% higher in HCR vs. LCR, but the difference was not statistically significant (p>0.05). Despite a similar rate of respiration in the presence of 0.1 mM ADP, HCR rats demonstrated a higher rate of respiration in the presence of 0.1 mM ADP + 20 mM creatine (33% higher vs. LCR, p<0.05). Thus, mitochondria from HCR rats exhibit enhanced mitochondrial sensitivity to creatine (i.e., ability of creatine to decrease the Km for ADP). It is proposed that increased respiratory sensitivity to ADP in the presence of creatine can effectively increase muscle sensitivity to ADP during exercise (when creatine is increased) and may, in part, be responsible for the increased running performance in HCR rats. Supported by NIH HL 64270, RR 17718, AR 40155, and AR 53219.