京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20663号 / 医博第4273号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤渕 航, 教授 木村 剛, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Muscle fatigue is an intrinsic myoprotective process that prevents damaging ATP depletion during intense or prolonged exercise by limiting ATP demand when ATP production becomes insufficient. One mechanism of fatigue involves a reduction in membrane excitability with the opening of ATP-sensitive K+ (KATP) and ClC-1 Cl- channels, resulting in submaximal sarcoplasmic reticulum Ca2+ release and reduced force generation, but the intracellular signalling pathways for this process is unknown. As a first step toward understanding this process, the objective of this study was to test the hypothesis that adenosine receptors (ARs) and AMPK trigger fatigue when a metabolic stress occurs during muscular activity. Compared to control conditions, a pan-activation of ARs with 10 µM adenosine and NECA initially reduced the fatigue rate during the first 60 s of a 3 min fatigue bout triggered with 1 tetanic contraction every s. An activation of the A1 adenosine receptor (A1R) with 10 and 20 µM ENBA resulted in faster rate of fatigue; an effect blocked by 5 µM DPCPX, an A1R antagonist. At 10 and 20 µM, adenosine, NECA, and ENBA activated AMPK via an increased in T172 phosphorylation. At 10 µM, MK8722, an AMPK agonist, initially caused a reduction in fatigue rate during the first 60 s followed by an increased fatigue rate during the last 2 min of the fatigue bout. Co-activation of ARs and AMPK did not give rise to either an additive or synergistic effect. FDB from AMPK α1-/- and α2-/- mice had faster fatigue rate and greater increased in unstimulated force compared to FDB from AMPK α1+/+ and α2+/+ mice. It is suggested that ARs and AMPK play a role in the mechanism of fatigue when a metabolic stress develops during muscle activity.

This work examines the synthesis of poly(ADP-ribose) in normal and SV40-transformed monolayer cultures of 3T3 cells as a function of growth and DNA fragmentation. A review of the relevant literature is given in the introduction of this work. Poly(ADP-ribose) synthesis has been implicated in transcription, replication, repair, differentiation and regulation of cell growth. The results of this study suggest that poly(ADP-ribose) synthesis is involved in some aspect of cell-growth control and DNA repair.

Objective: The aim was to discriminate among several hypotheses of preconditioning of isolated rabbit cardiomyocytes and to determine if ischaemic preincubation would evoke a protective response. Methods: Isolated myocytes were subjected to 5 min of preincubation, in the presence or absence of glucose, and incubated in the presence of 1 mM iodoacetic acid during the final sustained ischaemic period. In a second series, the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA), ingenol 3, 20-dibenzoate, and thymeleatoxin were added during preincubation. In a third series, preincubation periods were substituted by brief ischaemic pelleting of cells. Final prolonged ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury was determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 5 min glucose-free preincubation. Addition of iodoacetic acid into the final ischaemic pellet increased the rates of rigor contracture and injury, but did not abolish the protective response. Direct protein kinase C activation with PMA, a non-selective phorbol ester, and ingenol, an ε, δ-PKC isozyme selective activator, protected cells, but thymeleatoxin, an α,β,γ-PKC isozyme selective activator, did not. A 10 min ischaemic preincubation preconditioned, but the protection was not enhanced when ischaemia was extended to 30 min, or when PMA was included during the initial ischaemic preincubation. Adenosine partially inhibited the response. Conclusions: (1) Preconditioning of isolated myocytes is not dependent on glycolysis or glucose transport. (2) Preconditioning appears dependent on activation of the ε-PKC isoformn. (3) Ischaemia is capable of preconditioning isolated myocytes in vitro, and initiation of this effect is modified by simultaneous additional of adenosine but not by direct protein kinase C activation with PMA. Induction of protection by PMA and ingenol shows that protection requires protein kinase C activation, but direct potassium channel activation by regulatory G proteins is not critical.Cardiovascular Research 1994;28:1700-1706.

