RESUMO
BACKGROUND: Dopamine (DA) signalling in the brain is necessary for feeding behaviour, and alterations in the DA system have been linked to obesity. However, the precise role of DA in the control of food intake remains debated. On the one hand, food reward and motivation are associated with enhanced DA activity. On the other hand, psychostimulant drugs that increase DA signalling suppress food intake. This poses the questions of how endogenous DA neuronal activity regulates feeding, and whether enhancing DA neuronal activity would either promote or reduce food intake. METHODS: Here, we used designer receptors exclusively activated by designer drugs (DREADD) technology to determine the effects of enhancing DA neuronal activity on feeding behaviour. We chemogenetically activated selective midbrain DA neuronal subpopulations and assessed the effects on feeding microstructure in rats. RESULTS: Treatment with the psychostimulant drug amphetamine or the selective DA reuptake inhibitor GBR 12909 significantly suppressed food intake. Selective chemogenetic activation of DA neurons in the ventral tegmental area (VTA) was found to reduce meal size, but had less impact on total food intake. Targeting distinct VTA neuronal pathways revealed that specific activation of the mesolimbic pathway towards nucleus accumbens (NAc) resulted in smaller and shorter meals. In addition, the meal frequency was increased, rendering total food intake unaffected. The disrupted feeding patterns following activation of VTA DA neurons or VTA to NAc projection neurons were accompanied by locomotor hyperactivity. Activation of VTA neurons projecting towards prefrontal cortex or amygdala, or of DA neurons in the substantia nigra, did not affect feeding behaviour. CONCLUSIONS: Chemogenetic activation of VTA DA neurons or VTA to NAc pathway disrupts feeding patterns. Increased activity of mesolimbic DA neurons appears to both promote and reduce food intake, by facilitating both the initiation and cessation of feeding behaviour.
Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/fisiologia , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologia , Mesencéfalo/citologia , Mesencéfalo/fisiologia , Anfetamina/farmacologia , Animais , Modelos Animais de Doenças , Inibidores da Captação de Dopamina/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Masculino , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Piperazinas/farmacologia , Ratos , RecompensaRESUMO
Following the first point-prevalence study in Dutch nursing homes conducted each November from 2007 to 2009, we conducted a follow-up point-prevalence study of healthcare-associated infections (HCAIs) each November from 2010 to 2017. Similar methods and criteria were used. Resident characteristics were recorded, data collection was performed by the attending elderly care physicians via an online survey, as well as via a specifically designed App from 2012. As of the same year, information on incontinence was added. Between 2010 until 2017 on average 1786 residents per year were included, ranging from 1571 to 2185. HCAI prevalence with respect to age (mean: 83 years) and sex (31% men and 69% women) were similar over all the years. The overall mean prevalence rate in the first four years was 6.7% versus 2.2% in the last six years. Urinary tract infection was the most prevalent HCAI (1.5%). Most HCAIs occurred among residents of rehabilitation units. The prevalence of HCAI varied by nursing home (0.0-37.0%). The average use of antibiotics was stable over the years (6.0%) irrespective of HCAI rate. Use of incontinence materials was on average 73.5% with 64.3% of residents being reported as incontinent. Those implementing improvement of infection control and surveillance within a new setting do need to continue for multiple years before seeing the success of their endeavour.
Assuntos
Infecção Hospitalar/epidemiologia , Casas de Saúde , Fatores Etários , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Estudos Transversais , Uso de Medicamentos , Feminino , Seguimentos , Humanos , Masculino , Países Baixos/epidemiologia , Fatores SexuaisRESUMO
OBJECTIVE: Cardiac energetics and performance depend on the expression level of the fast (alpha-) and slow (beta-) myosin heavy chain (MHC) isoform. In ventricular tissue, the beta-MHC isoform predominates, whereas in atrial tissue a variable mixture of alpha- and beta-MHC is found. In several cardiac diseases, the slow isoform is upregulated; however, the functional implications of this transition in human myocardium are largely unknown. The aim of this study was to determine the relation between contractile properties and MHC isoform composition in healthy human myocardium using the diversity in atrial tissue. METHODS: Isometric force production and ATP consumption were measured in chemically skinned atrial trabeculae and ventricular muscle strips, and rate of force redevelopment was studied using single cardiomyocytes. MHC isoform composition was determined by one-dimensional SDS-gel electrophoresis. RESULTS: Force development in ventricular tissue was about 5-fold more economical, but nine times slower, than in atrial tissue. Significant linear correlations were found between MHC isoform composition, ATP consumption and rate of force redevelopment. CONCLUSION: These results clearly indicate that even a minor shift in MHC isoform expression has considerable impact on cardiac performance in human tissue.
