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Biomedical subjects

J N Finkelstein

Publications and source records attributed to J N Finkelstein.

At least 91 records · Page 5Linked to original sources
PubMed1984

Characterization of the plasma and mitochondrial membrane potentials of alveolar type II cells by the use of ionic probes.

The lipophilic cation triphenylmethylphosphonium (TPMP+) and the potassium analog Rb+, were used to monitor the membrane potential (delta psi) of freshly isolated rabbit type II alveolar epithelial cells. Type II cells were found to accumulate TPMP+ rapidly at 37 degrees C in Hanks' balanced-salt solution with 5 microM tetraphenyl boron, but this accumulation was partially due to non-membrane potential dependent binding of TPMP+ to the cell. Lysophosphatidylcholine (lysoPC) was found to abolish delta psi and permitted correction for bound TPMP+ or Rb+. TPMP+ remaining in the cell following correction for binding represents the sum of mitochondrial and plasma membrane potential dependent accumulation. The accumulation of Rb+ by the type II cell was found to be independent of the mitochondrial membrane potential and indicated a trans-plasma membrane Rb+ distribution potential of -62.9 +/- 4 mV. A similar value was obtained by estimating the plasma membrane potential dependent accumulation of TPMP+ in type II cells whose mitochondria were depolarized with carbonylcyanide m-chlorophenylhydrazone (CCCP). The release of TPMP+ due to CCCP treatment also permitted an estimation for the trans-mitochondrial membrane potential of -141.8 +/- 10 mV. These techniques of membrane potential measurements were found to be sensitive to changes in delta psi induced by a number of inhibitors and ionophores. The ability to measure the membrane potential of the type II pneumocyte, and the changes caused by various agents, should be useful in characterizing the functional responses of this pulmonary surfactant producing cell.

Animals↗
PubMed1984

Lysophosphatidylcholine cell depolarization: increased membrane permeability for use in the determination of cell membrane potentials.

Current techniques for the determination of cellular membrane potentials based on the uptake of a radiolabeled lipophilic cation, [3H]triphenylmethylphosphonium, and the cyanine dye, DiOC5(3), were analyzed in terms of the proportions of these probes which are accumulated due to potential-dependent and potential-independent forces. Measurements were made of probe uptake in two model systems: rabbit type II pneumocytes and human promyelocytic HL60 cells. For both cell types, the membrane potential-independent component of triphenylmethylphosphonium uptake was found to be a function of several variables, including the length of exposure of the cells to the transport facilitator tetraphenylboron, the concentration of tetraphenylboron, and the integrity of the cell membrane. To accurately determine the magnitude of the potential-independent component of probe uptake by type II and HL60 cells, the cell-permeabilizing agent lysophosphatidylcholine was used. The ability of lysophosphatidylcholine to depolarize cell membranes and accurately predict membrane potential-independent accumulation was found to be equal to or superior to several other techniques commonly used to achieve membrane depolarization (e.g. gramicidin, valinomycin plus high external potassium). Lysophosphatidylcholine cell treatment was found to be a simple, rapid, and accurate technique to increase cell membrane permeability and allow equilibration of intra- and extracellular ions. The method is shown to be useful for determining membrane potential-independent accumulation of both radiolabeled and fluorescent probes of membrane potential.

Animals↗
PubMed1984

Radiation induced secretion of surfactant from cell cultures of type II pneumocytes: an in vitro model of radiation toxicity.

