RESUMO
The effect of the proteases trypsin, thermolysin and papain on the cardiac membrane protein phospholamban was examined before or after phosphorylating the protein with the catalytic subunit of cyclic AMP-dependent protein kinase. The sensitivity of phospholamban to digestion by trypsin and thermolysin was greatly reduced by phosphorylation, suggesting that phospholamban undergoes a conformational change upon phosphorylation. It is suggested that this change in conformation is the mechanism by which phospholamban phosphorylation relieves its inhibition of the sarcoplasmic reticulum Ca2+-ATPase pump.
Assuntos
Proteínas de Ligação ao Cálcio , Papaína/farmacologia , Termolisina/farmacologia , Tripsina/farmacologia , Animais , Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Ligação ao Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cobaias , Miocárdio/análise , Fosforilação , Conformação Proteica/efeitos dos fármacos , Proteínas Quinases/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Phosphorylation of phospholamban in cardiac sarcolemma is implicated in the increased influx of Ca2+ through the slow calcium channel induced by catecholamines. A method is described for the preparation of highly purified sarcolemmal vesicles from rat heart, and this has been used to examine the phosphorylation of phospholamban in 32Pi-perfused rat hearts. Phospholamban phosphorylation is increased 3-fold after 30 s of perfusion with 0.1 microM isoprenaline. The time course of this increase precedes the inotropic response by 5-10 s.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Canais Iônicos/metabolismo , Isoproterenol/farmacologia , Miocárdio/metabolismo , Sarcolema/metabolismo , Animais , Cálcio/metabolismo , Técnicas In Vitro , Perfusão , Fosforilação , RatosRESUMO
1. The calcium sensitivity of force production of cardiac muscle fibres is altered by certain drugs. The sites of action of three such compounds (pimobendan, sulmazole, isomazole) within the myofibril have been investigated. Calmodulin antagonists, perhexilene and bepridil, which have been shown to alter the calcium dependence of myofibrillar ATPase activity and oxmetidine, an H2-receptor antagonist which binds to calmodulin, were also studied. 2. The rates of dissociation of calcium from both the regulatory and high affinity sites on bovine isolated cardiac troponin C (cTnC) were measured in a stopped-flow fluorimeter. The rates of dissociation were found to be 136.5 +/- 16 s-1 and 1.3 +/- 0.20 s-1 (mean +/- s.e.mean, n = 11 determinations; conditions: 100 mM KCl, 10 mM MOPS, 3 mM MgCl2, 0.1 mM dithriothreitol, pH 7.0, 15 degrees C). Sulmazole, isomazole and perhexiline (final concentration of 50 microM) had no effect on the rate of Ca2+ dissociation from the regulatory Ca2+ site, indicating that these compounds do not act on cTnC directly. 3. The rate of dissociation of Ca2+ from the regulatory site was slightly reduced (approximately 20%) by pimobendan (50 and 100 microM) and was somewhat increased by oxmetidine (28% at 100 microM). 4. Bepridil (25 microM) reduced the rate of dissociation by 50%, indicating a direct effect of bepridil on TnC. 5. Sulmazole, isomazole, perhexiline, pimobendan (50 microM) and bepridil (25 microM) were without effect on the rate of dissociation of Ca2+ from the high affinity Ca2+/Mg2+ sites. Oxmetidine caused 24% decrease in the rate of Ca2+ dissociation from these sites. 6. The rate of dissociation of Ca2+ from the regulatory site on the complex of troponin-tropomyosin (TnTm) was measured. Sulmazole and pimobendan (50 microM) were without effect on the rate of dissociation of Ca2+ from the regulatory site in the protein complex, and isomazole (50 microM) caused only a slight reduction (23%). Perhexiline (50 microM) or bepridil (10 microM) reduced the rate of Ca2 dissociation by about 50%. The rate of dissociation of Ca2+ from the high affinity Ca2 +/Mg2 + sites was not altered by sulmazole, isomazole, or pimobendan (50 microM), but was decreased - 35% by perhexiline (50 microM) or bepridil (10 microM).
