Beta-adrenergic receptor properties of a pulmonary alveolar type II cell preparation from the adult rat

The pulmonary alveolar type II cell synthesizes and secretes phosphatidylcholine (PC), a major component of surfactant, above basal level in response toβ-adrenergic stimulation. The investigation of the specific receptor which mediates these events was the topic of this study. Freshly isolated type II cells from adult rats were disrupted in a French pressure cell, and crude particulate fractions were recovered and used in assays for binding of the radioligand (−)-3-[¹²⁵I]-iodocyanopindolol. The receptor had high affinity forβ-adrenergic agents, and specific binding to the receptor was saturable and reversible. The K_D value obtained by kinetic means (19.6 pM) was in close agreement with that obtained by Scatchard (21.5 pM) and Hill (21.3 pM) analyses of steady-state binding data. The Scatchard correlation coefficients and Hill plot coefficients were close to 1, indicative of a single class of binding sites which displays no cooperativity. The specificity for catecholamine agonists and stereoselectivity observed were appropriate for aβ-adrenergic receptor. Use of selective drugs identified the presence of bothβ ₁- andβ ₂-adrenergic receptor subtypes (1:3, respectively) on this cell type. This work was presented in part at the Southern Section, American Federation for Clinical Research in New Orleans, January 1984.     

Beta-adrenergic receptor properties of a pulmonary alveolar type II cell preparation from the adult rat

The pulmonary alveolar type II cell synthesizes and secretes phosphatidylcholine (PC), a major component of surfactant, above basal level in response to beta-adrenergic stimulation. The investigation of the specific receptor which mediates these events was the topic of this study. Freshly isolated type II cells from adult rats were disrupted in a French pressure cell, and crude particulate fractions were recovered and used in assays for binding of the radioligand (-)-3-[125I]-iodocyanopindolol. The receptor had high affinity for beta-adrenergic agents, and specific binding to the receptor was saturable and reversible. The KD value obtained by kinetic means (19.6 pM) was in close agreement with that obtained by Scatchard (21.5 pM) and Hill (21.3 pM) analyses of steady-state binding data. The Scatchard correlation coefficients and Hill plot coefficients were close to 1, indicative of a single class of binding sites which displays no cooperativity. The specificity for catecholamine agonists and stereoselectivity observed were appropriate for a beta-adrenergic receptor. Use of selective drugs identified the presence of both beta 1- and beta 2-adrenergic receptor subtypes (1:3, respectively) on this cell type.     

The alveolar type III cell

The origin, topography, and ultrastructural morphology of the alveolar and bronchiolar type III cell in various species are reviewed. The type III cell has two distinctive features that are not present in other lung epithelial cells: a microvillous brush border and bundles of fine filaments. Regarding its topography, the cell appears to have a preferential although variable localization in different species. The morphology of this cell as revealed by our transmission and scanning electron microscopic studies in the rat lung is described and discussed in relation to the various supposed functions, that is, absorptive, chemoreceptive, and contractile.     

Phosphatidylcholine synthesis, secretion, and reutilization during differentiation of the surfactant-producing type II alveolar cell from fetal rabbit lungs.

