Regenerative growth of respiratory bronchioles in dogs.

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.

Transcranial Doppler sonography during acute 80 degrees head-down tilt (HDT) for the assessment of cerebral autoregulation in humans.

Cerebrovascular hemodynamics during postural changes have been sparsely investigated despite the fact that abnormal responses may contribute to the risk of stroke. The aim of this study was to determine the effect of acute 80 degrees head-down tilt (HDT) on cerebrovascular hemodynamics in humans using transcranial Doppler sonography (TCD). In 13 healthy volunteers (2 female, 11 male, age 19-37 years, mean age 26.8 years) left midcerebral artery blood flow velocities (CBFVs) were continuously monitored using TCD during 180 sec in horizontal position and during 60 sec of 80 degrees HDT. Simultaneously, systolic, diastolic, mean CBFVs, pulsatility index (PI), heart rate, beat-to-beat blood pressure (BP) and transcutaneous pCO2 were measured. In five volunteers, the procedure was repeated the next day to test the repeatability of the results. Mean BP increased slightly, but not significantly during tilt (from 80.5 +/- 7.7 mmHg to 85.9 +/- 14.1 mmHg; p > 0.05). Heart rate decreased significantly during the first 20 sec of HDT (from 66.8 +/- 9.9 min-1 to 60 +/- 11 min-1; p < 0.05). Transcutaneous pCO2 was within physiological ranges during the whole procedure (mean pCO2 minimum 39.5 +/- 2.9 mmHg, mean pCO2 maximum 42.2 +/- 3.3 mmHg). Mean CBFV did not change significantly during tilt (from 70.1 +/- 19.1 cm sec-1 to 66.6 +/- 14.1 cm sec-1; p > 0.05). PI, however, increased significantly with a more pronounced increase during the first 20 sec than the last 40 sec of tilt (PIsupine 0.92 +/- 0.11; PItilt(0-20 sec) 1.15 +/- 0.18; PItilt(21-60 sec) 1.03 +/- 0.16; p = 0.001; p = 0.017). The HDT results were found to be reproducible in the five volunteers. During 80 degrees-HDT mean BP and pCO2 did not change significantly. This observation combined with the significant decrease in heart rate during the first 20 sec of HDT, suggests that there is no sympathetic activation. The significant PI increase during HDT indicates a vasoconstriction of the cerebral resistance vessels. We assume that this vasoconstriction is due to the myogenic mechanism of cerebrovascular autoregulation triggered by a rapid, passive intracranial blood volume influx during HDT.

Transcranial Doppler sonography during acute 80° head-down tilt (HOT) for the assessment of cerebral autoregulation in humans.

Cerebrovascular hemodynamics during postural changes have been sparsely Investigated despite the fact that abnormal responses may contribute to the risk of stroke. The aim of this study was to determine the effect of acute 80° head-down tilt (HDT) on cerebrovascular hemodynamics in humans using transcranial Doppler sonography (TCD). In 13 healthy volunteers (2 female, 7 1 male, age 19-37 years, mean age 26.8 years) left midcerebral artery blood flow velocities (CBFVs) were continuously monitored using TCD during 180 sec in horizontal position and during 60 sec of 80° HDT. Simultaneously, systolic, diastolic, mean CBFVs, pulsatility index (PI), heart rate, beat-to-beat blood pressure (BP) and transcutaneous pC02 were measured. In five volunteers, the procedure was repeated the next day to test the repeatability of the results. Mean BP increased slightly, but not significantly during tilt (from 80.5± 7.7 mmHg to 85.9 ±14.1 mmHg; p < 0.05). Heart rate decreased significantly during the first 20sec of HDT (from 66.8± 9.9 min-1 to 60± 11 min-1; p> 0.05). Transcutaneous pC02 was within physiological ranges during the whole procedure (mean pC02 minimum 39.5±2.9 mmHg, mean pC02 maximum 42.2±3.3 mmHg). Mean CBFV did not change significantly during tilt (from 70.1 ± 7 9.1 cm sec-1 to 66.6 ± 14.1 cm sec-1; p < 0.05). PI, however, increased significantly with a more pronounced increase during the first 20 sec than the last 40sec of tilt (Plsupine 0.92±0.11; Pltnt(0-20sec) 1.15±0.18; Pltnt(2i~6o sec) 1-03±0.16; p = 0.001; p = 0.017). The HDT results were found to be reproducible in the five volunteers. During 80°-HDT mean BP and pC02 did not change significantly. This observation combined with the significant decrease in heart rate during the first 20 sec of HDT, suggests that there is no sympathetic activation. The significant PI increase during HDT indicates a vasoconstriction of the cerebral resistance vessels. We assume that this vasoconstriction is due to the myogenic mechanism of cerebrovascular autoregulation triggered by a rapid, passive intracranial blood volume influx during HDT. [Neurol Res 1999; 21: 457-462].