The pathogenesis of pneumonitis and fibrosis secondary to lung irradiation is incompletely understood. The role of the type II alveolar epithelial pneumocyte in these processes has been under investigation. The type II pneumocyte has been shown in vivo to respond to radiation induced injury with release of pulmonary surfactant. The effect of irradiation on cell cultures of type II pneumocytes was studied to determine if this could be reproduced in vitro. Type II pneumocytes were found to release surfactant material with a threshold of radiation dose between 1000 and 1500 rad. This is similar to the dosage range over which the same effect has been demonstrated in vivo. Experimental results support the concept that the release of surfactant is not due to either cell disruption or non-specific release of phospholipid from cell membranes. Irradiation appears to trigger membrane receptor mediated surfactant release. In addition, irradiation abolishes the ability of cells to subsequently respond to a physiologic agonist, suggesting radiation induced damage to the secretory mechanism. These studies establish that surfactant release in response to irradiation in vivo is a direct effect on type II pneumocytes. Cell cultures of type II pneumocytes can serve as a laboratory model of lung cell radiation toxicity.

Animals↗
PubMed1984

Arterial influx, efflux and tissue content of albumin in rabbits in vivo.

The uptake, in vivo, of 125I-labelled rabbit serum albumin by the thoracic intima-media of rabbits was determined at different times after single injection of the tracer. Plasma decay of radioactivity was monitored until sacrifice. Kinetic analysis of the plasma decay by a three compartment model gave the values of the fractional rates of catabolism and of transfers between intra- and two extravascular compartments. The uptake data were analysed by a similar compartmental model involving transfers between plasma and arterial tissue. kin, the fractional rate of influx into the tissue and kout, the fractional rate of efflux from the tissue were calculated by a procedure proposed earlier. The values of kin and kout (0.055 microliter X cm-2 X h-1 and 0.2 h-1), converted to proper units, are closer to the fractional rates for the slowly exchanging pool, probably representing tissues with non-fenestrated endothelium. On the basis of the model and a normal plasma level of albumin, it can be calculated that the steady state albumin content of intima-media should be about 11 micrograms X cm-2. Direct determination of arterial albumin content by electroimmunoassay gave a value consistent with this.

Animals↗
PubMed1984

Pulmonary surfactant: an interdisciplinary approach.

Pulmonary surfactant research is considered from the perspective of interdisciplinary correlations based on information acquired from biophysics and physical chemistry, biochemistry, cell biology, physiology, and medicine. Current views of the important molecular constituents of the pulmonary surfactant system are described and related to the biophysical surface properties necessary to generate appropriate effects on respiratory physiology. The fundamental importance of multidisciplinary characterizations of lung surfactant, together with correlations between these descriptions, is stressed throughout. The primary advantage of such an approach is that it provides broad but coordinated principles and data with which to test hypotheses of lung surfactant function and roles in respiratory physiology and pathophysiology. This perspective is used to examine available information about the functional composition of lung surfactant, its surface tension-lowering properties at physiological temperature and humidity, and considerations relevant to the formulation of effective exogenous surface-active mixtures in the treatment of surfactant-deficient states. Also discussed is the biophysical state in vivo of pulmonary surfactant at the alveolar level, including current knowledge of the alveolar hypophase as well as the concept of functional surfactant acting in predominantly dry alveoli in the normal lung.

Animals↗
PubMed1984

Monoclonal antibodies to surface antigens of rabbit type II pneumocytes.

Techniques for the production of monoclonal antibodies to cell surface antigens of type II pneumocytes are reported. Using these techniques, over 200 hybridomas were produced from spleen cell fusions of 8 mice. Of these, 25 expressed activity toward the type II pneumocyte cell surface. Many antibodies cross-reacted with a variety of cells and membranes from other organs and species, suggesting that these antibodies were ubiquitous to membrane antigens. Most of the antibodies cross-reacted strongly with dog lung membranes, suggesting the existence of common mammalian lung determinants. Five hybridomas produced supernatant antibody with considerable specificity for type II pneumocytes. Ascites fluid antibody was produced to these 5 hybridomas. Immunofluorescent staining of type II pneumocyte cell surfaces could be demonstrated with all 5 of these antibodies. This initial study demonstrates the feasibility of producing monoclonal antibodies to cell surface antigens of the type II pneumocyte.

