Search PubMed⌕ Search

Biomedical subjects

S Matalon

Publications and source records attributed to S Matalon.

At least 73 records · Page 4Linked to original sources
PubMed1995

Regulation of fluid-phase endocytosis in alveolar macrophages.

We investigated whether fluid-phase endocytosis in rabbit alveolar macrophages (AM) was regulated by alterations in intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Suspensions of freshly isolated AM were incubated with anionic dextrans (mol mass = 10 kDa), coupled to fluorescein isothiocyanate (FITC), at either 37 or 4 degrees C. There was a rapid increase in AM-associated fluorescence, quantified by laser flow-cytometry and video microscopy during the first hour of incubation at 37 degrees C, which was directly proportional to the amount of tracer present in the medium. In contrast, at 4 degrees C, AM fluorescence was similar to autofluorescence. Incubation of AM with forskolin (50 microM) or 3-isobutyl-1-methyl xanthine (IBMX; 0.1 mM) increased their cAMP content by 67 +/- 2 and 52 +/- 5% (mean +/- SE; n = 4) and decreased FITC-dextran uptake by 29 +/- 4 and 31 +/- 4% (n = 3). On the other hand, incubation of AM with 0.5 mM IBMX inhibited FITC-dextran uptake by 62 +/- 4% (n = 3), without any further increase in cAMP. Incubation of AM with 0.4 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP), a cell-permeable analogue of cAMP, decreased FITC-dextran uptake by 48 +/- 5% (n = 6). Pulse-chase experiments showed that the rate of FITC-dextran exocytosis was not affected by cAMP. We concluded that fluid-phase endocytosis in rabbit AM is regulated by cAMP and by an additional, cAMP-independent mechanism of IBMX.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Interaction of surfactant mixtures with reactive oxygen and nitrogen species.

Increased concentrations of partially reduced oxygen and nitrogen reactive species damage the alveolar epithelium and either cause or exacerbate surfactant deficiency. For this reason, there is a quest to identify surfactant replacement mixtures, which in addition to repleting depleted surfactant stores can also reduce the steady-state concentrations of reactive species in the alveolar space. Herein, we evaluated the ability of natural lung surfactant (NLS) and two mixtures (Exosurf and Survanta) used clinically for the correction of surfactant deficiency to scavenge hydroxyl radical-type species (.OH), generated either by the decomposition of peroxynitrite or by Fenton reagents (FeCl3 + H2O2). Exosurf or Survanta decreased .OH only when present at high lipid concentrations (6.5 mM). On the other hand, 40 microM of NLS decreased .OH concentrations from 75 +/- 2 to 53 +/- 2 microM (P < 0.05), most likely because of the interaction of .OH with protein sulfhydryl groups. Similarly, 40 microM of NLS incubated with a bolus of H2O2 (400 microM) decreased the H2O2 concentration in the supernatant by approximately 50%, due to the presence of catalase-type activity. In contrast to NLS, neither Exosurf nor Survanta scavenged H2O2, even when present at millimolar lipid concentrations. We concluded that Exosurf and Survanta contain limited antioxidant activity compared with NLS.

Animals↗
PubMed1995

Hyperoxic lung injury reduces exogenous surfactant clearance in vivo.

We studied the rate of clearance of treatment doses of radiolabeled calf lung surfactant extract, which was instilled into the lungs of young adult rabbits exposed to air (control) or 100% oxygen for 64 h. More than 75% of the instilled surfactant remained lung-associated at all time points up to 24 h post-instillation in both groups; however, significantly more of the labeled phosphatidylcholine (PC) remained in the alveolar wash of oxygen-exposed rabbits (44 +/- 9% in 100% O2 versus 27 +/- 5% in controls at 6 h and 27 +/- 2% in 100% O2 versus 6 +/- 1% in control rabbits at 24 h, p < 0.05). Less of the labeled PC could be found in type II pneumocytes isolated from the oxygen exposed animals than in control animals, both at 6 h (24 +/- 2 cpm/10(6) cells in O2 versus 38 +/- 7 cpm/10(6) cells in control) and 24 h (42 +/- 5 cpm/10(6) cells in O2 versus 70 +/- 12 cpm/10(6) cells in control) post-instillation. Type II cells from animals exposed to 100% oxygen also demonstrated significantly lower PC synthesis rates than cells from lungs of control animals. Interestingly, clearance of exogenous surfactant in rabbits exposed to 100% oxygen for 48 h, an exposure that does not cause significant type II pneumocyte dysfunction, was not different from control. We concluded that injury to type II pneumocytes may result in decreased clearance of instilled surfactant from the alveolar space and may be important in determining dosing regimens for the use of surfactant therapy in adult respiratory distress syndrome.