Cardiomyocytes isolated from rabbit hearts were preconditioned in vitro by 10 min of ischemia or treatment with 100 μM adenosine. Protection was assessed as average integrated mortality following osmotic swelling and determination of viability by trypan blue exclusion over 60-180 min ischemia. Repetitive submaximal stimulations with 1 μM adenosine amplified the protective response. Treatment with adenosine only at the onset of prolonged ischemia afforded a dose-dependent protection. The PKC inhibitor calphostin C (500 nM) blocked preconditioning and, when added during ischemic incubation of non-preconditioned cells, significantly increased injury. The memory of adenosine-induced preconditioning decayed over a 60 min post-incubation period. Light activation of calphostin C initially added to preconditioned ischemic cells in the dark indicated that a 10 min period of PKC activity at the onset of ischemia affords full protection. The reversible PKC inhibitors chelerythrine (5 μM) or staurosporine (100 nM) added only to bracket induction of ischemia, reduced but did not abolish protection. Protection was abolished when either drug was present during induction and a subsequent 30 min post-incubation period. Staurosporine included during initiation and post-incubation but washed out in the final 5 min of post-incubation allowed significant protection to occur. It is concluded that a single adenosine receptor-stimulation induces protection as it preconditions, and PKC activity appears to be required for both induction and protection. Memory may reside in post-receptor amplification of an initial protective response.

Background: Adenosine A2a receptors form a mutually inhibitory heteromeric complex with dopamine D2 receptors such that each receptor exhibits lower sensitivity to its agonist after the opposing receptor agonist is bound. This study analyzed the effects of CGS 21680, an adenosine A2A agonist, on nicotine conditioned place preference (CPP) in adolescence using a rodent model of schizophrenia (SZ). Methods: Rats were treated from postnatal day (P) 1 to P21 with saline or the dopamine D2/D3 agonist quinpirole (NQ treatment) and raised to P41. After an initial preference test, rats were conditioned with saline or nicotine (0.6 mg/kg base) from P43 to P51. CGS 21680 (0.03 or 0.09 mg/kg) was given 15 minutes before nicotine was administered. The post-conditioning test was administered on P52. On P53, the nucleus accumbens (NAcc) was analyzed for brain-derived neurotrophic factor (BDNF) and glial cell-lined neurotrophic factor (GDNF). Results: Results revealed that NQ treatment enhanced nicotine CPP, and both doses of CGS 21680 alleviated this enhancement. Nicotine also resulted in a CPP in controls, which was alleviated by both doses of CGS 21680. BDNF closely followed the behavioral results: CGS 21680 alleviated the enhancement in NAcc BDNF in NQ-treated animals, and eliminated the increase in NAcc BDNF produced by nicotine in controls. NQ-treated animals conditioned to nicotine resulted in an increase of NAcc GDNF, but this was eliminated by CGS 21680. Both BDNF and GDNF correlated with CPP performance. Conclusions: Results revealed that an adenosine A2A agonist decreased the rewarding aspects of nicotine and its accompanying neural plasticity changes in a model of SZ.

Schizophrenia (SZ) is a neurological disorder that presents with paranoia, hallucinations, and negative affect. A neurobiological hallmark of SZ is increased dopamine D2 receptor sensitivity. Antipsychotics that treat SZ have demonstrated limited efficacy as well as harmful and sometimes fatal side effects. Additionally, nicotine abuse is greatly increased in individuals diagnosed with SZ compared to the normal population. Treatment of this comorbidity is important, because cigarette smoking diminishes the quality of life in individuals with SZ and shortens their lifespan. The adenosine system is a potential novel target for SZ treatment. Adenosine A2a receptors form a heteromeric complex with dopamine D2 receptors that is mutually inhibitory, meaning each receptor exhibits lower sensitivity to its agonist after the opposing receptor agonist is bound. This study investigated the efficacy of an adenosine A2a agonist, CGS 21680, in alleviating the rewarding aspects of nicotine in the neonatal quinpirole rodent model of SZ. Rats were treated neonatally from postnatal (P)day 1 through 21 with the dopamine D2/D3 agonist quinpirole, raised to P41, and tested on conditioned place preference (CPP). CPP is a behavioral measure of the rewarding properties of a drug through temporal pairing of a drug with a distinct environmental context. Rats were given a drug free pre-conditioning preference test, and then conditioned to saline or nicotine (0.6 mg/kg base) from P43-51. Groups receiving CGS 21680 (0.03 or 0.09 mg/kg) were given the agonist 15 min before nicotine was administered. A drug-free post-conditioning test was administered on P52 to determine preference. The following day, brain tissue was analyzed for brain-derived neurotrophic factor (BDNF) and glial cell-lined neurotrophic factor (GDNF). BDNF is a ubiquitous neurotrophic factor involved in synaptic growth throughout the brain, whereas GDNF is important in dopamine plasticity. Results revealed that neonatal quinpirole enhanced nicotine CPP, replicating previous work, and both doses of CGS 21680 alleviated this enhancement. Nicotine resulted in a CPP in controls, and both doses of CGS 21680 also alleviated this preference. Therefore, CGS 21680 alleviated the rewarding aspects of nicotine. BDNF analyses in the nucleus accumbens (Nacc), a brain area that mediates drug reward, revealed that BDNF closely followed the behavioral results: CGS 21680 alleviated the enhancement in Nacc BDNF in neonatal quinpirole-treated animals, and eliminated the increase in Nacc BDNF produced by nicotine in controls. GDNF analyses revealed that neonatal quinpirole animals conditioned to nicotine resulted in an increase of GDNF in the NAacc, but this was eliminated by CGS 21680. This project revealed that an adenosine agonist with antispsychotic properties may have utility towards decreasing the rewarding aspects of nicotine and its accompanying neural plasticity changes in a model of SZ.

