<p>Brief episodes of ischemia and reperfusion render the myocardium more resistant to necrosis from a subsequent, otherwise lethal ischemic insult. This phenomenon is called ischemic preconditioning(IP). Today, much is known about the signalling pathways involved in IP; however, the details of the final steps leading to cardioprotection, remain elusive. Adenosine (a catabolite of ATP) plays a major role in the signalling pathways of IP. Following IP there is an unexplained discrepancy between an increased adenosine production (evidenced by increased 5’-nucleotidase activity) and the successively lower adenosine levels observed in the interstitial space. We propose that this discrepancy in adenosine production vs. availability may be due to an increased metabolic utilisation of adenosine by the IP myocardium. According to our hypothesis, IP induces/activates a metabolic pathway involving deamination of adenosine to inosine. Inosine is further catalysed (in presence of Pi) to hypoxanthine and ribose-1-phosphate. Ribose-1-phosphate can be converted to ribose-5-phosphate in a phosphoribomutase reaction. Ribose-5-phosphate is an intermediate of the hexose monophosphate pathway also operative under anaerobic conditions. Hence the ribose moiety of adenosine can be utilised to generate pyruvate and ultimately ATP (via lactate formation) n.b. without any initial ATP investment. Such cost-effective adenosine utilisation may at least partly explain the cardioprotective effect of IP. Objectives & Methods: In the current studies we investigated the role of adenosine metabolism according to the suggested metabolic pathway by addition of adenosine and inhibition of its metabolism during IP as well as by comparing tissue and interstitial levels of key energy-metabolites following different protocols of IP. Furthermore, we studied the importance of the IP protocol with regard to the number of ischemia and reperfusion cycles for the cardioprotective effect of IP. In addition, the validity of the microdialysis technique for experimental in vivo studies of myocardial energy metabolism was evaluated. For these purposes the microdialysis technique, tissue biopsies, and planimetric infarct size estimation in an open chest porcine heart-model was used. Results: Addition of adenosine via microdialysis probes enhanced the interstitial release of inosine, hypoxanthine and lactate in the myocardium of IP-subjects during prolonged ischemia. This finding did not occur in non-preconditioned subjects. Similar addition of deoxyadenosine a non-metabolizable adenosine receptor-agonist, did not evoke the same metabolic response. Purine nucleoside phosphorylase (PNP) is responsible for the conversion of inosine to hypoxanthine being a key enzyme in the above mentioned metabolic pathway. Inclusion of 8' aminoguanosine (a competitive inhibitor of PNP) decreased interstitial hypoxanthine release (as a token of PNP inhibition) and increased the release of taurine (marker of cellular injury) in the ischemic IP myocardium. Addition of inosine (a natural substrate of PNP) reverted these changes. Four IP cycles protected the heart more than one IP cycle as evidenced by morphometric and energy-metabolic data.Proportionally more hypoxanthine was found in the myocardium of IP subjects during prolonged ischemia. The ratio of tissue levels of inosine/hypoxanthine (used as an indicator of PNP activity) was significantly smaller in the IP groups. In addition, myocardial interstitial levels of energy-related metabolites (lactate, adenosine, inosine, and hypoxanthine) obtained by the microdialysis technique correlated with tissue biopsy levels of corresponding metabolites. Conclusions: IP activated a metabolic pathway favouring metabolism of exogenous adenosine to inosine, hypoxanthine and eventually lactate. Inhibition of adenosine metabolism following IP (via inhibition of PNP-activity resulted in enhanced cellular injury.</p><p>PNP-activity is proportionally higher in IP-myocardium. Metabolic utilisation of adenosine in IP-myocardium (as outlined above) may represent a costeffective way to produce ATP and at least partly explain the cardioprotective effect of IP. IP protects the myocardium in a graded fashion. Furthermore, we confirmed the validity of the microdialysis technique (in the current setting) for studying dynamic changes of myocardial energy metabolism.</p>

