RESUMEN
By keeping intracellular Na+ (aiNa) low, the Na,K-pump can prevent Ca2+ overload of cardiomyocytes. We therefore examined whether Ca2+ stimulates Na,K-pump activity in sheep cardiac Purkinje fibers. By removing Ca2+, Mg2+ and K+, the fibers depolarized and aiNa rose to 70 mM. After addition of 6 mM Mg2+ and lowering extracellular Na2+ to 29 mM, 30mM Rb+ was added, and over 10-15 min aiNa recovered to 3-7 mM. Two load-recovery cycles were conducted in 10 fibers. During one of the cycles Ca2+ (0.1-1.0 mM) was added before Rb+, causing a contracture. During recovery aiNa fell faster during Ca2+ contracture than in control cycles. Between 30 and 20 mM the rates were -10.0+/-1.6 and -5.4+/-0.6 mM/min, respectively (P<0.05). In Ca2+-exposed fibers tension fell almost parallel with aiNa. Na, K-pump reactivation caused membrane potential (Vm) to hyperpolarize transiently to -70 mV. Ca2+ did not affect membrane conductance. For a given aiNa during reactivation, Vm was more negative during Ca2+ contracture and depolarized faster (P<0.05). Intracellular pH (pHi) fell from 7.11+/-0.05 to 6.92+/-0.08 (n.s.) during control load-recovery cycles and was 6.83+/-0.14 at the end of the Ca2+ cycles. ATP content of the fibers did not change significantly through two complete load-recovery cycles, but creatine phosphate (CrP) fell by about 40%. By fitting the data to a model incorporating the Hill equation we show that during Ca2+-induced contracture maximum Na,K-pump rate (Vmax) was increased by about 40% and aiNa that causes 50% pump activation (k0.5) was lowered from 21. 2+/-1.6 to 15.5+/-1.4 mM.
Asunto(s)
Calcio/farmacología , Ramos Subendocárdicos/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfato/análisis , Animales , Estimulación Eléctrica , Activación Enzimática/efectos de los fármacos , Glucógeno/análisis , Concentración de Iones de Hidrógeno , Activación del Canal Iónico/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Microelectrodos , Fosfocreatina/análisis , Potasio/metabolismo , Ramos Subendocárdicos/enzimología , Ovinos , Sodio/metabolismo , Sodio/farmacologíaRESUMEN
The protein product of the myotonic dystrophy (DM) gene is a putative serine-threonine protein kinase (DM kinase). Previous reports have characterized the DM gene product as various 50-62-kDa proteins. The predicted protein size from DM cDNA sequence is 69 kDa. We therefore expressed a full-length recombinant human DM kinase protein and compared its size and expression to heart, cardiac Purkinje fibers, and skeletal muscle from normal and DM subjects. Recombinantly expressed DM kinase and endogenous DM kinase in human heart, displayed two immunoreactive DM kinase proteins with apparent molecular sizes of 71 and 80 kDa, suggesting that these prior reports are incorrect. In cardiac Purkinje fibers the 71-kDa protein was the major form, and in skeletal muscle the 80-kDa protein was the major form. Immunostaining showed DM kinase localized to neuromuscular junctions in skeletal muscle and intercalated discs in heart and Purkinje fibers. DM subjects showed low abundance of DM kinase in heart and skeletal muscle, suggesting haplotype insufficiency as a potential mechanism for disease expression. These studies describe differential expression of two protein forms of DM kinase, which are localized to specialized cellular structures associated with impulse transmission.
Asunto(s)
Expresión Génica , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/biosíntesis , Animales , Secuencia de Bases , Línea Celular , Membrana Celular/enzimología , Chlorocebus aethiops , Clonación Molecular , Cartilla de ADN , ADN Complementario , Humanos , Immunoblotting , Inmunohistoquímica , Riñón , Datos de Secuencia Molecular , Peso Molecular , Músculo Esquelético/enzimología , Miocardio/enzimología , Distrofia Miotónica/enzimología , Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Ramos Subendocárdicos/enzimología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Fracciones Subcelulares/enzimología , TransfecciónRESUMEN
1. The effects of phorbol esters on canine Purkinje fibers were examined using conventional microelectrode techniques. 2. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and 4-beta-phorbol-12,13-dibutyrate (PDB), which are specific activators of protein kinase C (PKC), decreased the action potential amplitude and the maximum rate of depolarization (Vmax) at 3 x 10(-7) M or higher. These phorbol esters had little effect on the resting potential. 3. PDB (1-3 x 10(-7) M) also reduced the contractile force, accompanied with initial increase (in 5 out of 8 experiments), whereas TPA did not decrease it to any significant extent. 4. An inactive analog of phorbol esters, 4-alpha-phorbol-12,13-didecanoate (PDD), decreased the action potential amplitude and Vmax, and slightly increased the action potential duration. However, PDD failed to produce any inotropic effect. 5. Post-rest potentiation of the contractile force after a rest from stimulation for 30 sec was inhibited in the presence of 3-10 x 10(-7) M TPA or 3 x 10(-7) M PDB. 6. Isoproterenol 10(-7) M augmented the action of PDB 3 x 10(-7) M. 7. These results suggest that activation of PKC may modulate myocardial Ca2+ homeostasis and influence the excitation-contraction process.
