RESUMEN
Loss of lung units due to pneumonectomy stimulates growth of the remaining lung. It is generally believed that regenerative lung growth involves only alveoli but not airways, a dissociated response termed "dysanaptic growth." We examined the structural response of respiratory bronchioles in immature dogs raised to maturity after right pneumonectomy. In another group of adult dogs, we also examined the effect of preventing mediastinal shift after right pneumonectomy on the response of respiratory bronchioles. In immature dogs after pneumonectomy, the volume of the remaining lung increased twofold, with no change in volume density, numerical density, or mean diameter of respiratory bronchiole, compared with that in the control lung. The number of respiratory bronchiole segments and branch points increased proportionally with lung volume. In adult dogs after pneumonectomy, prevention of mediastinal shift reduced lung strain at a given airway pressure, but lung expansion and regenerative growth of respiratory bronchiole were not eliminated. We conclude that postpneumonectomy lung growth is associated with proliferation of intra-acinar airways. The proportional growth of acinar airways and alveoli should optimize gas exchange of the regenerated lung by enhancing gas conductance and mixing efficiency within the acinus.
Asunto(s)
Bronquios/fisiopatología , Regeneración/fisiología , Animales , Peso Corporal , Bronquios/patología , Broncografía , Perros , Pulmón/diagnóstico por imagen , Pulmón/fisiopatología , Mediciones del Volumen Pulmonar , Masculino , Mediastino/diagnóstico por imagen , Neumonectomía , Periodo Posoperatorio , Radiografía TorácicaRESUMEN
Previous studies suggest that the desensitization and downregulation of beta 1-adrenergic receptors (beta 1-AR) in the failing heart are the result of the elevated plasma catecholamine levels associated with this disease. To examine norepinephrine (NE)-induced regulation of cardiac adrenergic receptors, rats were infused with l-NE (200 micrograms.kg-1.h-1 for 7 days) or vehicle (0.001 N HCl) by implantation of osmotic minipumps. The technique of coverslip autoradiography was used to quantify alpha 1-adrenergic receptors (alpha 1-AR), beta 1-AR, and beta 2-AR in different tissue compartments of rat hearts. For measurement of beta-AR binding, sections were incubated with 70 pM [125I]iodocyanopindolol (ICYP) alone or in the presence of 5 microM dl-propranolol or 5 x 10(-7) M CGP-20712A (a beta 1-antagonist) and then set up for autoradiography. [3H]prazosin (1 nM) with or without phentolamine was used to study alpha-AR binding. Chronic infusion of NE induced a greater downregulation of beta 2-AR compared with beta 1-AR in all regions studied, including atrial and ventricular myocytes, coronary arterioles, and connective tissue. An 18% loss of beta 1-AR was seen only in atrial myocytes; beta 1-AR density actually increased 28% in ventricular myocytes following NE infusion. There was a 15% decrease in alpha 1-AR in ventricular myocytes, whereas no change in alpha 1-AR density was seen in myocardial arterioles. Our study demonstrates that beta 2-AR are more susceptible to NE-induced downregulation than beta 1-AR. Thus other mechanisms may be involved in the selective downregulation of beta 1-AR in certain forms of heart failure.
Asunto(s)
Miocardio/metabolismo , Norepinefrina/farmacología , Receptores Adrenérgicos alfa/efectos de los fármacos , Receptores Adrenérgicos beta/efectos de los fármacos , Animales , Autorradiografía , Ventrículos Cardíacos , Masculino , Miocardio/patología , Necrosis , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos beta/metabolismoRESUMEN
We have previously demonstrated that a treatment regimen of slow-release sodium fluoride (SRNaF) and continuous calcium citrate increases lumbar bone mass, improves cancellous bone material quality, and significantly reduces vertebral fracture rate in osteoporotic patients. In order to assess whether such treatment also improves trabecular structure, we quantitated cancellous bone connectivity before and following 2 years of therapy with SRNaF in 23 patients with osteoporosis and vertebral fractures. In addition, we performed bone histomorphometry on the same sections used for connectivity measurements. There was a significant increase in L2-L4 bone mineral density during therapy (0.827 +/- 0.176 g/cm2 SD to 0.872 +/- 0.166, p = 0.0004). Significant histomorphometric changes were represented by increases in mineral apposition rate (0.6 +/- 0.4 microns/d to 1.1 +/- 0.7, p = 0.0078) and adjusted apposition rate (0.4 +/- 0.3 microns/d to 0.6 +/- 0.4, p = 0.016). On the other hand, trabecular spacing significantly declined (from 1375 +/- 878 microns to 1052 +/- 541, p = 0.05). Two-dimensional quantitation of trabecular struts on iliac crest histological sections disclosed significant increases in mean node number per mm2 of cancellous tissue area (0.22 +/- 0.12 vs. 0.39 +/- 0.27, p = 0.0077), the mean node to free-end ratio (0.23 +/- 0.21 vs. 0.41 +/- 0.46, p < 0.05), and in the mean node to node strut length per mm2 of cancellous area (0.098 +/- 0.101 vs. 0.212 +/- 0.183, p < 0.01). There were no significant changes in any of the measurements associated with free-end number or free-end to free-end strut length.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Densidad Ósea/efectos de los fármacos , Osteoporosis Posmenopáusica/tratamiento farmacológico , Osteoporosis/tratamiento farmacológico , Fluoruro de Sodio/farmacología , Fracturas de la Columna Vertebral/tratamiento farmacológico , Absorciometría de Fotón , Adulto , Anciano , Citratos/administración & dosificación , Citratos/farmacología , Citratos/uso terapéutico , Ácido Cítrico , Simulación por Computador , Preparaciones de Acción Retardada , Femenino , Estudios de Seguimiento , Fracturas Espontáneas/complicaciones , Fracturas Espontáneas/tratamiento farmacológico , Fracturas Espontáneas/prevención & control , Humanos , Ilion/efectos de los fármacos , Ilion/fisiología , Procesamiento de Imagen Asistido por Computador , Masculino , Persona de Mediana Edad , Osteoporosis/complicaciones , Osteoporosis Posmenopáusica/complicaciones , Fluoruro de Sodio/administración & dosificación , Fluoruro de Sodio/uso terapéutico , Espectrometría de Fluorescencia , Fracturas de la Columna Vertebral/complicaciones , Fracturas de la Columna Vertebral/prevención & controlRESUMEN