Brief episodes of ischemia and reperfusion render the myocardium more resistant to necrosis from a subsequent, otherwise lethal ischemic insult. This phenomenon is called ischemic preconditioning(IP). Today, much is known about the signalling pathways involved in IP; however, the details of the final steps leading to cardioprotection, remain elusive. Adenosine (a catabolite of ATP) plays a major role in the signalling pathways of IP. Following IP there is an unexplained discrepancy between an increased adenosine production (evidenced by increased 5’-nucleotidase activity) and the successively lower adenosine levels observed in the interstitial space. We propose that this discrepancy in adenosine production vs. availability may be due to an increased metabolic utilisation of adenosine by the IP myocardium. According to our hypothesis, IP induces/activates a metabolic pathway involving deamination of adenosine to inosine. Inosine is further catalysed (in presence of Pi) to hypoxanthine and ribose-1-phosphate. Ribose-1-phosphate can be converted to ribose-5-phosphate in a phosphoribomutase reaction. Ribose-5-phosphate is an intermediate of the hexose monophosphate pathway also operative under anaerobic conditions. Hence the ribose moiety of adenosine can be utilised to generate pyruvate and ultimately ATP (via lactate formation) n.b. without any initial ATP investment. Such cost-effective adenosine utilisation may at least partly explain the cardioprotective effect of IP. Objectives &amp; Methods: In the current studies we investigated the role of adenosine metabolism according to the suggested metabolic pathway by addition of adenosine and inhibition of its metabolism during IP as well as by comparing tissue and interstitial levels of key energy-metabolites following different protocols of IP. Furthermore, we studied the importance of the IP protocol with regard to the number of ischemia and reperfusion cycles for the cardioprotective effect of IP. In addition, the validity of the microdialysis technique for experimental in vivo studies of myocardial energy metabolism was evaluated. For these purposes the microdialysis technique, tissue biopsies, and planimetric infarct size estimation in an open chest porcine heart-model was used. Results: Addition of adenosine via microdialysis probes enhanced the interstitial release of inosine, hypoxanthine and lactate in the myocardium of IP-subjects during prolonged ischemia. This finding did not occur in non-preconditioned subjects. Similar addition of deoxyadenosine a non-metabolizable adenosine receptor-agonist, did not evoke the same metabolic response. Purine nucleoside phosphorylase (PNP) is responsible for the conversion of inosine to hypoxanthine being a key enzyme in the above mentioned metabolic pathway. Inclusion of 8' aminoguanosine (a competitive inhibitor of PNP) decreased interstitial hypoxanthine release (as a token of PNP inhibition) and increased the release of taurine (marker of cellular injury) in the ischemic IP myocardium. Addition of inosine (a natural substrate of PNP) reverted these changes. Four IP cycles protected the heart more than one IP cycle as evidenced by morphometric and energy-metabolic data.Proportionally more hypoxanthine was found in the myocardium of IP subjects during prolonged ischemia. The ratio of tissue levels of inosine/hypoxanthine (used as an indicator of PNP activity) was significantly smaller in the IP groups. In addition, myocardial interstitial levels of energy-related metabolites (lactate, adenosine, inosine, and hypoxanthine) obtained by the microdialysis technique correlated with tissue biopsy levels of corresponding metabolites. Conclusions: IP activated a metabolic pathway favouring metabolism of exogenous adenosine to inosine, hypoxanthine and eventually lactate. Inhibition of adenosine metabolism following IP (via inhibition of PNP-activity resulted in enhanced cellular injury. PNP-activity is proportionally higher in IP-myocardium. Metabolic utilisation of adenosine in IP-myocardium (as outlined above) may represent a costeffective way to produce ATP and at least partly explain the cardioprotective effect of IP. IP protects the myocardium in a graded fashion. Furthermore, we confirmed the validity of the microdialysis technique (in the current setting) for studying dynamic changes of myocardial energy metabolism.