Assuntos
Trifosfato de Adenosina/metabolismo , Função Atrial/fisiologia , Miocárdio/metabolismo , Função Ventricular/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fenômenos Biomecânicos , Eletroforese em Gel de Poliacrilamida , Átrios do Coração , Ventrículos do Coração , Humanos , Pessoa de Meia-Idade , Contração Miocárdica/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
Following our previous results which demonstrated that repeated short periods (2 min) of ischemia are capable of protecting the isolated rat heart from a subsequent global ischemia (30 min), in the present study we have concentrated on the metabolic changes occurring in rat hearts during six 2 min ischemia/3 min reperfusion cycles. Cardiac high-energy phosphates were monitored using 31P-NMR. Phosphocreatine levels fell (50-60%) during each ischemic period, and recovered to 70-80% of their initial values during reperfusion. P(i) rose by 59% during the first ischemic period, but increased less during subsequent ischemias (30% during the 6th occlusion, P < 0.05 vs. the first ischemic period) returning to baseline levels after each reperfusion. [ATP], pH, and [Mg2+] remained almost unaffected, but there was a decrease in HPLC-determined effluent ATP catabolites. The first occlusion led to a 95% drop in contractile function (P < 0.001 vs. baseline), but this recovered to 73% upon reperfusion (P < 0.02 vs. baseline), and was 65% at the end of the protocol. Phosphorylation potential (PP = [ATP]/([ADP].[P(i)]) correlated exponentially with total purine (r = 0.90) and with adenosine + inosine release (r = 0.81), and by the 6th ischemia/reperfusion cycle, exceeded that observed in controls by 21% (P < 0.05). We conclude that repeated short periods of ischemia do not lead to any significant alteration in the absolute myocardial ATP, but are associated with an enhanced cytosolic energy state in the heart, that enables the myocardium to reach a steady albeit lower functional state. Adenosine (+inosine) release may be involved in the regulation of the energy supply-demand balance.
Assuntos
Metabolismo Energético , Coração/fisiologia , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Organofosfatos/metabolismo , Adaptação Fisiológica , Animais , Estudos de Avaliação como Assunto , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Masculino , Contração Miocárdica , Reperfusão Miocárdica , Periodicidade , Fosfatos/análise , Isótopos de Fósforo , Nucleotídeos de Purina/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Through oxyradical formation xanthine oxidoreductase (XOD) could play a role in the etiology of cardiac damage. Its measurement poses problems, due to little substrate specificity, self-inactivation and endogenous inhibitors. Perfusion of guinea-pig hearts with hypoxanthine gave rise to only little xanthine release; in contrast rat hearts showed vivid xanthine production. Therefore, xanthine breakdown was hypothesized to exceed its formation in guinea-pig hearts. The kinetics of both substrates for XOD in cardiac homogenates were therefore compared with those obtained in perfused hearts. Oxypurine contents and effluent catabolites were determined by HPLC. Regardless of substrate, Vmax values in homogenates were about 38 and 13 mU/g for rat and guinea-pig heart, respectively. Km values were in the 3-5 microM range; therefore the hypothesis concerning the low xanthine release in guinea-pig hearts must be rejected. Activities in hearts perfused with hypoxanthine (50 microM) were 40 and 18 mU/g for rat and guinea pig, respectively; perfusion with xanthine produced < 50% of the activities observed with hypoxanthine (p < 0.002). Intracellular xanthine concentration, estimated from sorbitol distribution space and myocardial xanthine content was negative in both species, contrasting intracellular hypoxanthine levels, which approached extracellular concentrations. This disparate distribution indicates that hypoxanthine transport across the cell membrane far exceeds that of xanthine. Consequently, hypoxanthine is preferable to xanthine as substrate in perfused hearts to estimate XOD activity in situ.