A small RNA encoded within the nucleus is an essential subunit of a RNA processing endonuclease (RNase MRP) hypothesized to generate primers for mitochondrial DNA replication from the heavy strand origin of replication. Controversy has arisen, however, concerning the authenticity of an intramitochondrial pool of MRP RNA, and has called into question the existence of pathways for nucleo-mitochondrial transport of nucleic acids in animal cells. In an effort to resolve this controversy, we combined ultrastructural in situ hybridization and biochemical techniques to assess the subcellular partitioning of MRP RNA. Cryosections of mouse cardiomyocytes were hybridized with biotin-labeled RNA probes complementary to different regions of MRP RNA and varying in length from 115 to 230 nucleotides, followed by immunogold labeling. In addition, we transfected mouse C2C12 myogenic cells with constructs bearing mutated forms of the mouse MRP RNA gene and compared the relative abundance of the resulting transcripts to that of control RNAs within whole cell and mitochondrial fractions. In the former analysis we observed preferential localization of MRP RNA to nucleoli and mitochondria in comparison to the nucleoplasm and cytoplasm. In the latter series of studies we observed that wild-type MRP RNA partitions to the mitochondrial fraction by comparison to other RNA transcripts that are localized to the extramitochondrial cytoplasmic space (28S rRNA) or to the nucleoplasm (U1 snRNA). Deletions within 5' or 3' regions of the MRP RNA gene produced transcripts that remain competent for mitochondrial targeting. In contrast, deletion of the midportion of the coding region (nt 118 to 175) of the MRP RNA gene resulted in transcripts that fail to partition to the mitochondrial fraction. We conclude that an authentic intramitochondrial pool of MRP RNA is present in these actively respiring cells, and that specific structural determinants within the MRP RNA molecule permit it to be partitioned to mitochondria.
Asunto(s)
Endorribonucleasas/metabolismo , Mitocondrias Cardíacas/química , Mitocondrias Musculares/química , ARN/análisis , Animales , Elementos sin Sentido (Genética) , Secuencia de Bases , Línea Celular , Nucléolo Celular/química , Nucléolo Celular/ultraestructura , Citoplasma/química , Citoplasma/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Microscopía Inmunoelectrónica , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/ultraestructura , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/ultraestructura , Datos de Secuencia Molecular , ARN/genética , ARN/metabolismoRESUMEN
This study characterizes the sequential alterations of, and relations between, multiple electrolytes in cytoplasm, mitochondria, and whole cells during hypoxia and on reoxygenation in isolated neonatal rat ventricular myocytes. Subcellular electrolyte content and distribution were measured by electron probe x-ray microanalysis, membrane phospholipid degradation by tritiated arachidonic acid release, and cell morphology by electron microscopy. At 1-2 hours of hypoxia, the myocyte population showed a loss of cytoplasmic potassium, magnesium, and chlorine without alteration of cytoplasmic sodium or calcium. Mitochondria showed increased potassium with unchanged magnesium content. There was no morphological evidence of cell injury or tritiated arachidonic acid release. At 3-5 hours of hypoxia, the myocyte population showed a further loss of cytoplasmic potassium and magnesium and an increase in cytoplasmic sodium, chlorine, and calcium. At a single-cell level, the increase in cytoplasmic sodium preceded the increase in cytoplasmic calcium. Mitochondria showed increased sodium and chlorine and decreased magnesium before increased calcium content; potassium loss was manifest only at 5 hours of hypoxia. At 3-5 hours of hypoxia, there was also tritiated arachidonic acid release and morphological evidence of cell injury. Reoxygenation for 1 hour after 5 hours of hypoxia partially reversed the mean alterations of all electrolytes, except calcium, in the cytoplasm of the myocyte population, whereas analysis was required at a single-cell level to show a partial reversal in calcium levels in cytoplasm of reoxygenated cells. Reoxygenation for 1 hour after 5 hours of hypoxia partially reversed the mean alterations of all electrolytes, including calcium, in the mitochondria of the myocyte population. Recovery of potassium in the cytoplasm correlated with reduction of mitochondrial calcium content on reoxygenation and best predicted recovery of cellular homeostasis of sodium, chlorine, magnesium, and calcium. This study demonstrates that in this experimental model of hypoxia 1) initial losses of cytoplasmic potassium and magnesium occur in the absence of cell injury; 2) increases of sodium, chlorine, and calcium occur in association with cell injury, with sodium increasing before calcium; 3) membrane phospholipid degradation and electrolyte derangement, including increased calcium, may contribute to reversible and irreversible phases of cell injury; 4) analysis of calcium at a subcompartmental level and at a single-cell level is required to correlate reduction of calcium on reoxygenation with recovery of cell homeostasis; 5) reduction of calcium content in mitochondria may predict recovery of cell homeostasis; and 6) recovery of potassium on reoxygenation best predicts recovery of cell membrane function and cell homeostasis.