The control of extracellular adenosine is crucial to the regulation of synaptic transmission and neuroprotection. Equilibrative nucleoside transporters (ENTs) are highly expressed in the hippocampus and widely accepted as critical regulators of adenosine tone. However, the mechanisms regulating the surface distribution and transport function of ENTs are largely unknown. Since ENT1 and ENT2 contain consensus sequences for phosphorylation by protein kinase CK2, and because this protein has been reported to regulate synaptic plasticity and ENT function in non-neuronal systems, the present thesis outlines the hypothesis that CK2-induced phosphorylation of ENTs is important for their cellular localization and thus the regulation of adenosine tone and synaptic transmission. Here, a functional interaction between adenosine CK2, ENTs and AMPA receptors in the hippocampus is reported. Western blot analysis shows that a variety of CK2 inhibitors (DMAT, TBB and DRB) significantly reduced the density of ENT1 and ENT2 proteins in hippocampal membrane fractions, suggesting that CK2-mediated phosphorylation of ENTs promotes their surface localization. In contrast, it was found that the ENT1 inhibitor NBTI significantly increased in the membrane localization of ENT1, relative to the control. Moreover, ENTs were found to immunoprecipitate with GluR1 and GluR2-containing AMPA receptors; and CK2 inhibitors caused a decrease in the membrane localization of GluR2 and GluR1 AMPA receptors. These results suggest a novel signaling complex linking CK2-regulated adenosine transport to AMPA receptor trafficking in the rat hippocampus. Although the physiological significance of these findings requires further investigation, this thesis provides insight into an adenosine regulation pathway that may be important for the regulation of synaptic transmission and neuroprotection in the rat hippocampus.

Adenosine deaminase has been shown to consist of three molecular weight forms, A, B, and C. In higher mammals, the A and C forms are dominant while in lower mammals, the B and C forms are dominant. In this work, the B and C forms were isolated from the frog liver and several kinetic parameters were determined.Ammonium sulfate salt fractionation, starting at 40 percent and increased by 5 percent increments to 80 percent was used to separate the two forms. The B form adenosine deaminase was predominantly found in the 50 to 60 percent precipitate fractions while the C form was predominant in precipitate fractions containing more than 60 percent ammonium sulfate. The rechromatographed B and C forms were subjected to isoelectric focusing and thin layer electrophoresis. The B form separated into three activity bands while the C form separated into two activity bands`. Michaelis constant values were determined to be 4.61 X 10-5M and 2.00 X 10-5M for the B and C forms with adenosine as a substrate, respectively. The relative substrate specificity ratio showed that the B form was very specific for adenosine.In conclusion, the B form adenosine deaminase was found to be dominant in the frog liver. The Michaelis constant, relative substrate specificity ratio, thin layer electrophoresis and isoelectric focusing distinguished between the adenosine deaminase B and C forms. The technique of ammonium sulfate fractionation gave excellent separation between the B and C forms of adenosine deaminase.Ball State UniversityMuncie, IN 47306

L' ATP es una molecula clau en el metabolisme cel.lular, actuant com a donador d'energia lliure i acoplant reaccions endergoniques i exergoniques. L'ATP es sintetitzat a la mitocondria en un proces anomenat fosforil.lacio oxidativa despres d'una serie de reaccions a la cadena de citocroms que es troba en la membrana interna de l'organel.la. La font d'energia necessaria per aquesta sintesi s'obte en les cel.lules animals dels nutrients de l'ingestio, i de la llum solar en les plantes. Existeix una via alternativa de sintesi d'ATP extramitocondrial, la glicolisi, que s'inicia amb la fosforilacio de la glucosa per l'enzim hexokinasa.Interaccio llum-materiaL'energia electromagnetica l'ona de la qual oscil.la en