Animals↗
PubMed1994

Upregulation of sodium conductive pathways in alveolar type II cells in sublethal hyperoxia.

We investigated whether exposure of rats to sublethal hyperoxia (85% O2 for 7 days) raises the levels of proteins antigenically related to Na+ channels in alveolar type II (ATII) cells and, if so, whether this rise was accompanied by an increase in conductive Na+ transport in vitro. ATII cells were isolated from the lungs of these rats at the end of the exposure period. In Western blot studies, a polyclonal antibody raised against Na+ channel protein (NaAb), recognized in a specific manner a 135 +/- 10 kDa polypeptide in plasma membrane vesicles of ATII cells from both control and oxygen-exposed rats. However, higher levels of immunoreactivity were seen in ATII cells from oxygen-exposed rats. When ATII cells were patched in the whole cell mode using symmetrical solutions (150 mM Na(+)-glutamate), outward rectified Na+ currents were observed. When corrected for cell capacitance, both inward and outward currents of ATII cells from rats exposed to hyperoxia were significantly higher than control. Addition of either 1 microM amiloride or 1 microM 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride in the bath solution decreased the magnitude of outward currents of both control and hyperoxic ATII cells by approximately 50%. Taken together, these results indicate that exposure of rats to sublethal hyperoxia results in upregulation of ATII cell conductive pathways with low affinity to amiloride and increased Na+ transport. This may be an early adaptive response that limits the degree of alveolar edema in injured lungs.

Adaptation, Physiological↗
PubMed1994

Peroxynitrite inhibition of oxygen consumption and sodium transport in alveolar type II cells.

Active sodium (Na+) transport by alveolar type II (ATII) cells plays an important role in limiting the volume of alveolar fluid. Reactive oxygen and nitrogen species, released in the epithelial lining fluid by activated inflammatory cells or present in inspired gases, may damage Na+ transporters and decrease fluid reabsorption. To test this hypothesis we exposed ATII cells to xanthine and xanthine oxidase (1 or 10 mU/ml), or to boluses of peroxynitrite (0.1-1 mM final concentration) for 15 min and measured 1) cellular oxygen consumption (VO2); 2) amiloride-inhibitable 22Na+ uptake, as an index of Na+ movement through apically located Na+ channels; and 3) ouabain-sensitive 86Rb+ uptake, as an index of the activity of the basolaterally located Na(+)-K(+)-ATPase. After exposure of ATII cells to 0.5 or 1 mM peroxynitrite, amiloride-inhibitable 22Na+ uptake decreased to 68 +/- 7 and 56 +/- 11 of their control values, respectively (mean +/- SE; n > or = 6). Exposure to 0.5 mM peroxynitrite decreased ATII cell VO2 from 76 +/- 6 to 25 +/- 5 microM.h-1 x 10(6) cells-1 (mean +/- SE; n = 5). Cell viability and ouabain-sensitive 86Rb+ uptake remained at control levels for either peroxynitrite concentration. Exposure of ATII cells to 10 mU/ml xanthine oxidase decreased their VO2 from 94 +/- 8 to 63 +/- 6 (mean +/- SE; n = 5), but did not alter amiloride-inhibitable 22Na+ uptake. These findings indicate that physiological concentrations of peroxynitrite, but not of reactive oxygen species, decrease ATII cell Na+ transport by damaging apically located amiloride-sensitive Na+ channels.

Amiloride↗
PubMed1994

Regulation of low-amiloride-affinity sodium channels in alveolar type II cells.