Assuntos
Hipoxantinas/metabolismo , Miocárdio/enzimologia , Xantina Desidrogenase/metabolismo , Xantina Oxidase/metabolismo , Xantinas/metabolismo , Animais , Cobaias , Hipoxantina , Técnicas In Vitro , Perfusão , Ratos , Ratos Wistar , Especificidade por Substrato , XantinaRESUMO
The coronary vasodilator adenosine can be formed in the heart by breakdown of AMP or S-adenosylhomocysteine (SAdoHcy). The purpose of this study was to get insight into the relative importance of these routes of adenosine formation in both the normoxic and the ischemic heart. A novel HPLC method was used to determine myocardial adenosine and SAdoHcy. Accumulation of SAdoHcy was induced in isolated rat hearts by perfusion with L-homocysteine thiolactone or L-homocysteine. The release of adenosine, inosine, hypoxanthine, xanthine and uric acid was determined. Additional in vitro experiments were performed to determine the kinetic parameters of S-adenosylhomocysteine hydrolase. During normoxia the thiolactone caused a concentration-dependent increase in SAdoHcy. At 2000 microM of the thiolactone an SAdoHcy accumulation of 0.49 nmol/min per g wet weight was found during normoxia. L-Homocysteine (200 microM) caused an increase of 0.37 and 4.17 nmol SAdoHcy/min per g wet weight during normoxia and ischemia, respectively. The adenosine concentration in ischemic hearts was significantly lower when homocysteine was infused (6.2 vs. 11.5 nmol/g; P less than 0.05). Purine release was increased 4-fold during ischemia. The Km for hydrolysis of SAdoHcy was about 12 microM. At in vitro conditions favoring near-maximal SAdoHcy synthesis (72 microM adenosine, 1.8 mM homocysteine), the synthesis rate in homogenates was 10 nmol/min per g wet weight. From the combined in vitro and perfusion studies, we conclude that S-adenosylhomocysteine hydrolase can contribute significantly to adenosine production in normoxic rat heart, but not during ischemia.
Assuntos
Adenosina/biossíntese , Hidrolases/metabolismo , Miocárdio/metabolismo , Adenosil-Homocisteinase , Animais , Doença das Coronárias/metabolismo , Homocisteína/análogos & derivados , Homocisteína/metabolismo , Técnicas In Vitro , Cinética , Masculino , Perfusão , Ratos , Ratos EndogâmicosRESUMO
Using (13)C NMR, we tested the hypothesis that protection by preconditioning is associated with reduced glycogenolysis during ischemia. Preconditioned rat hearts showed improved postischemic function and reduced ischemic damage relative to ischemic controls after 30 min stop-flow ischemia and 30 min reperfusion (contractility: 30+/-10 vs. 2+/-2%; creatine kinase release: 41+/-4 vs. 83+/-15 U/g; both P<0.05). Preconditioning decreased preischemic [(13)C]glycogen by 24% (a 10% decrease in total glycogen), and delayed ischemic [(13)C]glycogen consumption by 5-10 min, reducing ischemic glycogenolysis without changing acidosis relative to controls. Upon reperfusion, glycogen synthesis resumed only after preconditioning. Glutamate (13)C-isotopomer analysis showed recovery of Krebs cycle activity with higher anaplerosis than before ischemia (23+/-4 vs. 11+/-3%, P<0.05), but in controls reperfusion failed to restore flux. Compared to control, preconditioning before 20 min ischemia increased contractility (86+/-10 vs. 29+/-14%, P<0.05) and restored preischemic anaplerosis (13+/-3 vs. 39+/-9%, P<0.05). Preconditioning is associated with reduced glycogenolysis early during ischemia. However, protection does not rely on major variations in intracellular pH, as proposed earlier. Our isotopomer data suggest that preconditioning accelerates metabolic and functional recovery during reperfusion by more efficient/active replenishment of the depleted Krebs cycle.