una longitud d'ona de nanometres es anomenada llum. En aquestes frequencies, l'energia promou excitacio electronica de certs atoms i molecules. Existeix una interaccio diferent de la llum amb molecules, produida per el camp electromagnetic que per definicio la llum provoca en qualsevol medi. El camp electric resultant desplaca els electrons dels enllacos quimics produint una polaritzacio del medi sense que existeixi absorcio de l'energia. Aquest es un mecanisme interactiu que existeix sempre, i es l'unic que es dona en molecules que son transparents (no absorbeixen) per a una determinada frequencia de la llum, com es el cas de l'ATP per l'energia visible i infravermella propera. Experiments, resultats i discussioQuan l'ATP es excitat amb fotons ultraviolats, es produiex una fluorescencia en longituts d'ona visibles. L'io magnesi s'ha utilitzat per estudiar com la llum visible i infravermella propera produiex un desplacament de carregues electriques a la molecula d'ATP. La construccio d'un interferometre de Michelson ha servit per observar l'interaccio no absortiva de la llum i l'ATP. La mesura directa de l'index de refraccio d'una solucio d'ATP dona informacio sobre les caracteristiques electriques del medi. L'observacio de que aquest index canvia despres d'irradiar la solucio amb longituds d'ona visibles i infravermelles properes, confirmen que la llum provoca canvis electrics significatius en l'ATP. En aquest treball tambe s'ha estudiat el comportament bioquimic de l'ATP irradiat quant forma part de dues reaccions quimiques diferents: la de la luciferina-luciferasa i la de la hexoquinasa. En tots dos casos, l'us d'aquest ATP irradiat ha produit una alteracio dels parametres cinetics estudiats, V0 i k en la reaccio de la luciferina-luciferasa, i km i vmax en la reaccio de l'hexoquinasa.ConclusionsAquesta interaccio no absortiva de la llum amb l'ATP es la primera descrita per a una biomolecula. El mecanisme aporta noves dades per explicar els efectes observats en el metabolisme cel.lular despres de l'irradiacio d'organismes, teixits i cultius cel.lulars amb llum visible i infravermella propera. / ATP is a key molecule in cellular metabolism. In this thesis, I examined the effects of visible (635 and 655 nm) and near-infrared (810 and 830 nm) light on ATP in solution. I also examined were the biochemical behavior of light-exposed ATP in the luciferine-luciferase reaction and hexokinase reaction, the initial step in glycolysis that begins extra mithocondrial ATP synthesis. Irradiated groups in the luciferine-luciferase reaction showed an improvement in the kinetic parameters V0 and k, and more ATP molecules reacted with the enzyme when they were excited by light. When irradiated ATP was added to the hexokinase reaction, the experimental groups showed significant differences in the Michaelis-Menten kinetic parameters (km for ATP and vmax) and the rate of product synthesis was greater. Changes in both reactions were wavelength and dose dependant. When ATP was excited with UV photons, it fluoresced. This fluorescence decreased when Mg2+ was added, probably because the ion binds the phosphates, which are the part of the molecule responsible for light emission. Irradiating the ATP-Mg2+ solution with 655 nm and 830 nm light increased the fluorescence resulting from a displacement of charges in the phosphor-oxygen bond that repels Mg2+. The refraction of light in an ATP solution was observed by the Michelson interferometer and by directly measuring the refractive index. The refractive index changed after red and near-infrared light interaction due to a change in the electrical permittivity of the medium. Since ATP in water is transparent to visible and near-infrared light, and is therefore not a chromophore for those wavelengths, I conclude that the observed light interaction with ATP is not due to photon absorption but to the electromagnetic disturbance produced by the light, which leads to a polarization of the dielectric molecule that is ATP. This interaction of visible and near-infrared electromagnetic energy with ATP offers new perspectives for explaining light interaction at subcellular level.