Assuntos
Cálcio/fisiologia , Miocárdio/metabolismo , Tropomiosina/metabolismo , Troponina/metabolismo , Animais , Bepridil/farmacologia , Cálcio/metabolismo , Bovinos , Corantes Fluorescentes , Coração/efeitos dos fármacos , Imidazóis/farmacologia , Técnicas In Vitro , Cinética , Perexilina/farmacologia , Piridazinas/farmacologia , Relação Estrutura-Atividade , Troponina CRESUMO
1. The effects of siguazodan (SK&F 94836) a selective phosphodiesterase (PDE) inhibitor with inotropic and vasodilator activity, were studied on human platelets. 2. Siguazodan selectively inhibited the major cyclic AMP-hydrolysing PDE in human platelet supernatants. The inhibited enzyme has been variously termed cyclic GMP-inhibited PDE or PDE-III. 3. In platelet-rich plasma (PRP), siguazodan inhibited U46619-induced aggregation more potently than that induced by ADP and collagen. Treatment of the PRP with aspirin had no effect on the potency of siguazodan. 4. In washed platelets, siguazodan increased cyclic AMP levels and reduced cytoplasmic free calcium [( Ca2+]i). ADP decreased the ability of siguazodan to raise cyclic AMP and this may explain its lower potency in inhibiting responses to ADP. 5. Siguazodan has anti-platelet actions over the same concentration range that it is an inotrope and vasodilator.
Assuntos
Plaquetas/efeitos dos fármacos , Guanidinas/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Piridazinas/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Difosfato de Adenosina/metabolismo , Aspirina/farmacologia , Plaquetas/enzimologia , Plaquetas/metabolismo , Cálcio/metabolismo , Cromatografia por Troca Iônica , Colágeno/farmacologia , AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Testes de Função Plaquetária , Endoperóxidos Sintéticos de Prostaglandinas/farmacologiaRESUMO
Perfused rat hearts prelabelled with 32P were made ischaemic by reducing the medium flow from 12 ml/min to 0.5 ml/min. There was a rapid decrease in the contractile performance, but no significant changes in the phosphorylation state of troponin I, myosin P-light chain, an 11 K protein or in the proportion of phosphorylase in the a form occurring up to 5 min of ischaemia. Control hearts stimulated with a bolus of isoproterenol showed a large increase in the contractile force and in the phosphorylation of troponin I, 11 K protein, and phosphorylase, respectively. These responses were progressively reduced by increasing periods of ischaemia. The reduction and loss of increased phosphorylation of these proteins on exposure to isoproterenol was parallelled with an inhibition of cyclic AMP accumulation in the ischaemic heart. Phosphorylation of the myosin P-light chain remained unchanged under all the conditions studied.
Assuntos
Doença das Coronárias/metabolismo , Coração/fisiopatologia , Isoproterenol/farmacologia , Miocárdio/metabolismo , Proteínas/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Coração/efeitos dos fármacos , Cinética , Contração Miocárdica/efeitos dos fármacos , Radioisótopos de Fósforo , Fosforilases/metabolismo , Fosforilação , Ratos , Ratos Endogâmicos , Troponina/metabolismo , Troponina IRESUMO
BACKGROUND: To examine changes to whole body and regional lean mass (LM) and fat mass (FM) over 33 months of intermittent androgen suppression therapy (IAST). METHODS: Phase II cohort study of 72 prostate cancer patients without metastatic bone disease. Patients received flutamide 250 mg tid and leuprolide 22.5 mg three monthly depot for the 9-month initial treatment phase (iTREAT), at which point patients ceased therapy providing PSA <4 ng ml(-1) with continued monitoring for further 2 years (POST). AST was recommenced when PSA exceeded pretreatment level or ≥ 20 ng ml(-1). Body composition was assessed using dual energy X-ray absorptiometry at baseline, completion of treatment phase, and 1 and 2 years post treatment phase (months 21 and 33). RESULTS: LM decreased by 1.3 kg and FM increased by 2.3 kg (P<0.001) following iTREAT. During the POST period, there were no further adverse effects on LM or FM, but also no recovery to pretreatment levels. Patients who failed to recover testosterone by month 33 experienced a significant increase in FM compared with those who recovered eugonadal levels of testosterone (10 nmol ml(-1); P = 0.019). Change in testosterone was moderately correlated to changes in % FM (r = -0.314, P<0.028) and LM (r = 0.300, P<0.036) during POST phase. Waist circumference progressively increased over time and by 2 years, POST had not recovered to baseline levels. CONCLUSIONS: Loss of LM and gain in FM during the 9-month iTREAT was not reversed during 2-year POST, although further deterioration was not observed. Subgroup analysis identified those recovering testosterone showed some body composition improvements. These findings suggest potential benefits of IAST, where testosterone levels are able to recover, to reduce the ongoing adverse effects on body composition, such as the acceleration of sarcopenia and risks associated with metabolic disease.