Animals↗
PubMed1983

Properties of freshly isolated type II alveolar epithelial cells.

The biochemical characteristics of type II alveolar epithelial cells dissociated from adult rabbit lung by instillation of low concentrations of an elastase trypsin mixture are reported. Cells studied immediately (within 4 h) after isolation were found to incorporate the radioactively labelled precursors [U-14C]glucose, [methyl-3H]choline and [3H]palmitate into cellular phosphatidylcholine at rates 2-10-fold higher than previously reported for cells not subject to short-term cell culture. Secretion of phosphatidylcholine was stimulated by beta-adrenergic agonists. Measurement of specific activities of enzymes of phospholipid biosynthesis in subcellular fractions of isolated lung cells showed a significant enrichment of acyl coenzyme A-lysophosphatidylcholine acyltransferase, an enzyme believed to be involved in pulmonary surfactant phosphatidylcholine remodeling, in the endoplasmic reticulum of type II cells. These observations support the utility of freshly isolated type II cells as a model system for the study of the functions of the alveolar epithelium.

Animals↗
PubMed1983

De novo fatty acid synthesis by freshly isolated alveolar type II epithelial cells.

Fatty acid synthesis was studied in freshly isolated type II pneumocytes from rabbits by 3H2O and (U-14C)-labeled glucose, lactate and pyruvate incorporation and the activity of acetyl-CoA carboxylase. The rate of lactate incorporation into fatty acids was 3-fold greater than glucose incorporation; lactate incorporation into the glycerol portion of lipids was very low but glucose incorporation into this fraction was approximately equal to incorporation into fatty acids. The highest rate of de novo fatty acid synthesis (3H2O incorporation) required both glucose and lactate. Under these circumstances lactate provided 81.5% of the acetyl units while glucose provided 5.6%. Incubations with glucose plus pyruvate had a significantly lower rate of fatty acid synthesis than glucose plus lactate. The availability of exogenous palmitate decreased de novo fatty acid synthesis by 80% in the isolated cells. In a cell-free supernatant, acetyl-CoA carboxylase activity was almost completely inhibited by palmitoyl-CoA; citrate blunted this inhibition. These data indicate that the type II pneumocyte is capable of a high rate of de novo fatty acid synthesis and that lactate is a preferred source of acetyl units. The type II pneumocyte can rapidly decrease the rate of fatty acid synthesis, probably by allosteric inhibition of acetyl-CoA carboxylase, if exogenous fatty acids are available.

Acetyl-CoA Carboxylase↗
PubMed1983

Comparative adsorption of natural lung surfactant, extracted phospholipids, and artificial phospholipid mixtures to the air-water interface.

Adsorption to the air-water interface of natural lung surfactant obtained by bovine lung lavage is compared and contrasted with the adsorption of mixtures of synthetic phospholipids and of extracted mixed lung lipids containing minimal protein. Surface pressure-time (pi-t) adsorption isotherms are measured at 35 degrees C for the surfactant mixtures as a function of the presence or absence of divalent metal cations (Ca2+ and Mg2+) and of heating to 45 degrees C or 90 degrees C. The effect of aqueous dispersion technique (sonication or mechanical vortexing) on the adsorption process is also studied for the extracted or synthetic phospholipid mixtures. The results imply that the protein component is necessary for the optimal adsorption of natural lung surfactant. However, by taking advantage of different methods available for phospholipid dispersion in an aqueous phase in vitro, it is possible to formulate dispersions of extracted lung phospholipids containing of order 1% protein which adsorb as well as the complete surfactant system. These results suggest that protein concentrations in surfactant mixtures can be minimized for applications such as exogenous lung surfactant replacement for the neonatal Respiratory Distress Syndrome (RDS). However, for situations which may involve alterations in endogenous surfactant function such as in lung injury, effects involving pulmonary surfactant protein and protein-lipid interactions may be of functional significance.