Evidence indicates that pulmonary pool sizes of choline and related intermediates available for synthesis of phosphatidylcholine, the major component of the surfactant, change during gestation. Furthermore, recent data suggest that the type II lung cells that produce the surfactant potentially can reutilize components of this material. However, the relationship of the de novo synthetic mechanism to the secretion and reutilization of phosphatidylcholine has not been established. This is particularly true in the case of the fetal lung where, although alterations in precursor pool sizes, including choline, have been demonstrated, few or no data are available concerning how phosphatidylcholine synthesis affects or is affected by secretion and reutilization of this phospholipid by fetal type II cells. The present study was undertaken to determine the effect of availability of choline on de novo synthesis of phosphatidylcholine by isolated fetal rabbit type II cells during the differentiation process. In addition, differentiating type II alveolar cells were used to examine the hypothesis that these cells incorporate phospholipid from the extracellular environment and the quantity and/or composition of this phospholipid differently affects cellular secretion or de novo phosphatidylcholine synthesis. Assuming that the cells did not discriminate between radioactive and nonradioactive choline, elevation of extracellular choline increased the synthesis of cellular phosphatidylcholine and disaturated phosphatidylcholine in a dose-dependent manner to 0.08 mM choline. Cells induced to differentiated by exposure to fibroblast-conditioned medium synthesized more total and disaturated phosphatidylcholine at all extracellular choline concentrations. Incubation of the fetal type II cells with dipalmitoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylcholine significantly depressed the incorporation of [3H]choline into cellular phosphatidylcholine after 24 or 48 h, but not necessarily at both times. Dipalmitoylphosphatidylcholine depressed the secretion of [3H]choline-labeled phosphatidylcholine after incubation for 24 h. 1-Palmitoyl-2-oleoylphosphatidylcholine stimulated the secretion of tritium-labeled phosphatidylcholine at a concentration of 25 micrograms/mL after 48 h. Comparison of the phospholipid effect by incubating the cells with 50 ng of 14C-labeled phospholipid for 24 h showed that 1-palmitoyl-2-oleoylphosphatidylcholine significantly reduced the synthesis of 3H-labeled phosphatidylcholine compared to dipalmitoylphosphatidylcholine. In contrast, 1-palmitoyl-2-oleoylphosphatidylcholine stimulated secretion of 3H-labeled phosphatidylcholine compared with the disaturated moiety. The differentiation state of the cells altered the magnitude of the cellular secretion response but not the character.(ABSTRACT TRUNCATED AT 400 WORDS)     

An autoantibody reactive with nuclei of polymorphonuclear neutrophils: a cell differentiation marker

An autoantibody that reacted with nuclei of polymorphonuclear neutrophils (PMN) was detected at titers of greater than 10 in sera of 25 of 50 patients with rheumatoid arthritis and 36 of 50 with autoimmune chronic active hepatitis but in none of 160 controls comprising 24 patients with alcoholic cirrhosis, 36 with multiple myeloma, and 100 healthy subjects. Through the use of enriched populations of hemopoietic cells, this antibody was shown to be cell- specific, reacting only with the nucleus of the mature neutrophil. It was unreactive with nuclei of progenitor cells in the myeloid series and with nuclei of eosinophils, monocytes, lymphocytes, and thymocytes. It reacted with a determinant that appeared to be a differentiation antigen. This cell-specific autoantibody may prove to be of value in analytical studies of granulocyte maturation.     

Neutral maltase: a marker of cell differentiation in human myeloid leukaemias?

S ummary . The neutral maltase activity was determined in granulocytes from normal subjects and patients with chronic or acute myeloid leukaemias. Whereas the enzyme activity is present in normal granulocytes, in chronic or acute leukaemias these cells have markedly decreased and no detectable activity respectively. These data suggest that the variations of the neutral maltase activity observed in myeloid leukaemic cells may be related either to enzymatic modifications occurring in the course of leukaemogenesis or conversely to the stage of cell differentiation reached by the neoplastic granulocytes.     

Effect of chronic ethanol ingestion on alveolar type II cell: glutathione and inflammatory mediator-induced apoptosis