Asunto(s)
Forbol 12,13-Dibutirato/farmacología , Ésteres del Forbol/farmacología , Proteína Quinasa C/metabolismo , Ramos Subendocárdicos/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Calcio/metabolismo , Carcinógenos/farmacología , Perros , Homeostasis/efectos de los fármacos , Microelectrodos , Contracción Miocárdica/efectos de los fármacos , Ramos Subendocárdicos/enzimologíaRESUMEN
The results with the K+ channel blocking compounds are encouraging because they show a slight structural change in a parent blocking compound can dramatically alter the type of K+ channel blocked by a drug. Our work continues in an effort to determine a more detailed relationship between structural features of a blocker and the type of K+ channel effected. We hope to be able to reveal the requirements for potent and specific blockers of each of these channel types. Selective blockade of these, and other, K+ channels in heart and smooth muscle cells, a goal suggested by the preliminary results presented here, may provide useful tools for more detailed studies of K+ channels. Our experiments also show that temperature can be used to separate the beta-adrenergic regulation of ICa and IDR suggesting different modulatory mechanisms. The results with forskolin show this separation exists at the level of elevated cAMP. This suggests that the K+ and Ca++ channel proteins may be phosphorylated by the same cAMP-dependent protein kinase but with different temperature-sensitive kinetics. It will be interesting to see if this temperature-sensitivity extends to phosphorylation of the channel during intracellular application of cAMP-dependent protein kinase.
Asunto(s)
Miocardio/metabolismo , Canales de Potasio/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Antiarrítmicos/farmacología , Bovinos , Cobayas , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/efectos de los fármacos , Técnicas In Vitro , Isoproterenol/farmacología , Potenciales de la Membrana/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Ramos Subendocárdicos/efectos de los fármacos , Ramos Subendocárdicos/enzimología , Compuestos de Amonio Cuaternario/farmacología , TemperaturaRESUMEN
In comparison to the working myocytes no remarkable differences in the localization of adenylate cyclase (AC) and guanylate cyclase (GC) were found. The sarcolemmal plasma membrane of the AV node and the Purkinje fibers was the main site of the activity of these two enzymes. GC activity was additionally found at peripheral junctional couplings. Stimulation by hormones and specific activators for both AC and GC was demonstrated with difficulty. The reasons may be more the cytochemical preparation technique than the specificity of the AC and GC in the conducting tissue.
Asunto(s)
Adenilil Ciclasas/metabolismo , Guanilato Ciclasa/metabolismo , Sistema de Conducción Cardíaco/enzimología , Animales , Nodo Atrioventricular/enzimología , Histocitoquímica , Técnicas In Vitro , Ramos Subendocárdicos/enzimología , Conejos , Sarcolema/enzimologíaRESUMEN
Canine Purkinje fibers were isolated by microdissection and analyzed for four enzymes of glycogen metabolism and eight related metabolites. Purkinje fiber glycogen levels were very high, confirming earlier reports. Glycogen synthesizing enzymes, glycogen synthase and UDP glucose pyrophosphorylase, were on the average 47 and 70% higher, respectively, in Purkinje fibers than in myocardium. Phosphorylase activity was approximately equal in the two tissue types, and phosphoglucomutase was 31% lower in Purkinje fibers. The metabolites of glycogen 6-phosphate were all higher in Purkinje fibers (P less than 0.001), but glucose 1,6-bisphosphate was lower by 50%. Phosphocreatine and ATP remained high in Purkinje fibers during 2 min of ischemia, while the phosphocreatine level in myocardium was falling by 75%. The results of this study suggest that the high glycogen synthetic capability, high precursor levels, and overall lower metabolic rate in Purkinje fibers compared with myocardium may explain the much higher glycogen levels.
