Effects of photobiomodulation and caffeine treatment on acute kidney injury in a hypoxic ischemic neonatal rat model.

Hypoxic ischemic encephalopathy (HIE) occurs in 2-5/1000 births, with acute kidney injury (AKI) occurring in 40%. AKI increases morbidity and mortality. Caffeine, an adenosine receptor antagonist, and photobiomodulation (PBM), working on cytochrome c oxidase, are potential treatments for AKI. To examine effects of caffeine and PBM on AKI in rats, Day 7 pups underwent a HIE intervention (Modified Rice-Vannucci model) replicating pathology observed in humans. Caffeine was administered for 3 days and/or PBM for 5 days following HIE. Weights and urine for biomarkers (NGAL, albumin, KIM-1, osteopontin) were collected prior to HIE, daily post intervention and at sacrifice. Both treatments reduced kidney injury seen on electron microscopy, but not when combined. HIE elevated urinary NGAL and albumin on Days 1-3 post-HIE, before returning to control levels. This elevation was significantly reduced by PBM or caffeine. KIM-1 was significantly elevated for 7 days post-HIE and was reduced by both treatments. Osteopontin was not altered by HIE or the treatments. Treatments, individually but not in combination, improved HIE-induced reductions in the enzymatic activity of mitochondrial complexes II-III. PBM and caffeine also improved weight gain. PBM and caffeine reduces AKI diagnosed by urinary biomarkers and confirmed by EM findings.

Identification of a ryanodine receptor in rat heart mitochondria.

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.

The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins.

Early in programmed cell death (apoptosis), mitochondrial membrane permeability increases. This is at least in part due to opening of the permeability transition (PT) pore, a multiprotein complex built up at the contact site between the inner and the outer mitochondrial membranes. The PT pore has been previously implicated in clinically relevant massive cell death induced by toxins, anoxia, reactive oxygen species, and calcium overload. Here we show that PT pore complexes reconstituted in liposomes exhibit a functional behavior comparable with that of the natural PT pore present in intact mitochondria. The PT pore complex is regulated by thiol-reactive agents, calcium, cyclophilin D ligands (cyclosporin A and a nonimmunosuppressive cyclosporin A derivative), ligands of the adenine nucleotide translocator, apoptosis-related endoproteases (caspases), and Bcl-2-like proteins. Although calcium, prooxidants, and several recombinant caspases (caspases 1, 2, 3, 4, and 6) enhance the permeability of PT pore-containing liposomes, recombinant Bcl-2 or Bcl-XL augment the resistance of the reconstituted PT pore complex to pore opening. Mutated Bcl-2 proteins that have lost their cytoprotective potential also lose their PT modulatory capacity. In conclusion, the PT pore complex may constitute a crossroad of apoptosis regulation by caspases and members of the Bcl-2 family.

Complexes between porin, hexokinase, mitochondrial creatine kinase and adenylate translocator display properties of the permeability transition pore. Implication for regulation of permeability transition by the kinases.

Complexes between hexokinase, outer membrane porin, and the adenylate translocator (ANT) were recently found to establish properties of the mitochondrial permeability transition pore in a reconstituted system. The complex was extracted by 0.5% Triton X-100 from rat brain membranes and separated by anion exchanger chromatography. The molecular weight was approximately 400 kDa suggesting tetramers of hexokinase (monomer 100kDa). By the same method a porin, creatine kinase octamer, ANT complex was isolated and reconstituted in liposomes. Vesicles containing the reconstituted complexes both retained ATP that could be used by either kinase to phosphorylate external creatine or glucose. Atractyloside inhibited this activity indicating that the ANT was involved in this process and was functionally reconstituted. Exclusively from the hexokinase complex containing liposome internal malate or ATP was released by addition of Ca2+ in a N-methylVal-4-cyclosporin sensitive way, suggesting that the hexokinase porin ANT complex might include the permeability transition pore (PTP). The Ca2+ dependent opening of the PTP-like structure was inhibited by ADP (apparent I(50), 8 microM) and ATP (apparent I(50), 84 microM). Also glucose inhibited the PTP-like activity, while glucose-6-phosphate abolished this effect. Although porin and ANT were functionally active in vesicles containing the creatine kinase octamer complex, Ca2+ did not induce a release of internal substrates. However, after dissociation of the creatine kinase octamer, the complex exhibited PTP-like properties and the vesicles liberated internal metabolites upon addition of Ca2+. The latter process was also inhibited by N-methylVal-4-cyclosporin. The activity of peptidyl-prolyl-cis-trans-isomerase (representing cyclophilin) was followed during complex isolation. Cyp D was co-purified with the hexokinase complex, while it was absent in the creatine kinase complex. The inhibitory effect of N-methylVal-4-cyclosporin on the creatine kinase complex may be explained by direct interaction with the creatine kinase dimer that appeared to support octamer formation.

The molecular structure of mitochondrial contact sites. Their role in regulation of energy metabolism and permeability transition.