Adsorption↗
PubMed1983

Surfactant release as an early measure of radiation pneumonitis.

The immediate release of surfactant into lung alveoli following irradiation has been studied as a potential indicator for the later development of radiation pneumonitis. Utilizing single dose radiation exposure to the whole thorax in male LAF1/J mice, steep dose response curves for lavaged alveolar surfactant were identified at 7 and 28 days after exposure. Seven days after irradiation there was no elevation with doses up to and including 12 Gy; above this dose a detectable increase occurred. At 28 days the surfactant recovered by lavage was elevated compared to the levels seen at day 7 for all doses; doses greater than 12 Gy produced surfactant values significantly greater than those found in mice treated with 12 Gy or less. The radiation pulmonary lethality dose response curve assessed four months later indicated an LD50 value of approximately 13 Gy. The early biochemical effect and the later radiation pneumonitis lethalities therefore closely coincided. The evidence strongly indicates that alveolar surfactant release uncovered hours to days after radiation exposure may be an early biochemical marker that predicts for subsequent pneumonitis radiation injury.

Animals↗
PubMed1982

De novo fatty acid synthesis in developing rat lung.

The rate of de novo fatty acid synthesis in developing rat lung was measured by the rate of incorporation of 3H from 3H2O into fatty acids in lung slices and by the activity of acetyl-CoA carboxylase in fetal, neonatal and adult lung. Both tritium incorporation and acetyl-CoA carboxylase activity increased sharply during late gestation, peaked on the last fetal day, and declined by 50% 1 day after birth. In the adult, values were only one-half the peak fetal rates. In vitro regulation of acetyl-CoA carboxylase activity in fetal lung was similar to that described in adult non-pulmonary tissues: activation by citrate and inhibition by palmitoyl-CoA. Similarly, incubation conditions that favored enzyme phosphorylation inhibited acetyl-CoA carboxylase activity in lung while dephosphorylating conditions stimulated activity. Incorporation of [U-14 C]glucose into lung lipids during development was influenced heavily by incorporation into fatty acids, which generally paralleled the rate of tritium incorporation into fatty acids. The relative utilization of acetyl units from exogenous glucose for overall fatty acid synthesis was greater in adult lung than in fetal or neonatal lung, suggesting that other substrates may be important for fatty acid synthesis in developing lung. In fetal lung explants, de novo fatty acid synthesis was inhibited by exogenous palmitate. Taken together, these data suggest that de novo synthesis may be an important source of saturated fatty acids in fetal lung but of lesser importance in the neonatal period. Furthermore, the regulation of acetyl-CoA carboxylase activity and fatty acid synthesis in lung may be similar to non-pulmonary tissues.

Acetyl-CoA Carboxylase↗
PubMed1982

Sequential effects of irradiation on the pulmonary surfactant system.

This study examines the effect of irradiation on lung surfactant synthesis and secretion in mice. Animals were irradiated with 650, 1300, or 1950 rad and morphological and biochemical indices of surfactant system function were followed for 18 weeks. No changes were seen at 650 rad; the results at 1300 and 1950 rad were virtually identical. Increased amounts of alveolar surfactant phospholipid were measureable by 24-hours. This persisted for four weeks and returned to normal by 18 weeks. Tissue surfactant phospholipid was initially reduced, returned to normal by four weeks and was increased at 18 weeks. At 18 weeks there was increased incorporation of surfactant precursor and increased production of alveolar surfactant. These biochemical changes were reflected in morphologic alterations showing release of lamellar body contents into alveoli in the first week and an increase in lamellar bodies in type II pneumocytes by 18 weeks. Elevated tissue protein levels and morphologic evidence of increased collagen formation were also found at 18 weeks. These findings indicate effects of irradiation on the pulmonary surfactant system and have important implications for the pathogenesis and potential therapy of radiation pneumonitis.

Animals↗
PubMed1982

Isolation of type II pneumocytes by laser flow cytometry.