Asunto(s)
Electrólitos/metabolismo , Hipoxia/metabolismo , Oxígeno/farmacología , Fracciones Subcelulares/metabolismo , Animales , Animales Recién Nacidos , Separación Celular , Microanálisis por Sonda Electrónica , Ventrículos Cardíacos , Miocardio/citología , Miocardio/metabolismo , Miocardio/ultraestructura , RatasRESUMEN
Oxygen-derived free radical production has been documented to occur on reperfusion of the ischemic myocardium. Intracellular ionic calcium ([Ca++]i) levels in isolated adult rat cardiac myocytes exposed to free radicals were evaluated using fura-2. The effect of different time periods of free radical exposure on altering [Ca++]i was examined. Myocytes were either exposed to the free radical generating system continuously or exposed for 5 or 10 minutes and then returned to the HEPES buffer. Myocytes maintained in HEPES buffer or the HEPES buffer containing purine and iron-loaded transferrin continued to stimulate, exhibited relatively uniform 340/380 nm ratios and maintained a rod shape. Continuous exposure to free radicals resulted in a significant increase in [Ca++]i. Myocytes became unresponsive to stimulation at 31 +/- 7 (SE) minutes and eventually exhibited contracture. Exposure to the free radical generating system for 10 minutes resulted in a response similar to continuous exposure. Myocytes exposed to the generating system for 5 minutes exhibited regular calcium transients for 55 +/- 5 minutes. Thus, even a brief period of free radical exposure alters calcium flux and may induce subsequent myocardial damage.
Asunto(s)
Calcio/fisiología , Corazón/efectos de los fármacos , Especies Reactivas de Oxígeno/farmacología , Animales , Calcio/metabolismo , Separación Celular , Estimulación Eléctrica , Electrofisiología , Ventrículos Cardíacos , Iones , Miocardio/citología , Miocardio/metabolismo , Concentración Osmolar , Ratas , Factores de TiempoRESUMEN
Intracellular calcium ([Ca2+]i) elevation may mediate cardiac arrhythmias. However, direct measurement of the rapid alterations of [Ca2+]i on a beat-to-beat basis using fast temporal resolution and without signal averaging in the spontaneously beating in vivo heart is lacking. Furthermore, data from an isolated spontaneously beating myocyte preparation that develops arrhythmia similar to that in the in vivo heart are unavailable. We measured rapid changes of [Ca2+]i with fast temporal resolution in isolated spontaneously beating neonatal rat ventricular myocytes with cell-to-cell communication and characterized the interrelation between [Ca2+]i and arrhythmia. An elevated extracellular calcium ([Ca2+]o) concentration of 10.8 mM induced premature beats, a rapid beating rate (tachyarrhythmia), and chaotic or fibrillatory beating activity in a small group of myocytes. [Ca2+]i levels during systole increased from the nanomolar to micromolar concentration range before arrhythmia development. Spontaneous oscillations of [Ca2+]i during diastole could evoke a spontaneous tachyarrhythmia. In the presence of [Ca2+]i elevation, a spontaneous tachyarrhythmia could induce severe [Ca2+]i overload. Reduction of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid AM (5 microM) in the presence of 10.8 mM [Ca2+]o reversed the arrhythmia. In single ventricular myocytes superfused with 10.8 mM [Ca2+]o, oscillations of membrane potential characteristic of transient inward current occurred that were prevented by ryanodine (0.1 microM), an inhibitor of Ca2+ flux across the sarcoplasmic reticulum. This study characterizes 1) an isolated multicellular myocyte model of arrhythmia similar to that evident in in vivo hearts, 2) elevation of [Ca2+]i with systolic [Ca2+]i levels of 1-3 microM and diastolic [Ca2+]i oscillations before the initiation of arrhythmia, 3) tachyarrhythmia as a cause of severe [Ca2+]i overload, which may be important in the perpetuation and degeneration of arrhythmias, and 4) reversal of arrhythmia with reduction of [Ca2+]i. The results in the isolated myocyte model may have relevance to the generation and perpetuation of certain cardiac arrhythmias associated with calcium overload.