BACKGROUND: In septic patients, chronic alcohol abuse increases the incidence of the acute respiratory distress syndrome, a syndrome that requires alveolar type II cell proliferation and differentiation for repair of the damaged alveolar epithelium. We previously showed in a rat model that chronic ethanol ingestion decreased the antioxidant glutathione (GSH) in type II cells and exacerbated endotoxin-mediated acute lung injury. We hypothesized that this GSH depletion by ethanol, particularly mitochondrial GSH, predisposed type II cells to inflammatory mediator-induced apoptosis. METHODS: Adult male rats were fed the Lieber-DeCarli diet for 2, 6, or 16 weeks. Alveolar type II cells were then isolated and treated with hydrogen peroxide or TNF-alpha. The effect on glutathione (cytosolic and mitochondrial), apoptotic events, and necrosis were determined. In other studies, rats were fed ethanol for 6 weeks and were treated with endotoxin and apoptosis of type II cells determined by the TUNEL method. RESULTS: Chronic ethanol ingestion alone resulted in a progressive decrease in mitochondrial GSH and a progressive increase in the basal apoptosis and necrosis rate (p < or = 0.05). Furthermore, there was a progressive increase in the sensitivity of the cells to H2O2 or TNF-alpha induced cytochrome c release, caspase 3 activation, apoptosis, and necrosis (p < or = 0.05). Finally, there was a 2-fold increase in apoptotic type II cells in vivo when chronic ethanol ingestion was superimposed on endotoxemia. CONCLUSIONS: These results suggested that chronic ethanol ingestion resulted in a progressive depletion of mitochondrial GSH and sensitization of type II cells to inflammatory mediator-induced apoptosis and necrosis. These effects may be particularly relevant during acute stress when proliferation and differentiation of these cells are critical to repair of the damaged alveolar epithelium and may have important ramifications for the treatment of acute respiratory distress syndrome in patients with a history of alcohol abuse.     

Establishment of pulmonary alveolar type II cell line from p53-deficient mice

We obtained a pulmonary alveolar type II epithelial cell line, MAC7, derived from the lung of p53-deficient mice (p53 −/−). When this cell line was passaged for long periods of time, the epithelial cells grew at a high rate for over 50th passage and never entered the non-growing senescent phase characteristic of the normal cells (p53 +/+). Each pulmonary epithelial outgrowth was characterized morphologically and immunocytochemically, and the growth and viability of the outgrowths were examined by MTT assay. Expression of surfactant-associated protein, SP-B, was detected, and transmission electron microscopy demonstrated a type II phenotype containing lamellar bodies and phospholipids. These findings indicate that MAC7 cells retain the morphological and physiological properties of alveolar type II epithelial cells. This cell line should be useful in experimental systems for studying lung pathology. Accepted for publication: 1 February 2001     

Bronchial aspirate alkaline phosphatase: a sensitive marker enzyme to evaluate bronchial cell damage.

A correlation between the number of bronchial cells and alkaline phosphatase levels in bronchial aspirates obtained from patients was demonstrated.     

Extracellular matrix-driven alveolar epithelial cell differentiation in vitro

During homeostasis and in response to injury, alveolar type II (AT2) cells serve as progenitor cells to proliferate, migrate, differentiate, and re-establish both alveolar type I (AT1) and AT2 cells into a functional alveolar epithelium. To understand specific changes in cell differentiation, we monitored morphological characteristics and cell-specific protein markers over time for isolated rat AT2 cells cultured on combinations of collagen, fibronectin and/or laminin-5 (Ln5). For all matrices tested, cultured AT2 cells displayed reduced expression of AT2 cell-specific markers from days 1 to 4 and increased expression of AT1-specific markers by day 3, with continued expression until at least day 5. Over days 5 to 7 in culture, cells took on an AT1-like phenotype (on collagen/fibronectin alone; collagen alone; or Ln5 alone), an AT2-like phenotype (on collagen/fibronectin/Ln5; or collagen/Ln5), or both AT1-like and AT2-like phenotypes (on collagen/fibronectin matrix with a subsaturating amount of Ln5). Cells transferred between matrices at day 4 of culture retained the ability to alter day 7 phenotype. We conclude that in vitro, (1) AT2 cells exhibited phenotype plasticity that included an intermediate cell type with both AT1 and AT2 cell characteristics independent of day 7 phenotype; (2) both collagen and Ln5 were needed to promote the development of an AT2-like phenotype at day 7; and (3) components of the extracellular matrix (ECM) contribute to phenotypic switching of alveolar cells in culture. The described tissue culture models provide accessible models for studying changes in alveolar epithelial cell physiology from AT2 cell progenitors to the establishment of alveolar epithelial monolayers that represent AT1-like, AT2-like, or a mix of AT1- and AT2-like cells.     