The isolation of a pure population of type II pneumocytes from adult rabbit lung by the technique of multiparameter laser flow cytometry is reported here. Lipophilic phosphine-3R was used to stain cell dissociated from lungs by elastase and trypsin. Type II cells were then sorted by simultaneous measurement of high phosphine-3R fluorescent staining of lamellar bodies and intermediate intensity of low-angle light scatter. We were able to obtain an essentially pure population (98%) at sorting rates of 5 X 10(5) lamellar-body-containing cells per hour. The cell sorter technique offers an attractive alternative to the density gradient isolation of type II cells, particularly in terms of detailed population analysis. In addition, refinements in cell sorter methods offer the potential to discriminate differentiating type II cells with few lamellar bodies that do not segregate on the basis of buoyant density.

Acridines↗
PubMed1982

Path dependence of adsorption behavior of mixtures containing dipalmitoyl phosphatidylcholine.

The adsorption of aqueous phospholipid dispersions containing dipalmitoyl phosphatidylcholine (DPPC) is investigated at 35-37 degrees C as a function of dispersion preparation technique. Systems studied in terms of surface pressure-time (pi-t) adsorption behavior were pure DPPC, 9:1 DPPC:dipalmitoyl phosphatidylethanolamine, 7:3 DPPC:egg phosphatidylglycerol (PG), and lipids extracted from cow lung lavage. The results show that pi-t characteristics can differ significantly depending on the technique by which the DPPC-containing mixtures are initially dispersed in 0.15 M NaCl solution. Examples of path dependence include the fact that DPPC, which will not adsorb at T = 35 degrees C when placed in powdered crystals on the subphase surface, exhibits measurable pi-t changes after subphase dispersion by sonication or by mechanical vortexing. For 7:3 DPPC:PG, dispersion by sonication on ice or by mechanical vortexing gives faster adsorption than dispersion by sonication without temperature control. The effect of heating to T = 45 degrees C, which is greater than the gel to liquid crystal transition temperature of DPPC (Tc = 41 degrees C), is found to be particularly detrimental to the adsorption of 7:3 DPPC:PG. Of the phospholipid mixtures studied, extracted cow lung lipids exhibited by far the greatest adsorption capability and also showed less path dependence than 7:3 DPPC:PG. Similarly, in terms of dispersion techniques investigated, sonication on ice tended to give the most rapid adsorption for a given phospholipid mixture.

Adsorption↗
PubMed1978

Pulmonary surfactant synthesis. A highly active microsomal phosphatidate phosphohydrolase in the lung.

Lung cell-free homogenate, which contains about twice the units of phosphatidate phosphohydrolase per mg of protein compared to liver, was fractionated by differential centrifugation and the fractions were assayed for phosphatidate phosphohydrolase and marker enzymes of endoplasmic reticulum, mitochondria, and lysosomes. Over 60% of the lung phosphatidate phosphohydrolase was associated with the endoplasmic reticulum, compared to 50% of the total liver enzyme. Thus a major portion of the more active lung enzyme is potentially involved in lipid biosynthesis by the endoplasmic reticulum. Less than 0.2% of the total lung enzyme was found in a lamellar body fraction, consistent with previous findings. The lung microsomal phosphohydrolase was specific for lipid substrates, showing equal activity towards phosphatidic acid or lysophosphatidic acid and relatively low activities towards glycerophosphates. It had a neutral pH optimum, similar to the liver enzyme, but differed somewhat in its relative activity at extremes of pH. Stability at 65 degrees C was greater for the lung enzyme. Fluroide inhibited lung (or liver) microsomal phosphatidate phosphohydrolase, while tartrate, MgCl2, or EDTA had no effect. The presence of a high activity of phosphatidate phosphohydrolase in lung endoplasmic reticulum is consistent with the rapid synthesis of pulmonary surfactant phosphatidylcholine.

Animals↗