Asunto(s)
Arritmias Cardíacas/etiología , Calcio/metabolismo , Corazón/fisiología , Miocardio/metabolismo , Animales , Animales Recién Nacidos , Arritmias Cardíacas/fisiopatología , Células Cultivadas , Electrofisiología , Potenciales de la Membrana , Miocardio/citología , Ratas , Rianodina/farmacología , Retículo Sarcoplasmático/metabolismoRESUMEN
New methods of tissue preparation were developed to study the morphology and distribution of calcium ions in duodenal enterocytes from normal, rachitic, and vitamin D-replete (either cholecalciferol [CC] or 1,25-dihydroxycholecalciferol [1,25-DHCC] treated) chicks. Frozen hydrated sections were prepared from cryofixed tissues by ultracryomicrotomy at -125 degrees C. Sections were subsequently freeze-dried by increasing the temperature to -100 degrees C. The latter temperature was maintained throughout both the structural and elemental analyses. In cells from normal, rachitic, and vitamin D-treated [CC] animals the brush border from lanthanum-infused tissues was electron dense and calcium-lanthanum positive by x-ray analysis. In the absence of lanthanum, i.e., sucrose-infused duodena, the microvilli were still calcium positive. In the terminal web region of normal and CC-treated enterocytes, numerous, apparently interconnected, tubules and vesicles were seen. Vacuole-like structures were also seen. Such structures were especially prominent in the enterocytes from the vitamin-treated [CC] animals. Except for the vacuoles, the tubules and vesicles were electron dense in the lanthanum-infused duodena, and clear in sucrose-infused tissues. In both instances, the structures were calcium positive. Similar, but even larger structures were seen below the terminal web. Here however, the tubules and vesicles seemed to be organized into multiple complex interconnecting networks, i.e., tubulo-vesicular complexes. Both the tubules and the vesicles seemed to be interconnected via smaller channel-like entities. The extensiveness of this structure was better appreciated in the enterocytes from lanthanum-infused tissues, where it appeared similar in structure and complexity to an en face view of the sarcoplasmic reticulum of skeletal muscle. These intestinal complexes were less well developed, decreased in number, and quite often absent, in the apical cytoplasm of absorptive cells from rachitic chicks. In the enterocytes from animals treated for 24 hours with 1,25-DHCC, the same highly developed tubulo-vesicular networks were again seen in the enterocyte apical cytoplasm. They were even more developed in the 1,25-DHCC-treated animals. All structures were intensely calcium positive in enterocytes from both the lanthanum- and the sucrose-infused preparations. Numerous endocytotic (pinocytotic) vesicles were seen at the lumenal plasmalemma. Similar structures were also apparent in the terminal web region of the 1,25-DHCC-treated enterocytes. Exocytotic vesicles were seen at the apical aspect of the lateral cell membrane, below the level of the junctional complex. All components of this unique system contained high concentrations of calcium.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Duodeno/ultraestructura , Animales , Calcio/metabolismo , Pollos , Citoplasma/efectos de los fármacos , Citoplasma/ultraestructura , Duodeno/efectos de los fármacos , Duodeno/metabolismo , Microanálisis por Sonda Electrónica , Secciones por Congelación , Técnicas Histológicas , Microscopía Electrónica de Rastreo , Raquitismo/metabolismo , Raquitismo/patología , Vitamina D/farmacologíaRESUMEN
Oxygen-derived free radicals have been implicated in damage to membrane phospholipids leading to alterations in membrane function. The purpose of this study was to investigate alterations in intracellular ionic calcium (Ca2+) levels and Ca2+ transients, cellular morphology, conjugated diene levels, arachidonate release, and lactate dehydrogenase release resulting from the exposure of cultured neonatal rat ventricular myocytes to a xanthine oxidase catalyzed free radical generating system capable of producing superoxide and hydroxyl radicals. The ability of alpha-tocopherol to prevent alterations due to free radical exposure was investigated. For measurements of Ca2+, myocytes grown on coverslips for 3-4 days were loaded with fura-2/AM and studied by microspectrofluorometry. Control myocytes superfused with a physiological buffer or buffer containing purine and iron-loaded transferrin exhibited Ca2+ transients associated with spontaneous contractions. For control, buffer perfused myocytes (n = 4), the fura-2 340/380 ratios were 0.5 +/- 0.1 (mean +/- S.E.) and 1.6 +/- 0.03 at the minimum and maximum, respectively, of the Ca2+ transient, after 1 h of perfusion. Exposure to the free radical generating solution (n = 14) altered intracellular Ca2+. The 340/380 minimum ratio was 639% of the control value after approximately 30-70 mins with cessation of normal Ca2+ transients. Bleb development was associated with increased Ca2+. Myocytes reperfused with control medium continued to exhibit an elevated minimum fura-2 ratio at 687% of control. Myocytes pretreated with 10 microM alpha-tocopherol (n = 13) for 18-24 h and exposed to free radicals did not exhibit increases in intracellular Ca2+, having a minimum 340/380 ratio of 0.5 +/- 0.1 after 60-90 mins, and although myocytes often ceased contracting, they resumed spontaneous Ca2+ transients with control medium reperfusion and also maintained normal structure. Exposure of myocyte cultures to free radical generating solutions resulted in increased levels of conjugated dienes and increased release of [3H]arachidonate and lactate dehydrogenase compared to control values after 1 h. alpha-Tocopherol treatment attenuated the increase in conjugated diene levels, and the release of [3H]arachidonate and lactate dehydrogenase. Thus, free radicals alter intracellular Ca2+, conjugated dienes and membrane structure indicating their ability to induce altered ionic homeostasis in association with myocardial membrane damage. alpha-Tocopherol decreased free radical mediated injury.