Spinal tuberculosis (TB) is associated with serious neurologic morbidity. It commonly presents as back pain, with or without systemic symptoms. Magnetic resonance imaging (MRI) is the most sensitive and specific imaging modality for spinal TB. The diagnosis of spinal TB is made with tissue biopsy and acid-fast bacilli (AFB) culture; however, tissue AFB smear and tissue TB deoxyribonucleic acid (DNA) polymerase chain reaction (PCR) can influence early clinical decision making. Ancillary tests such as the purified protein derivative (PPD) skin test, QuantiFERON®-TB Gold (QFT) or pleural adenosine deaminase (ADA) can be used in conjunction with radiology and clinical findings to initiate treatment while AFB tissue cultures are pending. Spinal TB responds well to early medical management and surgery is reserved for cases with neurologic complications.

This study presented evidence for the existence of a membrane-bound, sodium-activated ATPase which does not require potassium ions for its activity. Furthermore, the data suggest the presence of a soluble factor which activates this enzyme and may play a regulatory role in sodium transport.

Despite advancements in percutaneous coronary intervention, stents are still limited by a 2% annual rate of in-stent restenosis (ISR) related to neointimal (NI) tissue proliferation. Efforts to prevent ISR formation remain the focus of ongoing work. Adenosine (ADO) is a purine nucleoside with integral roles in vascular homeostasis, though it has limited clinical application. ADO signals primarily via four receptors with ADO receptor-A2B (ADOR-A2B) considered to play an integral role in vascular healing. Dipyridamole (DP) is a commercially approved therapy known to improve vascular events and modulate adenosine biology. Our objectives with this study included (i) assessing whether ADO could serve as a biomarker of cardiac events; (ii) determine if DP could mitigate NI formation in a pre-clinical stent model; and, (iii) quantify the mechanisms of DP-related vasculoprotection, specifically related to ADOR-A2B. We assessed the analytic and biologic variability of circulating ADO levels in humans and demonstrated that circulating ADO was not predictive of cardiac events at one year following invasive coronary angiography. We then assessed whether modulation of adenosine biology with DP had therapeutic efficacy in a pre-clinical model. Utilizing meta-analysis, we confirmed the sustained effects of DP on vascular patency rates in both pre-clinical and clinical studies. We refined a pre-clinical rabbit model of stent implantation with assessment of stent healing by intravascular optical coherence tomography – with excellent translation to clinical observations. We then assessed DP in a pre-clinical model, demonstrating reduction in ISR and improved stent healing with DP compared to control. Last, we sought to elucidate the mechanisms behind the observed DP effects, specifically related to ADOR-A2B. In vivo, DP therapy demonstrated reduced NI smooth muscle cell (SMC) content. In vitro assessment of DP demonstrated dose-dependent inhibition of SMC proliferation and migration with alteration of SMC phenotypic switching, while selective modulation of ADOR-A2B and ADOR-A2B knockdown support an ADOR-A2B-mediated component to the observed DP effects. Adenosine biology is integral to vascular homeostasis. In humans, circulating adenosine levels in humans are not predictive of one year cardiovascular events. However, DP may improve vascular healing post stent implantation and warrants clinical evaluation for stent healing. The observed DP benefits may, in part, stem from ADOR-A2B modulation. ADOR-A2B is a viable target for assessment of small molecule modulation as a novel therapeutic target to improve vascular outcomes.