The control of extracellular adenosine is crucial to the regulation of synaptic transmission and neuroprotection. Equilibrative nucleoside transporters (ENTs) are highly expressed in the hippocampus and widely accepted as critical regulators of adenosine tone. However, the mechanisms regulating the surface distribution and transport function of ENTs are largely unknown. Since ENT1 and ENT2 contain consensus sequences for phosphorylation by protein kinase CK2, and because this protein has been reported to regulate synaptic plasticity and ENT function in non-neuronal systems, the present thesis outlines the hypothesis that CK2-induced phosphorylation of ENTs is important for their cellular localization and thus the regulation of adenosine tone and synaptic transmission. Here, a functional interaction between adenosine CK2, ENTs and AMPA receptors in the hippocampus is reported. Western blot analysis shows that a variety of CK2 inhibitors (DMAT, TBB and DRB) significantly reduced the density of ENT1 and ENT2 proteins in hippocampal membrane fractions, suggesting that CK2-mediated phosphorylation of ENTs promotes their surface localization. In contrast, it was found that the ENT1 inhibitor NBTI significantly increased in the membrane localization of ENT1, relative to the control. Moreover, ENTs were found to immunoprecipitate with GluR1 and GluR2-containing AMPA receptors; and CK2 inhibitors caused a decrease in the membrane localization of GluR2 and GluR1 AMPA receptors. These results suggest a novel signaling complex linking CK2-regulated adenosine transport to AMPA receptor trafficking in the rat hippocampus. Although the physiological significance of these findings requires further investigation, this thesis provides insight into an adenosine regulation pathway that may be important for the regulation of synaptic transmission and neuroprotection in the rat hippocampus.

Platelet transfusions can be necessary during treatment of patients with thrombocytopenia or impaired platelet function. Platelet function in platelet concentrates (PC) deteriorate with storage time. Studying swirling is often used to control the quality of PC’s before transfusion but the method has some disadvantages. Therefore other methods can be useful, for example impedance aggregometry (IA, Multiplate® Analyzer) to measure platelet function.      In this study the change in platelet function over time was examined in buffy coat and apheresis platelets with IA where aggregation had been induced with adenosine diphosphate (ADP) and collagen. PC’s were tested on day 1, 4 and 7 after donation. One of the main aims of this study was to evaluate if dilution to a standardized platelet concentration (800x109 platelets/L) for IA of PC’s could be used, since platelet concentration has been shown to influence aggregation. The effect of pathogen inactivation (INTERCEPT) on platelet function and the importance of fibrinogen for aggregation were also studied.      The dilution of platelet samples reduced the range of measured values and was suitable to use with collagen but not ADP. The platelet function decreased significantly over time with both agonists. There was a significant difference between pathogen inactivated and gamma irradiated PC’s with collagen activation on day 1. Fibrinogen was shown to be of importance for platelet aggregation, but other factors in plasma seem to be necessary too.      In conclusion, IA is a suitable method for following change in aggregability over time in PC’s and sample dilution reduced variation in results.

published_or_final_version / Physiology / Master / Master of Medical Sciences

Lactic acid, which induces pH depression, leads to ATP efflux from muscle to extracellular space: it was reported that CFTR was involved in this process. However, the mechanism by which lactic acid activated CFTR and brought about the ATP release is still unknown. This study was performed to investigate (1) what channels may be involved or even conduct ATP release, and (2) how lactic acid activated CFTR. Expression of the possible channels that may conduct ATP release in L6 cells was investigated using RT-PCR: ClC-2, ClC-3, ClC-7, CACC, VDAC, connexin 40, connexin 43 and pannexin 3 were expressed in L6. Incubation of cultured L6 cells with lactic acid (10 mM) increased the extracellular ATP from 0.6 ± 0.06 to 1.1 ± 0.09 nM (P ? 