Asunto(s)
Glucógeno/metabolismo , Sistema de Conducción Cardíaco/enzimología , Ramos Subendocárdicos/enzimología , Adenosina Trifosfato/metabolismo , Animales , Perros , Glucosa-6-Fosfato , Glucofosfatos/metabolismo , Glucógeno Sintasa/metabolismo , Fosfocreatina/metabolismo , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Uridina Difosfato Glucosa/metabolismo , Uridina Trifosfato/metabolismoRESUMEN
Single canine Purkinje cells were isolated by microdissection and analyzed quantitatively for six enzymes of energy metabolism. Subendocardial Purkinje cells were clearly distinguishable morphologically and biochemically from adjacent myocardium and had enzyme levels comparable with extramural Purkinje cells. Oxidative enzymes, citrate synthase, malate dehydrogenase, and beta-hydroxyacyl CoA dehydrogenase were 40-60% lower in Purkinje cells than in myocardium. Lactate dehydrogenase was also 40% lower, but the other glycolytic enzymes, hexokinase and phosphofructokinase, were similar in level in myocardium and Purkinje cells. The results of this study show that it is completely practicable to apply quantitative histochemical analysis to biochemical study of the cardiac conducting system.
Asunto(s)
Sistema de Conducción Cardíaco/enzimología , Miocardio/enzimología , Ramos Subendocárdicos/enzimología , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Animales , Separación Celular , Citrato (si)-Sintasa/metabolismo , Perros , Femenino , Hexoquinasa/metabolismo , Histocitoquímica , L-Lactato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/metabolismo , Masculino , Músculos/enzimología , Fosfofructoquinasa-1/metabolismo , Ramos Subendocárdicos/citologíaRESUMEN
The previous observations of differences between different cardiac regions (ventricular myocardium, atrial myocardium, Purkinje fibre system) with respect to the maturation of the M-line region and the establishment of mature metabolic characteristics, have been extended. It was found that M-line maturation proceeds differently also between different regions of the conduction system. The M-line proteins, myomesin and MM-creatine kinase, were detected earlier, by means of immunohistochemistry, in the AV bundle and bundle branch cells than in the AV node cells. Also, a difference was observed in large foetuses. Striations in the AV node were less evident than in the AV bundle and the bundle branches in sections incubated with antibodies against myomesin as well as against MM-creatine kinase. Using enzyme histochemistry it was observed that the differences in metabolic properties between the AV node, the AV bundle and the bundle branches on the one hand, and the ordinary myocardium on the other, of adult hearts, are not established at the early stages. No clear difference in activity of succinate dehydrogenase was seen between the conduction tissues and the ordinary myocardium in the foetal hearts, while the conduction tissues showed a lower activity in the adult hearts. Furthermore, the pattern of activity of mitochondrial glycerol-3-phosphate dehydrogenase between the conduction tissues and the atrial and ventricular myocardium was quite different in early foetal stages compared with the adult stage.
Asunto(s)
Creatina Quinasa/análisis , Corazón Fetal/anatomía & histología , Glicerolfosfato Deshidrogenasa/análisis , Sistema de Conducción Cardíaco/embriología , Animales , Nodo Atrioventricular/anatomía & histología , Nodo Atrioventricular/embriología , Nodo Atrioventricular/enzimología , Bovinos , Conectina , Femenino , Técnica del Anticuerpo Fluorescente , Secciones por Congelación , Sistema de Conducción Cardíaco/anatomía & histología , Sistema de Conducción Cardíaco/enzimología , Histocitoquímica , Mitocondrias Musculares/enzimología , Proteínas Musculares/análisis , Ramos Subendocárdicos/anatomía & histología , Ramos Subendocárdicos/embriología , Ramos Subendocárdicos/enzimología , Succinato Deshidrogenasa/análisisRESUMEN
In this communication, the results of applying various histochemical techniques for the localization of oxidoreductases, transferases, hydrolases and isomerases in the human heart are presented. The Purkinje fibres of the atrioventricular conducting system of the human heart differ from the myocardium proper in containing a slightly higher activity of most of the glycolytic and gluconeogenetic enzymes investigated. The relatively higher activity of 6-phosphofructokinase, the key enzyme in anaerobic carbohydrate metabolism, is especially noteworthy. On the other hand, the activities of some of the enzymes that play a part in the aerobic energy metabolism is slightly less than those in the myocardium fibres. As for the activity of the NADPH regenerating enzymes, the activity of 6-phosphogluconate dehydrogenase and malate dehydrogenase (oxaloacetate-decarboxylating) is somewhat higher, and the activity of glucose-6-phosphate dehydrogenase similar, in the Purkinje fibres compared to that in the myocardial fibres. The activity of myosin ATPase is similar for both types of fibre. Likewise, the fibres of the conducting system and of the myocardium show a similar activity of acid phosphatase, beta-glucuronidase, non-specific naphthylesterase and peroxidase. The neurogenic function of the conducting system of the human heart was demonstrated by the high activity of acetylcholinesterase in the Purkinje fibres and in the atrioventricular node. All these histochemical findings in Purkinje fibres are similar at widely differing levels of the conducting system.