We determined the mechanisms by which beta-agonists increase sodium (Na+) currents across rat alveolar type II (ATII) cells grown in primary culture. When ATII cells were patched in the cell-attached mode using symmetrical Na+ solutions (150 mM Na(+)-glutamate), single-channel currents were observed for holding potentials between -80 and 30 mV (referenced to the pipette solution) with a single-channel conductance of 27 +/- 3 pS, a mean open time (tau 1) of 3.3 +/- 0.15 ms and an open probability (Po) of 0.36 +/- 0.06 (n = 7). Addition of 10 microM terbutaline into the bath increased tau 1 to 6.43 +/- 0.5 ms and Po to 0.62 +/- 0.06 (n = 7) without affecting channel conductance. Single-channel currents with a conductance of 25 +/- 2 pS were also recorded across ATII cells patched in the inside-out mode. Addition of 250 U/ml of protein kinase A (PKA), 1 mM ATP, and 5 mM MgCl2 in the bath solution (150 mM Na(+)-glutamate) increased the single channel tau 1 from 3.26 +/- 0.15 to 7.38 +/- 0.38 and Po from 0.41 +/- 0.06 to 0.72 +/- 0.07 (n = 6) without altering conductance. Addition of 1 microM amiloride or ethylisopropylamiloride (EIPA) in the pipette solution (150 mM Na(+)-glutamate) blocked single-channel activity almost completely. Ionic substitution experiments showed the relative permeability of Na+ to K+ and Na+ to Cl- to be 7:1 and 8:1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1994

Concurrent generation of nitric oxide and superoxide damages surfactant protein A.

The conditions under which nitric oxide (.NO) may modulate or promote lung injury have not been identified. We hypothesized that .NO-induced injury results from peroxynitrite, formed by the reaction of .NO with superoxide. The simultaneous generation of .NO and superoxide by 3-morpholinosydnonimine (SIN-1, 0.1-2 mM) resulted in oxidation of dihydrorhodamine, a marker of peroxynitrite production, and a dose-dependent decrease in the ability of SP-A to enhance lipid aggregation. Western blot analysis of SIN-1 exposed SP-A samples, overlaid with a polyclonal antibody against nitrotyrosine, were consistent with nitration of SP-A tyrosine residues. Superoxide dismutase (100 U/ml), L-cysteine (5 mM), xanthine oxidase (10 mU/ml) and xanthine (500 microM), or urate (100 microM) prevented the SIN-1-induced dihydrorhodamine oxidation and injury to SP-A. .NO alone, generated by S-nitroso-N-acetylpenicillamine plus 100 microM L-cysteine, or superoxide and hydrogen peroxide, generated by pterin and xanthine oxidase in the absence of iron, did not damage SP-A or oxidize dihydrorhodamine. We concluded that peroxynitrite, but not .NO or superoxide and hydrogen peroxide, in concentrations likely to be encountered in vivo, caused nitrotyrosine formation and decreased the ability of SP-A to aggregate lipids.

Humans↗
PubMed1994

Dependence of surfactant function on extracellular pH: mechanisms and modifications.

We investigated alterations in pH on the surface properties of natural lung surfactant and the calf lung surfactant extract (CLSE), suspended in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, using a pulsating bubble surfactometer. Increasing the pH value of the medium to > 7.4 decreased the ability of CLSE, but not of natural lung surfactant mixtures (2 mg phospholipid/ml), to achieve a low minimum surface tension during dynamic compression and enhanced their sensitivity to albumin inactivation. These detrimental effects on surface tension were reversed by addition of surfactant protein A (SP-A; 3% by weight) or by increasing the lipid concentration to 4 mg/ml. SP-A-induced lipid aggregation at pH 10 was not different than at pH 7.4. Alkalinization impaired the ability of CLSE to restore normal lung mechanics in excised surfactant-deficient rats lungs. These results indicate that cooperation between SP-A and the hydrophobic surfactant proteins has an important role in achieving low minimum surface tension at pH > or = 7.6.

Animals↗
PubMed1994

Manganese superoxide dismutase expression in alveolar type II epithelial cells from nonventilated and hypoperfused lungs.