Asunto(s)
Calcio/metabolismo , Lípidos de la Membrana/metabolismo , Miocardio/metabolismo , Fosfolípidos/metabolismo , Animales , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Células Cultivadas , Radicales Libres , Ventrículos Cardíacos/metabolismo , Oxígeno/metabolismo , RatasRESUMEN
The development of irreversible myocardial ischemic injury is associated with progressive degradation of membrane phospholipids, accumulation of arachidonate and other free fatty acids, and electrolyte derangements, including calcium accumulation. To study the relationship between arachidonate release and calcium loading during adenosine triphosphate (ATP) depletion in cardiac myocytes, the effects of two purported phospholipase inhibitors, mepacrine and U26,384, were evaluated. Cultured neonatal rat ventricular myocytes were pretreated for 90 minutes with 5 to 10 microM U26,384 (a steroidal diamine) or 10 to 50 microM mepacrine (an alkyl acridine) and then treated for 3 hours with 30 microM of the metabolic inhibitor, iodoacetic acid (IAA), with or without an additional dose of drug. IAA treatment resulted in a marked reduction in ATP level and a several-fold increase in free fatty acid radioactivity released from myocytes prelabeled with tritiated arachidonic acid (3H-AA). U26,384 produced substantial inhibition of the increased 3H-AA release, and was effective when given as a single pretreatment dose before IAA exposure or as continuous treatment before and during IAA exposure (for example, with 5 microM U26,384, the percentage of 3H-AA release versus IAA alone was 8% +/- 2% [SEM] [N = 15] for pretreatment only and 13% +/- 4% [N = 10] for continuous treatment). Mepacrine also resulted in significant reduction in 3H-AA release, but was more effective when given as continuous treatment (for example, with 50 microM mepacrine, the percentage of 3H-AA release versus IAA alone was 43% +/- 9% [N = 6] for pretreatment only and 22% +/- 7% [N = 9] for continuous treatment). More detailed analysis showed that U26,384 and mepacrine blocked the IAA-induced redistribution of 3H-AA into free fatty acids from other lipid species. Electron probe x-ray microanalysis of freeze-dried cryosections revealed marked electrolyte derangements in myocytes exposed to IAA, including a 24-fold increase in cellular Ca, a four fold increase in cellular Na, and a seven fold decrease in cellular K, and associated changes in cytoplasm and mitochondria. U26,384 treatment markedly reduced these electrolyte abnormalities, and maintained normal Ca levels in some protocols. Mepacrine treatment was less effective, but did produce normal Ca levels in 50% of myocytes. Prevention of IAA-induced cellular hypercontraction and blebbing also was observed. These data support the hypothesis that reduction of free fatty acid accumulation by inhibition of accelerated phospholipid degradation is associated with protection of myocytes from calcium loading and morphologic damage during inhibition of ene
Asunto(s)
Ácidos Araquidónicos/metabolismo , Calcio/metabolismo , Enfermedad Coronaria/metabolismo , Estrenos/farmacología , Corazón/efectos de los fármacos , Quinacrina/farmacología , Adenosina Trifosfato/metabolismo , Animales , Ácido Araquidónico , Células Cultivadas , Enfermedad Coronaria/patología , Microanálisis por Sonda Electrónica , Fosfolipasas A/antagonistas & inhibidores , RatasRESUMEN
Myocardial ischemic injury results in altered membrane integrity, energy depletion, and electrolyte shifts leading to accumulation of intracellular Ca. However, analysis of the direct effects of Ca accumulation is complicated by other concomitant cellular changes produced by ischemia. The purpose of this study was to examine the effects of Ca loading in rat papillary muscles produced by Na+-K+ pump inhibition in oxygenated K+-free buffer. Changes in contractile characteristics, high energy phosphate, and elemental concentrations of subcellular compartments were measured. Electron probe X-ray microanalysis was used to assess elemental concentrations in cryosections. After 3 h of Na+-K+ pump inhibition, resting tension (RT) increased to 164% and developed tension (DT) fell to 16.8% of control values. One hour after return to complete buffer, RT and DT partially recovered but remained significantly different from the 180 to 240-min values for the control muscles. Electron probe X-ray microanalysis showed increases in cytoplasmic and mitochondrial Na and Ca and a decrease in K during Na+-K+ pump inhibition. Mitochondrial Ca was greater than 100-fold greater than Ca in control mitochondria. Morphologically, the majority of cells showed ultrastructural damage. The mean ATP level was 20% of control. After 1 h of recovery, the cells appeared more heterogeneous, and the mean mitochondrial Ca decreased, whereas mean cytoplasmic Ca increased. Further statistical analysis showed a bimodal distribution for Na, Ca, K, Mg, and Cl, which coincided with the morphologically mixed population of cells. This suggests that replacement of extracellular K+ was associated with restored electrolyte gradients in some cells and the persistent or further alteration of electrolytes in others. These results suggest that variable Ca accumulation and associated ATP depletion without the compounding effects of ischemia lead to cell injury similar to reperfusion injury reported in ischemic myocardium.