Assuntos
Antagonistas de Androgênios/administração & dosagem , Composição Corporal/efeitos dos fármacos , Flutamida/administração & dosagem , Leuprolida/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Absorciometria de Fóton , Tecido Adiposo , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Masculino , Pessoa de Meia-Idade , TempoAssuntos
Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Animais , Proteínas de Transporte , AMP Cíclico/farmacologia , Epinefrina/farmacologia , Feminino , Coração/efeitos dos fármacos , Técnicas In Vitro , Peso Molecular , Perfusão , Fosforilação , Proteínas Quinases/metabolismo , RatosAssuntos
Cálcio/metabolismo , Fracionamento Celular/métodos , Músculo Liso Vascular/metabolismo , Proteínas Quinases/farmacologia , Sarcolema/metabolismo , Animais , Aorta Torácica/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Fosforilação , RatosAssuntos
Adenosina Trifosfatases/metabolismo , Cálcio/farmacologia , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Miofibrilas/metabolismo , Fosfoproteínas/metabolismo , Troponina/metabolismo , Animais , Bovinos , Cinética , Peso Molecular , Miofibrilas/efeitos dos fármacos , Proteínas Quinases/metabolismoAssuntos
Tecido Adiposo/metabolismo , Citratos/metabolismo , Miocárdio/metabolismo , Acetatos/metabolismo , Animais , Ácido Aspártico/metabolismo , Coenzima A/metabolismo , Glutamatos/metabolismo , Glicogênio/biossíntese , Glicólise , Hexoquinase/metabolismo , Técnicas In Vitro , Ligases/metabolismo , Magnésio/farmacologia , Perfusão , Fosfofrutoquinase-1/metabolismo , RatosRESUMO
1. Rat hearts were perfused with 32Pi, and contractile force was increased by positive inotropic agents (agents that increase contractility). The inhibitory subunit of troponin (troponin I) was then isolated by affinity chromatography in 8M-urea, and its 32P content measured. Incorporation of phosphate into the subunit was calculated on the basis of the [gamma-32P]ATP specific radioactivity in the hearts. 2. When hearts were perfused with 30 nM-DL-isoprenaline (N-isopropylnoradrenaline), there was an increase in contractile force over 30s which was paralleled by an increase in troponin I phosphorylation. When hearts were perfused for 25s with increasing concentrations of isoprenaline from 1 NM to 0.6 muM, there was again a parallel increase in contractile force and troponin I phosphorylation. The maximum phosphorylation observed was 1.5 mol of phosphate/mol of troponin I, which was reached after 25s with 0.1 muM-isoprenaline. 3. Hearts were stimulated with a 15s pulse perfusion of 30nM-DL-isoprenaline. There was an increase in contractile force which was followed by a return to the control value within 50s. Troponin I phosphorylation increased to a plateau value which was reached within 30s, and remained constant for 60s after the isoprenaline pulse. Phosphorylase a and 3':5'-cyclic AMP concentration showed changes similar to that of the contractile force. There was no change in 3':5'-cyclic GMP concentration. 4. When hearts stimulated with a 15S pulse of isoprenaline were subsequently perfused with 0.6 muM-acetylcholine, the changes in contractile force, phosphorylase a and 3':5'-cyclic AMP were very similar to those seen with the 15s pulse of isoprenaline alone. Troponin I phosphorylation increased to a maximum 30s after the end of the isoprenaline pulse, but then rapidly decreased during the subsequent 30s. This decrease was preceded by a 60% increase in the concentration of 3':5'-cyclic GMP. 5. Hearts were perfused with 0.2 muM-glucagon for periods up to 60s. Contractile force showed little change for the first 30s, but then increased rapidly. This was paralleled by changes in 3':5'-cyclic AMP concentration. Troponin I phosphorylation increased slowly, but the increase in contractile force had reached a maximum before significant phosphorylation had occurred. 6. It is concluded that under certain conditions, e.g. immediately after beta-adrenergic stimulation, there is a good correlation between contractile force and troponin I phosphorylation. However, under other conditions, e.g. when contractile force is decreasing after removal of beta-adrenergic stimulation or in the presence of glucagon, contractile force and troponin I phosphorylation are not well correlated. These results suggest that mechanisms for modifying cardiac contractility, other than troponin I phosphorylation, must be present in rat heart.
Assuntos
Isoproterenol/farmacologia , Proteínas Musculares/metabolismo , Contração Miocárdica/efeitos dos fármacos , Fosfatos/metabolismo , Troponina/metabolismo , Acetilcolina/farmacologia , Animais , Cromatografia de Afinidade , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Glucagon/farmacologia , Técnicas In Vitro , Masculino , Fosforilases/metabolismo , Ratos , Fatores de TempoRESUMO
When hearts from control and phosphorylase kinase-deficient (I strain) mice were perfused with 0.1 micrometer-DL-isoprenaline, there was a parallel increase in contraction, cyclic AMP concentration and troponin I phosphorylation. However, there was no increase in phosphorylase a in the I-strain hearts, whereas the control hearts showed a large increase. Assays of I-strain heart extracts showed a normal cyclic AMP-dependent protein kinase activity but no phosphorylase kinase activity. It is concluded that troponin I is phosphorylated in intact hearts by protein kinase and not phosphorylase kinase.