Asunto(s)
Sistema de Conducción Cardíaco/enzimología , Ramos Subendocárdicos/enzimología , Acetilcolinesterasa/metabolismo , Metabolismo Energético , Gluconeogénesis , Glucólisis , Histocitoquímica , Humanos , Miocardio/enzimología , Oxidorreductasas/metabolismoRESUMEN
A histochemical multi-step technique for the demonstration of phosphofructokinase activity in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the non-structurally bound enzyme into the medium during incubation. In the histochemical system the enzyme converts the substrate D-fructose-6-phosphate to D-fructose-1,6-diphosphate, which in turn is hydrolyzed by exogenous and endogenous fructose diphosphate aldolase to dihydroxyacetone phosphate and D-glyceral-dehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triosephosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase into 1,3-diphospho-D-glycerate. Concomitantly the electrons are transported via NAD+, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.
Asunto(s)
Músculos/enzimología , Miocardio/enzimología , Fosfofructoquinasa-1/metabolismo , Anaerobiosis , Animales , Bovinos , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Histocitoquímica , Humanos , NAD/metabolismo , Nitroazul de Tetrazolio/metabolismo , Ramos Subendocárdicos/enzimología , Ratas , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
Phosphorylase activity has been investigated histochemically under controlled conditions in the specialized tissue and the general myocardium of goat heart using glucose-1-phosphate as the substrate. It has been observed that the phosphorylase content of the nodal and conducting tissue is very high as compared to the general myocardium. The phosphorylase content of the cardiac neural elements is also high. Since the connective tissue which surrounds the nodal and conducting tissue has almost no phosphorylase activity, the nodal and conducting tissue is sharply demarcated by the histochemical reaction for phosphorylase. This reaction can, therefore, be used as a specific and expedient method for the localization of specialized tissue as an exploratory and confirmatory test.
Asunto(s)
Miocardio/enzimología , Fosforilasas/aislamiento & purificación , Animales , Nodo Atrioventricular/enzimología , Fascículo Atrioventricular/enzimología , Cabras , Histocitoquímica , Ramos Subendocárdicos/enzimología , Nodo Sinoatrial/enzimologíaRESUMEN
In this communication the results of applying various histochemical semipermeable membrane techniques to the localization of several enzymes in bovine and porcine heart are presented. The Purkinje fibers of the atrioventricular conducting system of the bovine heart differ from the myocardium proper in containing a greater activity of the glycolytic and gluconeogenetic enzymes--lactate dehydrogenase, glyceraldehyde-phosphate dehydrogenase, hexokinase, glucosephosphate isomerase and phosphoglucomutase, and less activity of the aerobic enzymes--NADH: nitroBT oxidoreductase and isocitrate dehydrogenase (NADP+). The metabolic reactions obtained with Purkinje fibers of the porcine heart are less pronounced. These histochemical findings are in accordance with the impression that Purkinje fibers, compared with the common myocardial fibers, have a higher rate of anaerobic metabolism and a lower rate of aerobic metabolism. The activity of the NADPH regenerating enzymes glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating), and the activity of acid hydrolases such as non-specific esterase and acid phosphatase is higher in the Purkinje fibers of both the bovine and porcine heart.
Asunto(s)
Sistema de Conducción Cardíaco/enzimología , Oxidorreductasas/metabolismo , Ramos Subendocárdicos/enzimología , Animales , Bovinos , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Histocitoquímica , Isoenzimas , L-Lactato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/metabolismo , NADH Tetrazolio Reductasa/metabolismo , PorcinosRESUMEN
Choline acetyltransferase is the enzyme that catalyzes the biosynthesis of acetylcholine, the neurotransmitter of the pre- and postganglionic parasympathetic system. To assess the extent of parasympathetic innervation, enzyme activity was measured in specialized and contractile regions throughout the guinea pig heart. Enzyme activity in the right atrial appendage was 137 nmol g(-1) hr(-1). Activity was greatest in the region of the sinoatrial node (187 nmol g(-1) hr(-1). Also, enzyme activity was high in the regions of the atrioventricular node (153 nmol g(-1) hr(-1), the proximal conduction bundles (133 nmol g(-1) hr(-1), and the base of the anterior papillary muscle of the right ventricle (179 nmol g(-1) hr(-1), which contains the moderator band and Purkinje fibers. In contrast, the enzyme activity in the inferior interventricular septum and the free walls of the right and left ventricles, which are more predominantly contractile tissue, was 67 +/- 6,108 +/- 14, and 56 +/- 11 nmol g(-1) hr(-1), respectively. This activity is significantly lower than in the right atrial appendage. These results suggest that the density of parasympathetic innervation is similar in all the components of the conduction system, from the sinoatrial node to Purkinje tissues. Furthermore, the parasympathetic innervation of regions specialized for conduction is up to four times more dense than that of contractile regions.