Neutral lipid trafficking regulates alveolar type II cell surfactant phospholipid and surfactant protein expression

Adipocyte differentiation-related protein (ADRP) is a critically important protein that mediates lipid uptake, and is highly expressed in lung lipofibroblasts (LIFs). Triacylglycerol secreted from the pulmonary circulation and stored in lipid storage droplets is a robust hormonal-, growth factor-, and stretch-regulated precursor for surfactant phospholipid synthesis by alveolar type II epithelial (ATII) cells. A549 lung epithelial cells rapidly take up green fluorescent protein (GFP)-ADRP fusion protein-associated lipid droplets (LDs) in a dose-dependent manner. The LDs initially localize to the perinuclear region of the cell, followed by localization in the cytoplasm. Uptake of ADRP-LDs causes a time- and dose-dependent increase in surfactant protein-B (SP-B) expression. This mechanism can be inhibited by either actinomycin D or cycloheximide, indicating that ADRP-LDs induce newly synthesized SP-B. ADRP-LDs concomitantly stimulate saturated phosphatidylcholine (satPC) synthesis by A549 cells, which is inhibited by ADRP antibody, indicating that this is a receptor-mediated mechanism. Intravenous administration of GFP-ADRP LDs to adult rats results in dose-dependent increases in lung ADRP and SP-B expression. These data indicate that lipofibroblast-derived ADRP coordinates ATII cells' synthesis of the surfactant phospholipid-protein complex by stimulating both satPC and SP-B. The authors propose, therefore, that ADRP is the physiologic determinant for the elusive coordinated, stoichiometric synthesis of surfactant phospholipid and protein by pulmonary ATII cells.     

Identification and characterization of a lysophosphatidylcholine acyltransferase in alveolar type II cells

Pulmonary surfactant is a complex of lipids and proteins produced and secreted by alveolar type II cells that provides the low surface tension at the air-liquid interface. The phospholipid most responsible for providing the low surface tension in the lung is dipalmitoylphosphatidylcholine. Dipalmitoylphosphatidylcholine is synthesized in large part by phosphatidylcholine (PC) remodeling, and a lysophosphatidylcholine (lysoPC) acyltransferase is thought to play a critical role in its synthesis. However, this acyltransferase has not yet been identified. We have cloned full-length rat and mouse cDNAs coding for a lysoPC acyltransferase (LPCAT). LPCAT encodes a 535-aa protein of approximately 59 kDa that contains a transmembrane domain and a putative acyltransferase domain. When transfected into COS-7 cells and HEK293 cells, LPCAT significantly increased lysoPC acyltransferase activity. LPCAT preferred lysoPC as a substrate over lysoPA, lysoPI, lysoPS, lysoPE, or lysoPG and prefers palmitoyl-CoA to oleoyl-CoA as the acyl donor. This LPCAT was preferentially expressed in the lung, specifically within alveolar type II cells. Expression in the fetal lung and in rat type II cells correlated with the expression of the surfactant proteins. LPCAT expression in fetal lung explants was sensitive to dexamethasone and FGFs. KGF was a potent stimulator of LPCAT expression in cultured adult type II cells. We hypothesize that LPCAT plays a critical role in regulating surfactant phospholipid biosynthesis and suggest that understanding the regulation of LPCAT will offer important insight into surfactant phospholipid biosynthesis.     