Assuntos
Glicogênio/metabolismo , Coração/fisiologia , Isquemia Miocárdica/metabolismo , Alanina/análise , Animais , Ciclo do Ácido Cítrico , Ácido Glutâmico/análise , Glicogênio/biossíntese , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ácido Láctico/análise , Espectroscopia de Ressonância Magnética , Masculino , Contração Miocárdica , Reperfusão Miocárdica , Ratos , Ratos Sprague-Dawley , Extratos de Tecidos/metabolismoRESUMO
Pathways producing and converting adenosine have hardly been investigated in human heart, contrasting work in other species. We compared the kinetics of enzymes associated with purine degradation and salvage in human and rat heart cytoplasm assaying for adenosine deaminase, nucleoside phosphorylase, xanthine oxidoreductase, AMP deaminase, AMP- and IMP-specific 5'-nucleotidases, adenosine kinase and hypoxanthine guanine phosphoribosyltransferase (HGPRT). Xanthine oxidoreductase was not detectable in human heart. The Km-values of the AMP-catabolizing enzymes were 2-5 times higher in human heart; the substrate affinity of the other enzymes was in the same order of magnitude in both species. The maximal activity (Vmax) of adenosine kinase was the same in both species, but HGPRT in man was only 12% of that in the rat. For human heart the Vmax-values of adenosine deaminase, nucleoside phosphorylase, AMP- and IMP-specific 5'-nucleotidases, and AMP deaminase were 25-50% of those for rat heart. We conclude that human heart is less geared to purine catabolism than rat heart as is evident from the lower activities of the catabolic enzymes. Maintenance of the nucleotide pool may thus play a more important role in human heart.
Assuntos
Monofosfato de Adenosina/metabolismo , Miocárdio/enzimologia , 5'-Nucleotidase/metabolismo , AMP Desaminase/metabolismo , Adenosina Desaminase/metabolismo , Adenosina Quinase/metabolismo , Animais , Humanos , Hipoxantina Fosforribosiltransferase/metabolismo , Inosina Monofosfato/metabolismo , Cinética , Masculino , Pentosiltransferases/metabolismo , Ratos , Ratos Wistar , Especificidade da Espécie , Xantina Oxidase/metabolismoRESUMO
High-energy phosphates in heart muscle deprived of oxygen are rapidly broken down to purine nucleosides and oxypurines. We studied the role of xanthine oxidase/dehydrogenase (EC 1.2.3.2/EC 1.2.1.37) in this process with novel high-pressure liquid chromatographic techniques. Under various conditions, including ischemia and anoxia, the isolated perfused rat heart released adenosine, inosine and hypoxanthine, and also substantial amounts of xanthine and urate. Allopurinol, an inhibitor of xanthine oxidase, greatly enhanced the release of hypoxanthine. From the purine release we calculated that the rat heart contained about 18 mU xanthine oxidase per g wet weight. Subsequently, we measured a xanthine oxidase activity of 9 mU/g wet wt. in rat-heart homogenate. When endogenous low molecular weight inhibitors were removed by gel-filtration, the activity increased to 31 mU/g wet wt. Rat myocardial xanthine oxidase seems to be present mainly in the dehydrogenase form, which upon storage at -20 degrees C is converted to the oxidase form.
Assuntos
Coração/fisiologia , Cetona Oxirredutases/metabolismo , Miocárdio/enzimologia , Xantina Desidrogenase/metabolismo , Xantina Oxidase/metabolismo , Animais , Doença das Coronárias/metabolismo , Vasos Coronários/fisiologia , Masculino , Perfusão , Nucleosídeos de Purina/metabolismo , Purinas/metabolismo , Ratos , Ratos EndogâmicosRESUMO
BACKGROUND: During ischemia, the intracellular calcium and inorganic phosphate (P(i)) concentrations rise and pH falls. We investigated the effects of these changes on force development in donor and failing human hearts to determine if altered contractile protein composition during heart failure changes the myocardial response to Ca(2+), P(i), and pH. METHODS AND RESULTS: Isometric force was studied in mechanically isolated Triton-skinned single myocytes from left ventricular myocardium. Force declined with added P(i) to 0.33+/-0.02 of the control force (pH 7.1, 0 mmol/L P(i)) at 30 mmol/L P(i) and increased with pH from 0.64+/-0.03 at pH 6.2 to 1.27+/-0.02 at pH 7.4. Force dependency on P(i) and pH did not differ between donor and failing hearts. Incubation of myocytes in a P(i)-containing activating solution caused a potentiation of force, which was larger at submaximal than at maximal [Ca(2+)]. Ca(2+) sensitivity of force was similar in donor hearts and hearts with moderate cardiac disease, but in end-stage failing myocardium it was significantly increased. The degree of myosin light chain 2 phosphorylation was significantly decreased in end-stage failing compared with donor myocardium, resulting in an inverse correlation between Ca(2+) responsiveness of force and myosin light chain 2 phosphorylation. CONCLUSIONS: Our results indicate that contractile protein alterations in human end-stage heart failure alter Ca(2+) responsiveness of force but do not affect the force-generating capacity of the cross-bridges or its P(i) and pH dependence. In end-stage failing myocardium, the reduction in force by changes in pH and [P(i)] at submaximal [Ca(2+)] may even be less than in donor hearts because of the increased Ca(2+) responsiveness.