Asunto(s)
Colina O-Acetiltransferasa/análisis , Sistema de Conducción Cardíaco/enzimología , Corazón/inervación , Miocardio/enzimología , Animales , Nodo Atrioventricular/enzimología , Cobayas , Atrios Cardíacos/enzimología , Tabiques Cardíacos/enzimología , Ventrículos Cardíacos/enzimología , Masculino , Músculos Papilares/enzimología , Sistema Nervioso Parasimpático/enzimología , Ramos Subendocárdicos/enzimología , Nodo Sinoatrial/enzimologíaRESUMEN
The ultrastructure of the rat cerebellar cortex and the activity of succinic dehydrogenase were examined during methionine sulphoximine (MSO)-provoked convulsions. The animals were killed 3, 6 and 12 hours after the injection of 600 mg/kg of MSO. Convulsions appeared 4--5 hours, status epilepticus developed 8-9 hours after the injection. Progressive ischaemic changes of Purkinje cells could be observed, with condensation of the nucleus and a density of the cytoplasmic matrix. The cisternae of the Golgi complex and endoplasmic reticulum showed some degree of dilation. The basis of Purkinje cells was surrounded by distorted axons and terminals that had lost in most cases the synaptic vesicles, and by clear spaces due to the swollen glial processes. Three to six hours after MSO injection, succinic dehydrogenase activity increased in the mitochondria of Purkinje cells. After the appearance of seizures the enzyme activity decreased. Twelve hours after the injection the enzyme activity recovered to a certain extent.
Asunto(s)
Cerebelo/ultraestructura , Convulsiones/patología , Animales , Cerebelo/enzimología , Histocitoquímica , Masculino , Metionina Sulfoximina , Ramos Subendocárdicos/enzimología , Ratas , Convulsiones/inducido químicamente , Convulsiones/enzimología , Succinato Deshidrogenasa/metabolismoAsunto(s)
Bloqueo Cardíaco/inmunología , Enfermedades Autoinmunes/complicaciones , Fascículo Atrioventricular/citología , Fascículo Atrioventricular/inmunología , Cardiomiopatías/etiología , Enfermedad de Chagas/complicaciones , Enfermedad de Chagas/inmunología , Enfermedad Crónica , Técnica del Anticuerpo Fluorescente , Bloqueo Cardíaco/etiología , Bloqueo Cardíaco/patología , Humanos , Inmunidad Celular , Ramos Subendocárdicos/citología , Ramos Subendocárdicos/enzimología , Ramos Subendocárdicos/inmunologíaRESUMEN
The effects of dimethyl quaternary propranolol (UM-272) on electrophysiologic properties of canine cardiac Purkinje fibers (PF) were studied using standard microelectrode techniques. In PF superfused with Tyrode's solution, the effects of UM-272, 10- minus 7 to 10- minus 5 M, were studied. In other experiments, UM-272, 3 mg/kg, was injected into donor animals whose blood was used to superfuse isolated PF. Antiarrhythmic concentrations of UM-272 decreased action potential (AP) amplitude, phase 0 upstroke velocity, membrane responsiveness, AP duration and the effective refractory period (the decrease in the effective refractory period was less than that in AP duration). Automaticity of spontaneously firing PF was suppressed. As perfusate potassium concentration was increased, the magnitude of UM-272 effect was accentuated. When the actions of equimolar concentrations of UM-272 and propranolol on PF AP were compared, propranolol attained a steady-state effect more rapidly and tended to depress the AP more markedly. UM-272 did not block epinephrine-induced increases in PF automaticity or the adenylate cyclase activation of PG homogenates induced by epinephrine. These studies indicate that UM-272 has direct effects on the PF AP similar to those of propranolol, but lacks the beta blocking properties of the latter.