Contact sites between the outer and peripheral inner membrane of mitochondria are involved in protein precursor uptake and energy transfer. Hexokinase and mitochondrial creatine kinase could be attributed by different techniques to the energy transfer contacts. Kinetic analyses suggested a functional interaction between the kinases, outer membrane pore protein, and inner membrane adenylate translocator (ANT). This suggestion was strongly supported by isolation of hexokinase and creatine kinase complexes that were constituted of kinase oligomers, porin and ANT. Phospholipid vesicles carrying reconstituted kinase-porin-ANT complexes enclosed internal ATP in contrast to vesicles containing free porin only. This indicated that unspecific transport through porin was regulated by its interaction with a specific antiporter, ANT. A direct interaction between porin and ANT in the hexokinase complex conferred the reconstituted system with permeability properties reminiscent of the mitochondrial permeability transition (PT) pore. In the creatine kinase complex this interaction between porin and ANT was replaced by contact of both with the kinase octamer. Thus PT-pore-like functions were not observed unless the creatine kinase octamer was dissociated, suggesting that the ANT was locked in the antiporter state by interaction with the octamer. Indeed, reconstituted pure ANT showed PT-pore-like properties concerning Ca2+ sensitivity. However, as cyclophilin was missing, sensitivity against cyclosporin was not observed.

The role of creatine kinase in inhibition of mitochondrial permeability transition.

Cyclosporin A sensitive swelling of mitochondria isolated from control mouse livers and from the livers of transgenic mice expressing human ubiquitous mitochondrial creatine kinase occurred in the presence of both 40 microM calcium and 5 microM atractyloside which was accompanied by a 2.5-fold increase over state 4 respiration rates. Creatine and cyclocreatine inhibited the latter only in transgenic liver mitochondria. Protein complexes isolated from detergent solubilised rat brain extracts, containing octameric mitochondrial creatine kinase, porin and the adenine nucleotide translocator, were reconstituted into malate loaded lipid vesicles. Dimerisation of creatine kinase in the complexes and exposure of the reconstituted complexes to >200 microM calcium induced a cyclosporin A sensitive malate release. No malate release occurred with complexes containing octameric creatine kinase under the same conditions.

Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore.

In vitro incubation of isolated hexokinase isozyme I or isolated dimer of mitochondrial creatine kinase with the outer mitochondrial membrane pore led to high molecular weight complexes of enzyme oligomers. Similar complexes of hexokinase and mitochondrial creatine kinase could be extracted by 0.5% Triton X-100 from homogenates of rat brain. Hexokinase and creatine kinase complexes could be separated by subsequent chromatography on DEAE anion exchanger. The molecular weight, as determined by gel-permeation chromatography, was approximately 400 kDa for both complexes. The Mr suggested tetramers of hexokinase (monomer 100 kDa) and creatine kinase (active enzyme is a dimer of 80 kDa). The composition of the complexes was further characterised by specific antibodies. Besides either hexokinase or creatine kinase molecules the complexes contained porin and adenylate translocator. It was possible to incorporate the complexes into artificial bilayer membranes and to measure conductance in 1 M KCI. The incorporating channels had a high conductance of 6 nS that was asymmetrically voltage dependent. The complexes were also reconstituted in phospholipid vesicles that were loaded with ATP. Complex containing vesicles retained ATP while vesicles reconstituted with pure porin were leaky. The internal ATP could be used by creatine kinase and hexokinase in the complex to phosphorylate external creatine or glucose. This process was inhibited by atractyloside. The hexokinase complex containing vesicles were furthermore loaded with malate or ATP that was gradually released by addition of Ca2+ between 100 and 600 microM. The liberation of malate or ATP by Ca2+ could be inhibited by N-methylVal-4-cyclosporin, suggesting that the porin translocator complex constitutes the permeability transition pore. The results show the physiological existence of kinase porin translocator complexes at the mitochondrial surface. It is assumed that such complexes between inner and outer membrane components are the molecular basis of contact sites observed by electron microscopy. Kinase complex formation may serve three regulatory functions, firstly regulation of the kinase activity, secondly stimulation of oxidative phosphorylation and thirdly regulation of the permeability transition pore.

Differential effects of creatine depletion on the regulation of enzyme activities and on creatine-stimulated mitochondrial respiration in skeletal muscle, heart, and brain.

Guanidinopropionic acid (GPA), an analogue of creatine (Cr), is known to inhibit Cr uptake by cells. The metabolic effects of chronic Cr depletion on brain, heart and soleus muscle of rats were studied. In GPA hearts and soleus muscle, total specific creatine kinase (CK) activity was decreased by approx. 40% compared to controls, whereas in brain this same activity was elevated by a factor of two. Immunoblot analysis of soleus mitochondria from GPA rats showed an approximate 4-fold increase in Mi-CK protein and a concomitant 3-fold increase in adenine nucleotide translocator (ANT) protein, when compared to control. In GPA-fed rats, the specific activities of adenylate kinase (ADK) and succinate dehydrogenase were significantly higher in brain and soleus (2-fold), but heart remained the same. However, hexokinase (HK) decreased by approx. 50% both in heart and soleus, indicating that muscle and brain follow different strategies to compensate the energy deficit caused by creatine depletion. Skinned muscle fibres from Cr-depleted soleus attained approx. only 70% maximum state 3 respiration with 0.1 M ADP in the presence of 10 mM Cr compared to 100% in control fibres. This defect in Cr stimulated respiration was also seen in isolated heart mitochondria, but was normal in those from brain. The observed deficit of Cr-stimulated respiration, the significant accumulation of Mib-CK and ANT, concomitant with the formation of Mib-CK rich intra-mitochondrial inclusions shown by electron microscopy, indicate that Mib-CK function and coupling to oxidative phosphorylation (OXPHOS), is impaired in these abnormal mitochondria. In addition, our results show tissue-specific metabolic compensations to Cr depletion.