Pulmonary surfactant transport in alveolar type II cells

Abstract:   Pulmonary surfactant (PS) is a mixture of several lipids (mainly phosphatidylcholine; PC) and four apoproteins (A, B, C and D). The classical hypothesis of PS transport suggests that PS is synthesized in the endoplasmic reticulum and transported to the lamellar body (LB) via the Golgi apparatus. However, recent studies have raised questions regarding this single route. This study examined, independently, the intracellular trafficking route of three different components of PS, that is, PC, SP-A and SP-B. Alveolar type II cells were isolated from Sprague–Dawley rats or Japanese white rabbits. The cells were cultured with either [³H]choline or [³⁵S]methionine/cysteine with or without brefeldin A, which disassembles the Golgi apparatus. LB was purified from disintegrated cells with sucrose density gradient centrifugation. [³H]PC was extracted from radiolabeled media, cells, and the LB fraction with Bligh–Dyer's method. [³⁵S]SP-A or [³⁵S]SP-B was immunoprecipitated from each sample with a specific antibody. [³H]PC was transported and stored to the LB via a Golgi-independent pathway. [³⁵S]SP-A was transported to the Golgi apparatus, underwent glycosylation, and was then constitutively secreted. The secreted [³⁵S]SP-A was re-uptaken into the LB. [³⁵S]SP-B was transported and stored to the LB via the Golgi-dependent pathway. These results indicate that, rather than a single route, surfactant components take different pathways to reside in the LB. These different pathways may reflect the different nature and role of each surfactant component such as surface tension-lowering activity and innate host defense.     

Alkaline phosphatase: a potential biomarker for stroke and implications for treatment

Stroke is the fifth leading cause of death in the U.S., with more than 100,000 deaths annually. There are a multitude of risks associated with stroke, including aging, cardiovascular disease, hypertension, Alzheimer’s disease (AD), and immune suppression. One of the many challenges, which has so far proven to be unsuccessful, is the identification of a cost-effective diagnostic or prognostic biomarker for stroke. Alkaline phosphatase (AP), an enzyme first discovered in the 1920s, has been evaluated as a potential biomarker in many disorders, including many of the co-morbidities associated with stroke. This review will examine the basic biology of AP, and its most common isoenzyme, tissue nonspecific alkaline phosphatase (TNAP), with a specific focus on the central nervous system. It examines the preclinical and clinical evidence which supports a potential role for AP in stroke and suggests potential mechanism(s) of action for AP isoenzymes in stroke. Lastly, the review speculates on the clinical utility of AP isoenzymes as potential blood biomarkers for stroke or as AP-targeted treatments for stroke patients.

     

Alkaline phosphatase from human uterine myoma. II. Kinetic and immunological properties

Thermostability of the purified alkaline phosphatase derived from human uterine muscle and myoma was established before and after desialization. Both enzymes were inhibited by sucrose, glucose and maltose in proportion to the carbohydrate concentration. L-Homoarginine inhibits the myoma enzyme in 90%, L-leucine, L-histidine and L-tryptophan in about 60%, and L-phenylalanine in less than 15%. The type of inhibition and Ki values were determined. Muscle and myoma enzymes cross-reacted with antisera against human liver and placental isoenzymes. Molecular and kinetic properties of the enzyme were compared with known human isoenzymes of alkaline phosphatase.     

Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D

Surfactant protein D (SP-D) is one of two collectins found in the pulmonary alveolus. On the basis of homology with other collectins, potential functions for SP-D include roles in innate immunity and surfactant metabolism. The SP-D gene was disrupted in embryonic stem cells by homologous recombination to generate mice deficient in SP-D. Mice heterozygous for the mutant SP-D allele had SP-D concentrations that were approximately 50% wild type but no other obvious phenotypic abnormality. Mice totally deficient in SP-D were healthy to 7 months but had a progressive accumulation of surfactant lipids, SP-A, and SP-B in the alveolar space. By 8 weeks the alveolar phospholipid pool was 8-fold higher than wild-type littermates. There was also a 10-fold accumulation of alveolar macrophages in the null mice, and many macrophages were both multinucleated and foamy in appearance. Type II cells in the null mice were hyperplastic and contained giant lamellar bodies. These alterations in surfactant homeostasis were not associated with detectable changes in surfactant surface activity, postnatal respiratory function, or survival. The findings in the SP-D-deficient mice suggest a role for SP-D in surfactant homeostasis.