Lungs that have been hypoxic and hypoperfused because of atelectasis and the resulting decrease in pulmonary arterial blood flow develop specific decreases in manganese superoxide dismutase (MnSOD) activity and are sensitive to oxidant injury during reoxygenation. Since the MnSOD protein is concentrated in mitochondria of alveolar epithelial type II cells (ATII), we hypothesized that expression of MnSOD would be decreased in these cells also as a result of hypoxia. To investigate whether regulation of MnSOD expression occurred before or after transcription, we determined whether MnSOD protein content or steady-state mRNA level changed after hypoxia as well. ATII cells were isolated by elastase digestion from lungs of adult rabbits after right lungs had been hypoxic and hypoperfused for 7 days because of unilateral atelectasis. MnSOD activity was measured by inhibition of cytochrome c reduction in the presence of 1 mM KCN, MnSOD protein content was measured on immunoblots, and MnSOD mRNA was quantified on slot blot autoradiograms. MnSOD activity was 8.4 +/- 1.9 U/mg protein in ATII cells from control lungs and 6.8 +/- 1.5 U/mg protein in ATII cells from hypoxic and hypoperfused lungs (n = 9, P = 0.037). MnSOD protein content was 5.1 +/- 1.4 micrograms/mg protein in ATII cells from control and 4.1 +/- 1.2 micrograms/mg protein in ATII cells from hypoxic and hypoperfused lungs (P = 0.021). ATII cell MnSOD mRNA/18S ribosomal RNA (ratio of arbitrary absorbance units) determined by RNA slot blots was 2.18 +/- 1.26 in ATII cells from control lungs and 2.94 +/- 0.88 in ATII cells from hypoxic lungs (n = 7, P > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury.

Activated alveolar macrophages and epithelial type II cells release both nitric oxide and superoxide which react at near diffusion-limited rate (6.7 x 10(9) M-1s-1) to form peroxynitrite, a potent oxidant capable of damaging the alveolar epithelium and pulmonary surfactant. Peroxynitrite, but not nitric oxide or superoxide, readily nitrates phenolic rings including tyrosine. We quantified the presence of nitrotyrosine in the lungs of patients with the adult respiratory distress syndrome (ARDS) and in the lungs of rats exposed to hyperoxia (100% O2 for 60 h) using quantitative immunofluorescence. Fresh frozen or paraffin-embedded lung sections were incubated with a polyclonal antibody to nitrotyrosine, followed by goat anti-rabbit IgG coupled to rhodamine. Sections from patients with ARDS (n = 5), or from rats exposed to hyperoxia (n = 4), exhibited a twofold increase of specific binding over controls. This binding was blocked by the addition of an excess amount of nitrotyrosine and was absent when the nitrotyrosine antibody was replaced with nonimmune IgG. In additional experiments we demonstrated nitrotyrosine formation in rat lung sections incubated in vitro with peroxynitrite, but not nitric oxide or reactive oxygen species. These data suggest that toxic levels of peroxynitrite may be formed in the lungs of patients with acute lung injury.

Adolescent↗
PubMed1993

Culture-induced alterations in alveolar type II cell Na+ conductance.

Changes in Na+ transport in rat alveolar type II (ATII) cells during culture were quantified and related to alterations in spatial distribution of proteins antigenically related to amiloride-sensitive Na+ channels. Adult rat ATII cells were cultured for periods ranging from 24 to 96 h. When patch clamped in the whole cell mode, both freshly isolated and cultured ATII cells exhibited outwardly rectified Na+ currents. At 0 and 24 h in culture, these currents were equally inhibited by amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (inhibitory constant approximately 1 microM). These conductive pathways were equally permeable to Na+ and K+. Immunocytochemical localization at 0 or 24 h in culture revealed the presence of plasma membrane antigenic sites; after 48 h, the appearance of intracellular antigenic sites increased significantly. A single band of molecular mass 135 kDa in membrane proteins of freshly isolated ATII cells was recognized in Western blots; at 48 h in culture, two lower bands with molecular masses of 75 and 65 kDa were detected in either membrane or cytoplasmic proteins. Photolabeling with 2'-methoxy-5'-nitrobenzamil showed that the 135-, 75-, and 65-kDa bands contained amiloride-binding sites. These results suggest the presence of low amiloride affinity conductive pathways in freshly isolated and cultured ATII cells. Culturing ATII cells resulted in internalization and possible breakdown of these pathways and decreased Na+ transport.

Amiloride↗
PubMed1993

Fetal lung epithelial cells contain two populations of amiloride-sensitive Na+ channels.