Assuntos
Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Fosforilase Quinase/deficiência , Proteínas Quinases/metabolismo , Troponina/metabolismo , Animais , AMP Cíclico/farmacologia , Técnicas In Vitro , Camundongos , Miocárdio/enzimologia , Fosfatos/metabolismoRESUMO
The derivation is given of the general case of a differential equation describing the labelling of a single metabolite pool that is subjected to constant rates of influx of radioactive material when the pool size is itself changing at constant rate. A programme, written in ALGOL, is briefly described that solves a set of these differential equations, and that can be used to simulate the radioactive labelling of a system of metabolite pools. A copy of the programme, together with examples of input and output, has been deposited as Supplementary Publication no. SUP 50001 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1970), 116, 7.
Assuntos
Computadores , Metabolismo , Modelos TeóricosRESUMO
1. The kinetic properties of the soluble and particulate hexokinases from rat heart have been investigated. 2. For both forms of the enzyme, the K(m) for glucose was 45mum and the K(m) for ATP 0.5mm. Glucose 6-phosphate was a non-competitive inhibitor with respect to glucose (K(i) 0.16mm for the soluble and 0.33mm for the particulate enzyme) and a mixed inhibitor with respect to ATP (K(i) 80mum for the soluble and 40mum for the particulate enzyme). ADP and AMP were competitive inhibitors with respect to ATP (K(i) for ADP was 0.68mm for the soluble and 0.60mm for the particulate enzyme; K(i) for AMP was 0.37mm for the soluble and 0.16mm for the particulate enzyme). P(i) reversed glucose 6-phosphate inhibition with both forms at 10mm but not at 2mm, with glucose 6-phosphate concentrations of 0.3mm or less for the soluble and 1mm or less for the particulate enzyme. 3. The total activity of hexokinase in normal hearts and in hearts from alloxan-diabetic rats was 21.5mumoles of glucose phosphorylated/min./g. dry wt. of ventricle at 25 degrees . The temperature coefficient Q(10) between 22 degrees and 38.5 degrees was 1.93; the ratio of the soluble to the particulate enzyme was 3:7. 4. The kinetic data have been used to predict rates of glucose phosphorylation in the perfused heart at saturating concentrations of glucose from measured concentrations of ATP, glucose 6-phosphate, ADP and AMP. These have been compared with the rates of glucose phosphorylation measured with precision in a small-volume recirculation perfusion apparatus, which is described. The correlation between predicted and measured rates was highly significant and their ratio was 1.07. 5. These findings are consistent with the control of glucose phosphorylation in the perfused heart by glucose 6-phosphate concentration, subject to certain assumptions that are discussed in detail.
RESUMO
The rate of exchange of phosphate bound to ventricular myosin light chain-2 (LC2-P) was measured in rat hearts perfused with [32P]Pi at various levels of perfusate Ca2+. Computer simulations of the light-chain labelling suggested the presence of two isotopically distinct pools of LC2-P, one large pool comprising 90% of the total and a small pool consisting of the remaining 10%. At control levels of perfusate Ca2+ the phosphate of the large pool turned over very slowly (t 1/2 congruent to 250 min), whereas that of the small pool turned over much more rapidly (t 1/2 congruent to 1 min). At high levels of perfusate free Ca2+ (5mM) the turnover of the phosphate of the small pool decreased markedly, whereas that of the large pool remained little changed. Conversely, at low perfusate free Ca2+ (0.2 mM), the turnover of the large pool decreased, whereas that of the small pool remained unchanged. The possible identity of these two pools is discussed. The total myosin-light-chain kinase activity of rat ventricle was found to be only 2-3-fold higher than the kinase activity expressed in the heart under control conditions. This, coupled with the very low turnover of most of the LC2-bound phosphate, implies that, in heart, there is insufficient myosin-light-chain kinase activity to cause a rapid rise in the overall level of light-chain phosphorylation, even under conditions of increased cytoplasmic Ca2+.