Asunto(s)
Contracción Miocárdica , Músculos Papilares/fisiología , Canales de Potasio/fisiología , Canales de Sodio/fisiología , Adenosina Trifosfato/metabolismo , Animales , Electrólitos/metabolismo , Microanálisis por Sonda Electrónica , Femenino , Microscopía Electrónica de Rastreo , Músculos Papilares/citología , Músculos Papilares/ultraestructura , Ratas , Ratas EndogámicasRESUMEN
This study tested the hypothesis that the initiating mechanism is a major determinant of the response to calcium (Ca) accumulation in myocardium. Cultured neonatal rat ventriculocytes were exposed to Na+, K+ pump inhibition with 1 mM ouabain and metabolic inhibition with 20 mM 2-deoxy-D-glucose and 1 mM cyanide (DOG-CN) for up to 2 h. Microspectrofluorometry of myocytes loaded with fura-2 showed that ouabain resulted in a relatively rapid increase in [Ca2+]i up to 2-3 microM (two to threefold above peak systolic level) and that DOG-CN produced an initial decrease and then a relatively slow increase in [Ca2+]i up to peak systolic level. Electron probe x-ray microanalysis (EPMA) showed prominent increases in Na and Ca and decreases in K and Mg in cytoplasm and mitochondria with both interventions, although the increases in Ca were greater with ouabain than DOG-CN. ATP was reduced by 58% after 1 and 2 h of ouabain and by 70 and 90% after 1 and 2 h of DOG-CN, respectively. Thus, ouabain produced greater calcium accumulation and less ATP reduction than DOG-CN. Upon return to normal medium for 30 min, myocytes showed recovery of most electrolyte alterations and resumption of normal Ca2+ transients after 1 h exposure to either ouabain or DOG-CN; however, recovery was less after 2 h of either treatment, with elevated [Ca2+]i maintained in many myocytes. We conclude that the severity of myocyte injury is influenced by the magnitude and duration of both ATP reduction and calcium accumulation.
Asunto(s)
Animales Recién Nacidos/fisiología , Calcio/fisiología , Metabolismo Energético/efectos de los fármacos , Miocardio/metabolismo , Canales de Potasio/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos/metabolismo , Calcio/metabolismo , Células Cultivadas , Cianuros , Desoxiglucosa , Microanálisis por Sonda Electrónica , Contracción Miocárdica , Miocardio/patología , Ouabaína , Canales de Potasio/metabolismo , Canales de Potasio/fisiología , Ratas , Canales de Sodio/metabolismo , Canales de Sodio/fisiologíaRESUMEN
Examination of anoxic isolated hepatocytes by light and electron microscopy indicated that initial morphologic changes were largely localized to the periphery of the cells. This early phase consisted of surface bleb formation but was not accompanied by alterations in parameters of plasma membrane integrity (leakage of cellular enzymes, exclusion of trypan blue). The time course of changes in structure was temporally related to alterations in the elemental distribution and content of various subcellular compartments. These studies, which employed electron probe X-ray microanalysis, demonstrated that rapid increases in the sodium and chlorine content and decreases in the potassium content of the cytoplasm, mitochondria and nucleus occurred, whereas no change in the calcium content of any subcellular compartment was detected. Concurrently, two cellular functions known to be dependent upon ion homeostasis, sodium-dependent taurocholate uptake and mitochondrial respiratory control, became markedly impaired. Reoxygenation within 30 min resulted in the restoration of both elemental distribution and the latter two functions to baseline. These data are consistent with the hypothesis that some early functional changes may be mediated by altered ion homeostasis. In contrast, additional studies indicated that sodium and water fluxes could be dissociated from the appearance of plasma membrane blebs. Thus, this study provides direct evidence that the structural and functional changes of early anoxic hepatocyte injury cannot be explained by a single mechanistic cascade, but apparently involve multiple mechanisms which may not be directly linked.