Assuntos
Cálcio/farmacologia , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/efeitos dos fármacos , Fosfatos/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional , Feminino , Insuficiência Cardíaca/patologia , Ventrículos do Coração/citologia , Humanos , Concentração de Íons de Hidrogênio , Masculino , Pessoa de Meia-Idade , Contração Miocárdica/efeitos dos fármacos , Cadeias Leves de Miosina/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Função VentricularRESUMO
Preconditioning is a powerful form of (myocardial) protection that follows brief sublethal ischemia. G-protein-coupled receptors constitute the trigger for entrance to the preconditioned state. In conjunction with other receptors, various membrane adenosine receptors play an important role in the transduction of extracellular signals, leading to protection by preconditioning, lasting 1-3 hr. Adenosine A(1)- and A(3)-receptors mediate inhibition of adenylate cyclase via a guanine nucleotide binding inhibitory protein (G(i/o)). A(2)-receptors couple to a comparable stimulatory protein (G(s)). Adenosine receptors are especially abundant in the central nervous system; in lesser numbers, they are found in many tissues, including the heart. A(1)-receptors are located on cardiomyocytes and vascular smooth muscle cells, A(2)-receptors on endothelial and vascular smooth muscle cells, and A(3)-receptors on ventricular myocytes. Ischemic preconditioning by endogenous adenosine takes place through A(1)- and A(3)-receptors. A(2A/B)-receptor activation results in vasodilation. The relevance of cellular mediators, such as 5'-nucleotidase, to generate adenosine for preconditioning is controversial. In contrast, the role of protein kinase C (PKC) is clearly established. Signals from different receptors converge at PKC, reaching a threshold activation of the kinase necessary to induce protection. Tyrosine and mitogen-activated protein kinases may play a role in addition to PKC. The exact products downstream responsible for the memory of preconditioning are elusive. A prime candidate for the end-effector of preconditioning is the K(ATP) channel. Preconditioning with adenosine-receptor agonists offers the possibility for treatment of coronary artery disease, but research in this field is still in its infancy.
Assuntos
Adenosina/farmacologia , Precondicionamento Isquêmico , Receptores Purinérgicos P1/fisiologia , 5'-Nucleotidase/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Canais Iônicos/fisiologia , Miocárdio/ultraestrutura , Proteína Quinase C/metabolismo , Transdução de SinaisRESUMO
There is scant literature about patients in the final phase of dementia. Uniform terminology and operational definition of the final phase of dementia is lacking. Furthermore, it is difficult to monitor these patients because existing assessment scales face bottom- or ceiling effects in this population. The aim of this study was to assess the prevalence and the characteristics of patients in the final phase of dementia in a group of 210 Dutch nursing home patients with dementia. Stage 7 of the Global Deterioration Scale of Reisberg et al. was used to operationally define the final phase of dementia. All patients were scored on a self-constructed assessment scale. Furthermore, treatment aspects and advance directives were registered.
Assuntos
Demência/diagnóstico , Demência/epidemiologia , Casas de Saúde , Atividades Cotidianas , Idoso , Idoso de 80 Anos ou mais , Demência/patologia , Feminino , Humanos , Masculino , Países Baixos , Prevalência , Testes Psicológicos , Índice de Gravidade de Doença , Fatores SexuaisRESUMO
Prevalence and characteristics of a group dementia patients in the final phase of dementia.There is scant literature about patients in the final phase of dementia. Uniform terminology and operational definition of the final phase of dementia is lacking. Furthermore, it is difficult to monitor these patients because existing assessment scales face bottom- or ceiling effects in this population. The aim of this study was to assess the prevalence and the characteristics of patients in the final phase of dementia in a group of 210 Dutch nursing home patients with dementia. Stage 7 of the Global Deterioration Scale of Reisberg et al. was used to operationally define the final phase of dementia. All patients were scored on a self-constructed assessment scale. Furthermore, treatment aspects and advance directives were registered.Twelve percent (26) of the dementia patients admitted to the psychogeriatric wards met the criteria for final phase of dementia. There was considerable variation in the characteristics of the patients. This study gives a start to further research on aspects of the final phase of dementia.