Active Na+ transport by the alveolar epithelium plays a major role in reabsorption of the fetal lung fluid after birth. We characterized the biochemical and physiological characteristics of Na+ conductive pathways in distal fetal lung epithelial (FLE) cells isolated from 20-day-old rat fetuses. We demonstrated that a polyclonal antibody to Na+ channel protein (NaAb) binds to the plasma membranes of FLE cells. In Western blot studies, this NaAb and an anti-idiotypic monoclonal antibody to the amiloride-binding subunit of the Na+ channel protein recognized 150- and 90-kDa polypeptides in plasma membrane vesicles of FLE. 22Na+ flux measurements across plasma membrane vesicles of FLE revealed the existence of electrogenic Na+ transport, which was twice as high as the corresponding adult value. One hundred micromolars of amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride inhibited 30, 40, and 70% of the electrogenic Na+ transport across plasma membrane vesicles of FLE cells, respectively. The half-maximum inhibition of electrogenic Na+ transport by these substances occurred between 0.3 and 1 microM. [3H]benzamil equilibrium binding studies in membrane vesicles of FLE cells revealed the existence of two binding sites that had dissociation constant values of 19 and 1,525 nM, respectively. These data indicate the presence of both high- and low-amiloride affinity Na+ conductive pathways (channels) in FLE cells.

Amiloride↗
PubMed1993

Mitigation of oxidant injury to lung microvasculature by intratracheal instillation of antioxidant enzymes.

We quantitated the ability of intratracheally administered liposome-encapsulated antioxidant enzymes to reduce reactive oxygen species injury to the pulmonary microvasculature. Cationic liposomes containing 3,500 U of Cu,Zn superoxide dismutase (Cu,Zn SOD) and 3,124 U of catalase were instilled into rabbits. The animals were killed 2-72 h later and their lungs were removed and perfused with Krebs Ringer with 5% wt/vol of fat-free bovine serum albumin. The pulmonary filtration co-efficient (Kf,c) was measured before and after adding 500 microM xanthine and 5 mU/ml xanthine oxidase (XO) into the lung perfusate. Two hours after a single intratracheal instillation of liposome-entrapped Cu,Zn SOD and catalase, lung antioxidant enzyme activities were 34 and 125% higher than the corresponding control values, remained virtually unchanged for up to 8 h post-instillation, and then decreased, reaching baseline values between 24 and 72 h. Addition of xanthine and XO into the lung perfusate of un-instilled rabbits, or rabbits that received liposomes with inactivated enzymes, caused a 100% increase in Kf,c (control value: 2 +/- 0.12 ml.min-1 x cmH2O-1 per 100 g dry lung weight). On the other hand, Kf,c values of rabbits lungs instilled with liposome-encapsulated active Cu,Zn SOD and catalase and challenged with xanthine and XO 8-24 h later remained at baseline levels. Instillation of liposomes containing either enzyme was equally effective in preventing the increase in Kf,c, indicating that both superoxide anions and hydrogen peroxide were necessary for the initiation of injury. We concluded that intratracheal instillation of liposome-encapsulated antioxidant enzymes caused a transient increase of lung antioxidant enzyme levels which protects the pulmonary microvasculature from free radical-initiated injury.

Animals↗
PubMed1993

Mechanisms of peroxynitrite-induced injury to pulmonary surfactants.

Activated alveolar macrophages secrete both nitric oxide and superoxide in the alveolar lining fluid which combine rapidly to form peroxynitrite, a potent oxidizing agent capable of damaging lipids and proteins in biological membranes. Peroxynitrite (1 mM) plus 100 microM Fe3+EDTA inhibited calf lung surfactant extract (CLSE) from reaching a minimum surface tension below 10 mN/m on dynamic compression. Peroxynitrite and its by-products reacted with the unsaturated lipid components of CLSE, as evidenced by the appearance of conjugated dienes and thiobarbituric acid products, and damaged all surfactant proteins. A mixture of the hydrophobic proteins [surfactant protein B (SP-B) and surfactant protein C (SP-C)] exposed to peroxynitrite became incapable of lowering phospholipid minimum surface tension on dynamic compression. Exposure of SP-A to peroxynitrite decreased its ability to cause lipid aggregation and to act synergistically with SP-B and SP-C in lowering surface tension of surfactant lipids. Western blot analysis of SP-A exposed to peroxynitrite was consistent with fragmentation and polymerization of the 28- to 36-kDa triplet band, and amino acid analysis revealed the presence of significant levels of 3-nitro-L-tyrosine. We conclude that peroxynitrite and its reactive intermediates inhibit pulmonary surfactant function by lipid peroxidation and damaging surfactant proteins.

Animals↗