Asunto(s)
Elementos Químicos/metabolismo , Hipoxia/metabolismo , Hígado/metabolismo , Animales , Aspartato Aminotransferasas/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Membrana Celular/patología , Cloro/metabolismo , Citoplasma/metabolismo , Microanálisis por Sonda Electrónica , Hipoxia/patología , L-Lactato Deshidrogenasa/metabolismo , Hígado/patología , Masculino , Microscopía Electrónica , Orgánulos/metabolismo , Orgánulos/patología , Fósforo/metabolismo , Potasio/metabolismo , Ratas , Ratas Endogámicas , Sodio/metabolismo , Ácido Taurocólico/metabolismoRESUMEN
We investigated the location and relative concentration in the human kidney of two enzymes involved in the processing of regulatory peptides, i.e., the angiotensin I-converting enzyme (ACE) and the neutral metalloendopeptidase 24.11 (NEP). Antibodies raised against these purified human kidney enzymes were used to determine their ultrastructural distribution by the immunogold procedure. Immunocytochemistry was performed on ultrathin frozen sections of fixed human kidney. Both enzymes were localized on the outside of the brush-border plasma membrane and, to a lesser extent, in vesicular organelles in the apical regions of epithelial cells in the proximal tubules. In addition, moderate amounts of NEP and some ACE were detected on the basal infoldings. No NEP or ACE was found in the distal tubules. Gold particles on the brush-border of proximal tubular epithelial cells were quantified with a computer based morphometry system. The results indicated about equal numbers of accessible antigenic sites for NEP and ACE on the brush-border at concentrations of antisera which yielded optimal labeling. The prominent localization of ACE and NEP on the brush-border membranes of proximal tubular epithelium suggests that these enzymes in the proximal tubules are involved in the cleavage of plasma-derived peptides after glomerular filtration.
Asunto(s)
Túbulos Renales Proximales/enzimología , Neprilisina/análisis , Peptidil-Dipeptidasa A/análisis , Adulto , Femenino , Secciones por Congelación , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Túbulos Renales Proximales/ultraestructura , Microscopía Electrónica , Microvellosidades/enzimologíaRESUMEN
Pathological calcification, observed in infarcted myocardium under certain conditions, is the most severe manifestation of abnormal calcium (Ca2+) homeostasis induced by ischemia and related forms of myocardial injury. Specialized techniques for measurement of intracellular electrolytes, i.e., electron probe X-ray microanalysis, and intracellular free Ca2+, i.e. carboxylate indicators including fura-2, are providing new insights into regulation of intracellular Ca2+ and the role of altered Ca2+ homeostasis in the pathogenesis of myocardial cell injury. Several lines of investigation indicate that increased intracellular Ca2+ develops in association with other electrolyte alterations, altered cell volume regulation, and altered membrane phospholipid composition during the progression of myocardial cell injury.
Asunto(s)
Calcio/fisiología , Enfermedad Coronaria/etiología , Hipoxia/etiología , Arritmias Cardíacas/metabolismo , Calcio/análisis , Calcio/farmacología , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Enfermedad Coronaria/metabolismo , Homeostasis , Humanos , Contracción Miocárdica/efectos de los fármacos , Miocardio/análisisRESUMEN
Transmission electron microscopy and ultracytochemistry were employed in an attempt to localize the enzyme calcium adenosine triphosphatase (Ca-ATPase) in the rod outer segments (ROS) of the toad retina. Utilizing a one-step incubation procedure, Ca-ATPase was identified as an electron-dense precipitate in the intradiskal spaces of the rod disks (vesicles) of the ROS. Analytical microscopy identified the reaction product as lead phosphate. The formation of the reaction product was dependent on the presence of ATP (the substrate) and calcium ions. However, calcium ions could be substituted for by magnesium ions. In addition, the reaction was vanadate sensitive. The latter is known to inhibit Ca-ATPase activity. Such data appear to indicate the presence of a Ca-Mg-ATPase in association with the rod disks. Since cyclic guanosine monophosphate (cyclic GMP), rather than calcium ions, is currently believed to be the primary intracellular messenger associated with phototransduction, the presence of an ROS Ca-ATPase may indicate other functions for this cation in the physiology and biochemistry of the visual process. Ca-ATPase might play a role in directional calcium fluxes between intracellular compartments.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Células Fotorreceptoras/enzimología , Segmento Externo de la Célula en Bastón/enzimología , Animales , Bufo marinus , Calcio/metabolismo , Femenino , Histocitoquímica/métodos , Microscopía ElectrónicaRESUMEN
To assess the potential of a porous hydroxyapatite matrix to serve as a bone graft substitute, bilateral 15 X 20 mm craniectomy defects were reconstructed in 17 dogs with blocks of implant and split-rib autografts. Specimens were retrieved at 3, 6, 12, 24, and 48 months, and undecalcified sections were prepared for microscopy and histometry. The implant and graft cross-sectional areas did not change with time, documenting their equivalent ability to maintain cranial contour. Bone ingrowth extended across the implant from one cranial shelf to the other in 15 specimens. Little apparent bone ingrowth was seen in most graft specimens. Two implants and three grafts were nonunited, possibly due to lack of fixation or the orientation of the histology sections. The implant specimens were composed of 39.3 percent hydroxyapatite matrix, 17.2 percent bone ingrowth, and 43.5 percent soft-tissue ingrowth. The graft specimens were composed of 43.7 percent bone and 56.3 percent soft tissue. This study supported the thesis that a porous hydroxyapatite matrix may function in part as a bone graft substitute. The brittle hydroxyapatite matrix undoubtedly became stronger with bone ingrowth, but the degree of cranial protection achieved was not measured in this study. The size of the cranial defect used in this study did not permit estimation of the distance over which bone ingrowth may be reliably expected. There remains a need for greater understanding of the causes of nonunion, the extent of predictable ingrowth depth, and the strength of the resultant implant-bone composite.