RESUMO
OBJECTIVE: Glycolysis-from-glycose may be more beneficial than glycogenolysis in protecting hearts against ischemia. We tested the hypothesis that ischemic preconditioning is mediated by increased exogenous glucose use during low-flow ischemia, an effect triggered by adenosine A1 receptor activation. METHODS: Langendorff rat hearts were subjected to 25 min low-flow ischemia (0.6 ml/min) and 30 min reperfusion. Prior to underperfusion, hearts (n = 6 per group) were subjected to two cycles of either preconditioning ischemia (PC), infusion of the adenosine A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 0.25 mumol/l), or PC in the presence of the adenosine antagonist 8-(p-sulfophenyl)theophylline (SPT; 50 mumol/l). Glycolysis-from-glucose during underperfusion was measured using D-[2-3H]glucose. RESULTS: At the end of reperfusion, recovery of rate-force product was enhanced in the PC and CCPA groups (62 and 67% of preischemic value) compared to the ischemic control hearts (IC, 32%; P < 0.05), whereas protection was abolished in the SPT hearts (20%; P < 0.05 vs. PC). PC improved total glycolysis-from-glucose during underperfusion by 31% (P < 0.05 vs. IC); SPT abolished this increase. CCPA reduced total lactate release and glucose uptake during ischemia by 47% and 61%, respectively (P < 0.05 vs. IC). Abolishment of the preconditioning-associated increase in glucose uptake during underperfusion, by switching to a low glucose buffer, resulted in a loss of functional protection. CONCLUSIONS: This study strongly suggests that increased exogenous glucose utilization during low-flow ischemia mediates ischemic preconditioning without increasing total anaerobic glycolytic flux. Although adenosine A1 receptor activation reduces ischemic injury, it does not facilitate the increased glucose uptake observed with ischemic preconditioning, suggesting a different mechanism of protection.
Assuntos
Adenosina/análogos & derivados , Glucose/metabolismo , Precondicionamento Isquêmico Miocárdico , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Agonistas do Receptor Purinérgico P1 , Adenosina/farmacologia , Animais , Glicólise , Masculino , Perfusão , Antagonistas de Receptores Purinérgicos P1 , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional , Teofilina/análogos & derivados , Teofilina/farmacologiaRESUMO
Quantitative determination of myocardial adenosine formation and breakdown is necessary to gain insight into the mechanism and regulation of its physiological actions. Deamination of adenosine was studied in isolated perfused rat hearts by infusion of adenosine (1 to 20 mumol X litre-1). All catabolites in the perfusates (inosine, hypoxanthine, xanthine and uric acid) were measured, as well as unchanged adenosine. Apparent uptake of adenosine was determined; it increased linearly with the concentration of adenosine infused. Adenosine was predominantly deaminated, even at low (1 mumol X litre-1) concentration. The inhibitory capacity of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was determined, while 5 mumol X litre-1 adenosine was infused. EHNA inhibited the apparent adenosine deaminase activity for 62 and 92% at 5 and 50 mumol X litre-1, respectively. When 50 mumol X litre-1 EHNA was infused into normoxic hearts, release of adenosine was significantly elevated, as was coronary flow. Induction of ischaemia increased total purine release four-to fivefold. Infusion of EHNA into ischaemic hearts did not alter total purine release, but adenosine release increased from 15 to 60% of total purines. However, when EHNA was present, a large part of total purine release still existed of inosine, hypoxanthine, xanthiner and uric acid. This was 83% during normoxia and 40% during ischaemia. These results suggest significant contribution of IMP and GMP breakdown to purine release from isolated perfused rat hearts.