0.05), indicating that lactic acid stimulated ATP efflux in vitro. The non-specific chloride channel inhibitor, DIDS, failed to abolish the lactic-acid-induced ATP release, suggesting that DIDS-sensitive chloride channels were not involved in the ATP efflux. Among the non-specific inhibitors of connexin channels, gadolinium inhibited acidosis-induced ATP efflux, but carbenoxolone failed to inhibit it, and so the role of connexins remains uncertain. The specific inhibitor of CFTR, CFTRinh-172, and the non-specific open-channel blocker of CFTR, glibenclamide, both abolished the acidosis-induced ATP release, but another specific inhibitor of CFTR, GlyH-101, which blocks CFTR from the external side, failed to abolish the ATP release, suggesting that acidosis-induced ATP is dependent on CFTR-activation, but does not involve ATP moving through the CFTR chloride channel. We hypothesize that, at low pH, the Na+/H+ exchanger (NHX) extruded H+ out of the cell and the resulting intracellular Na+ was transported out by Ca2+/Na+ exchanger (NCX); the localized increase in Ca2+ activated adenyl cyclase (AC), thus elevating intracellular cAMP; cAMP-activated-PKA then phosphorylated CFTR, which regulated an ATP release channel. KT-5720, an inhibitor of PKA, abolished the acidosis-induced ATP release, and forskolin, an agent that elevates cAMP, stimulated it, suggesting that the cAMP/PKA pathway was involved. The specific inhibitor of NCX, SN-6 and KB-R7943, both abolished the acidosis-induced ATP release, supporting a role for NCX in mediating this process. However, amiloride, the non-specific inhibitor of NHX failed to abolish ATP efflux. The whole cell Cl- currents were studied in L6 cells: lactic acid increased the whole cell currents from 2.33 ± 0.10 to 3.54 ± 0.34 nA (P ? 0.05), and this lactic-acid-induced increase in Cl- current could be inhibited by CFTRinh-172, suggesting that the CFTR Cl- channel was opened at low pH. Moreover, forskolin increased whole cell Cl- currents, which supported a role for the cAMP/PKA pathway in the lactic-acid-induced increase in CFTR current. These data confirm that CFTR is involved in the lactic-acid-induced ATP release from L6 cells. The roles of the NCX and cAMP/PKA pathway in activating CFTR at low pH are supported, but further studies are required to determine whether the NHX is involved in CFTR activation and whether connexins participate in ATP release. / published_or_final_version / Physiology / Master / Master of Philosophy

Contracting skeletal muscle releases ATP into the interstitial space where it is subsequently broken down to adenosine by the action of ecto-5’-nucleotidase. Both ATP and adenosine are vasodilators that contribute to the exercise hyperaemia. However, the mechanism for the release of ATP from muscle during exercise remains unknown. Cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from muscle at low intracellular pH: this study was performed to investigate whether CFTR was involved in the ATP release from skeletal muscle during contractions. Experiments were performed in rats anaesthetised with sodium pentobarbitone and breathing spontaneously. A microdialysis probe was placed in one gastrocnemius muscle: ATP was determined in interstitial microdialysate samples using a bioluminescence assay. The sciatic nerve was stimulated to induce two bouts of muscle contractions, separated by a recovery period of 40 mins; one of the inhibitors was administered prior to the second bout of contractions. Muscle contractions elevated the interstitial ATP by 1500 to 3000%. In the control experiments, no drug was given: both the contractile force and the increase in interstitial ATP were reproducible in repeated contraction bouts. Infusion of a specific inhibitor of CFTR, CFTRinh-172, did not alter the contractile force, but significantly lowered the interstitial ATP during muscle contractions, suggesting that CFTR was involved in the contraction-induced ATP release. Similarly, infusion of the Protein Kinase A inhibitor, KT5720, significantly reduced interstitial ATP during muscle contractions without altering contractile force, suggesting that CFTR in skeletal muscle is activated through the cAMP/PKA pathway. The increase in interstitial ATP during muscle contraction was also inhibited by the Na/H exchanger inhibitor, amiloride, or the Na/Ca exchanger inhibitor, SN6. It has been also shown that two gap junction hemichannel inhibitors, gadolinium and carbenoxolone, could attenuate the increase of ATP during muscle contraction. These data suggest that CFTR, activated through the cAMP/protein kinase A pathway, is involved in the ATP release during muscle contraction, and that activation of the Na/H exchanger and Na/Ca exchanger was also required, indicating that the signal transduction mechanism for CFTR activation during muscle contractions may be similar to that which is reported to occur at low pH. The preliminary data showed that the gap junction hemichannels might mediate the ATP release from skeletal muscle cells during muscle contraction. / published_or_final_version / Physiology / Master / Master of Philosophy