Asunto(s)
Hidroxiapatitas , Prótesis e Implantes , Cráneo/cirugía , Animales , Perros , Durapatita , Costillas/trasplante , Cráneo/ultraestructura , Cirugía Plástica , Cicatrización de HeridasRESUMEN
The effect of a pulsing electromagnetic field (PEMF) on bone ingrowth into porous hydroxyapatite (HA) and porous tricalcium phosphate (TCP) implanted in rabbit tibiae was studied. To quantitate the biological response, a recently developed method of surface measurement using a scanning electron microscope was used. The morphometrical findings in the HA pores demonstrated a significantly greater amount of bone and thicker bone trabeculae in the PEMF group as compared with the nonpulsed control group at 3 to 4 weeks postimplantation. No significant differences for these parameters were found in the TCP pores. Histologically, more bone and wider bone trabeculae were observed in the HA implants for the PEMF-treated animals at the early time periods when compared with those of the control animals. Alternatively, the histological findings of the TCP implants were similar between these two groups. These histological results tended to correlate with the morphometrical data. Together, these results suggest that accelerated bone formation and bone maturation occurred in response to PEMF in the HA pores but was without effect in the TCP pores. This stimulatory effect is most significant after 3-4 weeks of PEMF stimulation.
Asunto(s)
Desarrollo Óseo , Fosfatos de Calcio , Cerámica , Campos Electromagnéticos/métodos , Fenómenos Electromagnéticos/métodos , Prótesis e Implantes , Animales , Durapatita , Femenino , Hidroxiapatitas , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Electrónica de Rastreo , Modelos Biológicos , Conejos , Radiografía , Tibia/diagnóstico por imagen , Tibia/crecimiento & desarrollo , Tibia/ultraestructura , Factores de TiempoRESUMEN
The ultrahistochemical analysis of apical granules in the epithelial cells, i.e., granular cells, of the amphibian urinary bladder using the N,N-naphthaloylhydroxylamine procedure identified the presence of calcium in these structures. Subsequent analytical microscopy employing fresh-frozen ultrathin cryosections for X-ray microanalysis of the granules further confirmed the above histochemical findings. In addition to calcium, elemental analysis indicated the presence of magnesium, phosphorus, sulfur, silicon, potassium, and chlorine either within or in close proximity to the granules. The possibility that these granules function as subcellular compartments for the uptake and storage of calcium ions, in a way similar to mitochondria, and thus function in intracellular calcium homeostasis, is discussed. Additionally, a role for this cation in the secretion of granular glycoproteins, i.e., stimulus-secretion coupling, is hypothesized.
Asunto(s)
Bufo marinus/fisiología , Calcio/análisis , Gránulos Citoplasmáticos/ultraestructura , Vejiga Urinaria/citología , Animales , Calcio/metabolismo , Gránulos Citoplasmáticos/metabolismo , Epitelio/ultraestructura , Femenino , Histocitoquímica , Microscopía ElectrónicaRESUMEN
A histometry system has been developed to measure bone ingrowth into porous hydroxyapatite implants utilizing the backscattered electron image of thick sections. The system consists of a scanning electron microscope (SEM) with backscattered electron detector, digital beam controller, minicomputer based image digitization, and microcomputer based image processing, point counting, and lineal analysis. The SEM backscattered electron imaging mode yields high tissue contrast and sharp tissue boundaries, substantially reducing the lost cap and projection effect errors of thick sections. High-resolution digitization of the image substantially reduces the standard error of the estimates. By using the digitized image the tedious process of filtering artifacts and recording actual point counts, intersections, and intercept lengths is delegated to computer software. Performance of this system in a recent study demonstrated substantial ease of operator use and speed of measurement. In the absence of a digital beam controller an inexpensive video digitizer circuit board may be used to digitize photographic prints of the SEM images. The combination of increased accuracy, precision, operator ease, and speed suggests that this system can be useful for soft tissue-bone-implant histometry.