Assuntos
Adenosina Desaminase/metabolismo , Miocárdio/metabolismo , Nucleosídeo Desaminases/metabolismo , Consumo de Oxigênio , Purinas/biossíntese , Adenina/análogos & derivados , Adenina/farmacologia , Adenosina/farmacologia , Monofosfato de Adenosina/metabolismo , Animais , Circulação Coronária , Desaminação , Depressão Química , Coração/efeitos dos fármacos , Masculino , Miocárdio/enzimologia , Perfusão , Ratos , Ratos EndogâmicosRESUMO
OBJECTIVE: Cardiac injury, related to ischaemia and reperfusion, may be caused by the action of oxygen free radicals. Xanthine oxidoreductase activity may be an important free radical source. During cardiac ischaemia, the native dehydrogenase form may be converted to the oxidase form, which uses molecular oxygen to form superoxide radicals. Superoxide dismutase converts the radicals to H2O2, which is detoxified by catalase and glutathione peroxidase. In view of the large differences in xanthine oxidoreductase in various species, the activity of these antioxidant enzymes was investigated. METHODS: Normal rodent and porcine as well as explanted human hearts were perfused according to Langendorff. After a 30 minute stabilisation period, hypoxanthine was added to the perfusion buffer to estimate xanthine oxidoreductase. Hearts or biopsies were freeze clamped after 90 minutes. Effluent xanthine and urate were assayed with high performance liquid chromatography; tissue reduced glutathione content and the activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were determined spectrophotometrically. Apparent xanthine oxidoreductase was calculated as xanthine +2 x urate production. RESULTS: Xanthine oxidoreductase was (mU.g-1 protein, mean(SEM), n = 5-7): rat, 470(40); guinea pig, 270(41); pig < 1.5; and human, 5.4(1.0). Superoxide dismutase activities were (U.g-1 protein): rat, 13,370(1030); guinea pig, 10,100(1110); pig, 12,800(450); and human, 7400(450). Catalase activity (k < or = 10.g-1 protein) was low in all species studied. Glutathione peroxidase activity was 93(7) U.g-1 protein in rat heart, and 10 x lower in the other species. Glutathione reductase activity was (U.g-1 protein): rat, 15.0(1.6); guinea pig, 10.4(1.3); pig, 16.0(1.5); and human, 26.6(2.0). Tissue reduced glutathione concentrations were (mumol.g-1 protein): rat, 13.5(0.8); guinea pig, 18.5(0.9); pig, 11.1(2.9); and human 17.2(1.7). CONCLUSIONS: Considerable species differences in xanthine oxidoreductase activity exist, contrasting with the smaller variations in antioxidant enzyme activities. In the species examined catalase activities were very low. Rat hearts are far better protected against H2O2 than the other three species. Xanthine oxidoreductase induced free-radical damage probably plays a minor role in pig and human hearts. Human myocardium seems less protected against superoxide radicals.
Assuntos
Antioxidantes/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Xantina Oxidase/metabolismo , Alopurinol/farmacologia , Animais , Catalase/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Cobaias , Humanos , Miocárdio/enzimologia , Perfusão , Ratos , Especificidade da Espécie , Superóxido Dismutase/metabolismo , SuínosRESUMO
Although inosine has been used clinically to support the myocardium, no data are available on the fate of exogenous inosine in the human heart. We therefore infused six patients, catheterised for coronary angiography, with inosine (5 mg.kg-1.min-1 intravenously) for 6 minutes. Before infusion, the arterio-venous difference of inosine, hypoxanthine and xanthine across the heart was nil. During infusion, arterial inosine increased substantially, exceeding the coronary sinus concentration by a maximum of 200 (SEM 53) mumol.litre-1, p = 0.02, at the fourth minute. Arterial hypoxanthine and xanthine also increased, while the arterio-venous difference became 16(11) and 10(3) (p = 0.04) mumol.litre-1, respectively. Left ventricular dP/dtmax increased by 22(7)% (p = 0.04) at the end of infusion. Thus, there seemed to be substantial uptake of inosine by the human heart, followed by improvement in haemodynamics.