Assuntos
Miocárdio/metabolismo , Miosinas/metabolismo , Fosfatos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Modelos Biológicos , Perfusão , Fosforilação , Ratos , Ratos EndogâmicosRESUMO
Rat hearts perfused with 32Pi were made hypoxic by perfusion with medium gassed with N2/CO2 (19:1). There was a rapid decrease in tension development (25% of control within 40 s), but little change in the frequency of contraction, time to peak tension, or rate of relaxation. The phosphorylation of troponin-I, C-protein and myosin P-light chain was unaffected by 5 min of hypoxia, whereas the proportions of glycogen phosphorylase and pyruvate dehydrogenase in the active form increased slowly. When aerobically perfused hearts were challenged with a bolus (70 pmole) of D,L-isoprenaline, there was a large increase in contractile force, cyclic AMP concentration, phosphorylation of troponin-I and C-protein and activation of phosphorylase and pyruvate dehydrogenase. Hypoxia for 5 min caused a slight, progressive decrease in the response to isoprenaline of force, cyclic AMP and activation of phosphorylase and pyruvate dehydrogenase. In contrast, there was a larger decrease in the phosphorylation of troponin-I and C-protein, suggesting that the activity of cyclic AMP-dependent protein kinase towards the contractile proteins may be impaired by hypoxia. The phosphorylation of myosin P-light chain was unaltered by any condition. The response to hypoxia is compared to that of ischaemia, where a complete loss of the response to isoprenaline occurs after 5 min.
Assuntos
Proteínas Contráteis/metabolismo , Coração/efeitos dos fármacos , Hipóxia/fisiopatologia , Isoproterenol/farmacologia , Contração Miocárdica/efeitos dos fármacos , Animais , Cálcio/fisiologia , Catecolaminas/farmacologia , AMP Cíclico/metabolismo , Técnicas In Vitro , Miocárdio/enzimologia , Fosforilases/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Continuous perfusion of rat hearts with concentrations of forskolin between 0.1 and 12 microM resulted in transient increases in tension after 45 s, followed by a return to the control value after 5 min. In contrast, the content of cyclic AMP increased linearly with time over this period, reaching values up to 35 times control after 5 min. Increases in contractile force, intracellular cyclic AMP concentration and the proportion of phosphorylase in the a form were dependent on the concentration of forskolin when measured 45 s and 120 s after initiation of perfusion. In hearts perfused for 45 s with various concentrations of forskolin, the measured cyclic AMP-dependent protein kinase activity ratio and phosphorylase a content for a given measured intracellular cyclic AMP concentration were both much less than the corresponding values in hearts perfused for 30 s with various concentrations of isoprenaline. The phosphorylation of the contractile proteins troponin-I and C-protein also showed a concentration-dependent increase in hearts perfused with forskolin. There was a strong correlation between the cyclic AMP-dependent protein kinase activity ratios and the phosphorylation of the contractile proteins under all perfusion conditions. These results suggest that cyclic AMP is compartmented in perfused rat heart, and that much of the cyclic AMP produced in response to forskolin is unavailable to activate cyclic AMP-dependent protein kinase.
Assuntos
Colforsina/farmacologia , Proteínas Musculares/metabolismo , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Animais , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Coração/efeitos dos fármacos , Técnicas In Vitro , Perfusão , Fosforilase a/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Ratos , Ratos EndogâmicosRESUMO
1. Phosphoprotein phosphatases with activity towards the inhibitory subunit of troponin (troponin I), phosphorylase a and lysine-rich histone (fraction F1) have been fractionated from rat skeletal muscle by chromatography on Sephadex G-200 and polylysine-Sepharose. Six separate fractions were identified on the basis of substrate specificity and behaviour during chromatography. 2. All fractions showed similar Km values for any given protein substrate. The Km for troponin I (5 muM) was significantly lower than that previously reported. 3. Phosphatase activities towards troponin I and hosphorylase a did not show a requirement for bivalent-metal ions. Two of the fractions with only minor activity towards histone were activated by Mn2+. 4. Discontinuous polyacrylamide-gel-electrophoresis studies indicated that several of the fractions contained more than one phosphatase activity, and additionally showed that several of the activities could exist in different aggregation states. On the basis of these studies at least two phosphatases with activity only towards troponin I were identified. In addition, phosphorylase phosphatase (which has considerable activity towards troponin I) and a general phosphatase with activity towards all three substrates were found. 5. A fraction with mol.wt. of 150000 could be activated by freezing with 2-mercaptoethanol or by heating to 55 degrees C. This activation was accompanied by a decrease in mol.wt. to 25000. 6. The total amount of phosphatase with activity towards troponin I which was extracted would be sufficient to dephosphorylate all the troponin I present in skeletal muscle in approximately 10s.