Regulation of alpha 1-, beta 1-, and beta 2-adrenergic receptors in rat heart by norepinephrine.

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.

Effect of slow-release sodium fluoride on cancellous bone histology and connectivity in osteoporosis.

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)

Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis.

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.

Sinus histiocytosis of pelvic lymph nodes after hip replacement. A histiocytic proliferation induced by cobalt-chromium and titanium.

Six men who had undergone hip replacements for degenerative joint disease or trauma subsequently had radical prostatectomies or cystoprostatectomies with bilateral pelvic lymph node dissections for adenocarcinoma of the prostate or transitional cell carcinoma of the urinary bladder. The hip prostheses implanted in three patients were known to contain cobalt-chromium alloy and titanium. The pelvic lymph nodes ipsilateral to the hip prosthesis in five patients and the bilateral pelvic nodes in the only patient with bilateral hip prosthesis had dark brown or black cut surfaces. These lymph nodes did not contain carcinoma but showed florid sinus histiocytosis characterized by large polygonal histiocytes filling and expanding sinuses and interfollicular regions. The foamy histiocytes contained cobalt-chromium and titanium microparticles by light microscopy, ultrastructure, and energy-dispersive x-ray microanalysis. The lymph nodes uninvolved by the histiocytic reaction lacked the heavy metal microparticles. Four cases were found to have a small number of polyethylene particles, which might have contributed to the histiocytic response. By immunohistochemistry, the foamy cells displayed immunoreactivity for lysozyme, alpha-1-antitrypsin, alpha-1-antichymotrypsin, and cathepsin D, providing additional support for their histiocytic derivation. To our knowledge, this is the first time that microparticles of cobalt-chromium and titanium that migrate from hip prostheses to pelvic lymph nodes have been shown to elicit a distinctive type of florid sinus histiocytosis. Pathologists should be aware of this characteristic foreign-body tissue response to avoid confusion with other types of sinus histiocytosis or with metastatic carcinoma.

Barium sulfate crystals in a fine needle aspiration biopsy specimen from a neck mass in a patient with squamous cell carcinoma. A case report.

A fine needle aspirate of a neck mass in a 79-year-old man with a previous laryngectomy for squamous cell carcinoma was submitted for examination. In addition to cytologic findings of squamous cell carcinoma, there were numerous birefringent crystals of varying shapes and sizes. Energy dispersive x-ray analysis confirmed that these crystals were barium sulfate. Recognition of barium sulfate crystals in a fine needle aspiration or other cytologic specimens is important since barium granulomas may mimic neoplasms clinically. Barium sulfate may also indicate rupture of an organ or the presence of a fistula.

Subcellular electrolyte alterations during progressive hypoxia and following reoxygenation in isolated neonatal rat ventricular myocytes.

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.

Exposure to free radicals alters ionic calcium transients in isolated adult rat cardiac myocytes.

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.

Intracellular calcium transients and arrhythmia in isolated heart cells.

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.

Cryofixation, ultracryomicrotomy, and X-ray microanalysis of enterocytes from chick duodenum: vitamin-D-induced formation of an apical tubulovesicular system.

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)

Free radicals alter ionic calcium levels and membrane phospholipids in cultured rat ventricular myocytes.

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.

Association between inhibition of arachidonic acid release and prevention of calcium loading during ATP depletion in cultured rat cardiac myocytes.

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

Tension and electrolyte changes with Na+-K+ pump inhibition in rat papillary muscle.

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.

Relationship between calcium loading and impaired energy metabolism during Na+, K+ pump inhibition and metabolic inhibition in cultured neonatal rat cardiac myocytes.

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.

Anoxic hepatocyte injury: role of reversible changes in elemental content and distribution.

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.

Ultrastructural localization of angiotensin I-converting enzyme (EC 3.4.15.1) and neutral metalloendopeptidase (EC 3.4.24.11) in the proximal tubule of the human kidney.

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.

Altered calcium homeostasis in the pathogenesis of myocardial ischemic and hypoxic injury.

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.