Assuntos
Doença das Coronárias/metabolismo , Inosina/farmacocinética , Miocárdio/metabolismo , Adulto , Cateterismo Cardíaco , Doença das Coronárias/fisiopatologia , Feminino , Hemodinâmica , Humanos , Hipoxantina , Hipoxantinas/sangue , Inosina/sangue , Masculino , Pessoa de Meia-Idade , Xantina , Xantinas/sangueRESUMO
OBJECTIVE: We investigated whether the Frank-Starling mechanism is absent or preserved in end-stage failing human myocardium and if phosphorylation of contractile proteins modulates its magnitude through the sarcomere length-dependence of calcium sensitivity of isometric force development. METHODS: The effect of phosphorylation of troponin I and C-protein by the catalytic subunit of protein kinase A (3 microg/ml; 40 min at 20 degrees C) was studied in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at sarcomere lengths measured at rest of 1.8, 2.0 and 2.2 microm. Isometric force development was studied at various free-calcium concentrations before and after protein kinase A incubation at 15 degrees C (pH 7.1). RESULTS: Maximal isometric tension at 2.2 microm amounted to 39.6+/-10.4 and 33.7+/-3.5 kN/m2 in donor and end-stage failing cardiomyocytes, respectively. The midpoints of the calcium sensitivity curves (pCa50) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean delta pCa50=0.22). A reduction in sarcomere length from 2.2 to 1.8 microm caused reductions in maximum isometric force to 64% and 65% and in pCa50 by 0.10 and 0.08 pCa units in donor and failing cardiomyocytes, respectively. In donor tissue, the effect of protein kinase A treatment was rather small, while in end-stage failing myocardium it was much larger (delta pCa50=0.24) irrespective of sarcomere length. CONCLUSIONS: The data obtained indicate that the Frank-Starling mechanism is preserved in end-stage failing myocardium and suggest that sarcomere length dependence of calcium sensitivity and the effects of phosphorylation of troponin I and C-protein are independent.
Assuntos
Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Insuficiência Cardíaca/metabolismo , Contração Isométrica , Miocárdio/metabolismo , Sarcômeros/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Humanos , Técnicas In Vitro , Miocárdio/citologia , Fosforilação , Troponina I/metabolismoRESUMO
OBJECTIVE: Phosphorylation of the myosin light chain 2 (MLC-2) isoform expressed as a percentage of total MLC-2 was decreased in failing (21.1+/-2.0%) compared to donor (31.9+/-4.8%) hearts. To assess the functional implications of this change, we compared the effects of MLC-2 dephosphorylation on force development in failing and non-failing (donor) human hearts. METHODS: Cooperative effects in isometric force and rate of force redevelopment (K(tr)) were studied in single Triton-skinned human cardiomyocytes at various [Ca(2+)] before and after protein phosphatase-1 (PP-1) incubation. RESULTS: Maximum force and K(tr) values did not differ between failing and donor hearts, but Ca(2+)-sensitivity of force (pCa(50)) was significantly higher in failing myocardium (Deltap Ca(50)=0.17). K(tr) decreased with decreasing [Ca(2+)], although this decrease was less in failing than in donor hearts. Incubation of the myocytes with PP-1 (0.5 U/ml; 60 min) decreased pCa(50) to a larger extent in failing (0.20 pCa units) than in donor cardiomyocytes (0.10 pCa units). A decrease in absolute K(tr) values was found after PP-1 in failing and donor myocytes, while the shape of the K(tr)-Ca(2+) relationships remained unaltered. CONCLUSIONS: Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca(2+)-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca(2+)-sensitivity and impaired Ca(2+)-handling on diastolic function in human heart failure.
Assuntos
Cálcio/fisiologia , Miosinas Cardíacas/metabolismo , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/metabolismo , Adulto , Cálcio/farmacologia , Miosinas Cardíacas/fisiologia , Células Cultivadas , Eletroforese em Gel Bidimensional , Feminino , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Cadeias Leves de Miosina/fisiologia , Fosfoproteínas Fosfatases/farmacologia , Fosforilação , Proteína Fosfatase 1RESUMO
OBJECTIVE: The alterations in contractile proteins underlying enhanced Ca(2+)-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca(2+)-responsiveness in human heart failure. METHODS: Isometric force and its Ca(2+)-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca(2+)-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting. RESULTS: Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca(2+)-sensitivity of the contractile apparatus (pCa(50)) was significantly higher in failing myocardium (deltapCa(50)=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca(2+)-responsiveness. An inverse correlation was found between pCa(50) and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca(2+)-sensitivity to a larger extent in failing (deltapCa(50)=0.20) than in donor (deltapCa(50)=0.03) myocytes, abolishing the difference in Ca(2+)-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca(2+)-responsiveness. CONCLUSIONS: The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.