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L G Dobbs

Publications and source records attributed to L G Dobbs.

At least 37 records · Page 2Linked to original sources
PubMed1995

Evidence for G beta gamma-mediated cross-talk in primary cultures of lung alveolar cells. Pertussis toxin-sensitive production of cAMP.

In the presence of activated Gs alpha, the beta gamma complex of heterotrimeric G proteins (beta gamma) stimulates adenylyl cyclase (AC) in membranes prepared from cells expressing recombinant AC II or AC IV. Conditional stimulation of AC by beta gamma has been hypothesized to integrate cross-talk between Gs- and non-Gs-coupled regulation of cellular cAMP (Tang, W. J., and Gilman, A. G. (1991) Science 254, 1500-1503). Although observations in cells expressing recombinant receptors, G alpha s, and AC support this hypothesis (Federman, A. D., Conklin, B. R., Schrader, K. A., Reed, R. R., and Bourne, H. R. (1992) Nature 356, 159-161), this mechanism has not been investigated in differentiated cells. Expression of AC II has been reported only in lung, olfactory, and brain tissue. We found that rat lung alveolar type II cells express AC II and IV. Therefore, we hypothesized that beta gamma conditionally stimulates AC in type II cells. Consistent with this hypothesis, we found that the alpha 2-adrenergic agonist UK14304 did not affect basal cAMP in type II cells but potentiated terbutaline-stimulated cAMP accumulation. Treatment of cells with pertussis toxin partially inhibited terbutaline-stimulated cAMP accumulation and completely inhibited the effects of UK14304. In type II cell membranes, purified beta gamma tripled the terbutaline-stimulated increase in AC activity. In contrast, beta gamma inhibited AC activity in the absence of terbutaline. The addition of purified Go alpha blocked beta gamma-induced effects. In summary, type II cells expressing endogenous AC II and IV regulate cAMP accumulation and AC activity in a manner consistent with conditional stimulation by beta gamma. These observations support the overall hypothesis that conditional stimulation of AC by beta gamma integrates cross-talk between signal transduction systems in differentiated cells.

Adenylate Cyclase Toxin↗
PubMed1995

Cloning, characterization, and development expression of a rat lung alveolar type I cell gene in embryonic endodermal and neural derivatives.

We report here the identification and characterization of a novel gene, T1 alpha, expressed in high abundance in adult rat lung, fetal lung, and early fetal brain. T1 alpha was identified by a monoclonal antibody previously shown to be specific for an antigen expressed by alveolar epithelial type I cells. The cDNA for T1 alpha is 1.85 kb and identifies a single mRNA species of the same size on Northern blots of adult rat lung. The longest open reading frame of the cDNA is 498 bases which would encode a protein of approximately 18 kDa. The protein has a putative membrane spanning domain near the C-terminus but lacks consensus sequences for N-glycosylation. Northern blots and RT-PCR show high expression of T1 alpha in adult lung, with marginally detectable expression in adult brain, intestine, and kidney. RT-PCR analysis shows expression of T1 alpha in freshly isolated type I cells (50-60% purity) but not in highly purified type II cells or other lung cells. We believe therefore that T1 alpha is primarily if not uniquely expressed in alveolar type I cells in the adult rat. Polyclonal antisera against a 16-amino-acid peptide identified in the deduced sequence reacts with the apical membranes of adult type I cells in lung tissue sections but does not label other cell types. The above antiserum as well as the original monoclonal antibody recognize a single approximately 18-kDa protein derived from bacterial expression of a construct containing the T1 alpha open reading frame. By RT-PCR T1 alpha is detected in rat lung from Day 13.5 onward, but is detected by in situ hybridization earlier in lung, brain and neural derivatives, and foregut. Expression is down-regulated in all but lung tissues as development proceeds.

Amino Acid Sequence↗
PubMed1995

A type I cell-specific protein is a biochemical marker of epithelial injury in a rat model of pneumonia.

In this study we determined whether the alveolar fluid content of a specific epithelial type I cell protein, rTI40, can be used as a biochemical marker for lung injury. A model of alveolar epithelial injury was developed by instilling Pseudomonas aeruginosa bacteria (PA103) into the airspaces of anesthetized, ventilated rats. After 6 h, the alveolar fluid content of rTI40 from PA103-treated rats was increased over 80-fold in comparison to alveolar fluid from control rats (P < 0.05). This increase in rTI40 correlated with both morphological evidence of injury to alveolar epithelial type I cells and increased permeability of the alveolar epithelium to protein tracers. In contrast, the lactate dehydrogenase activity of alveolar fluid from PA103-treated rats was elevated only threefold over control values at 6 h (P < 0.05). In a second study using a less injurious strain of P. aeruginosa (PA103 exsA::omega), the alveolar fluid content of rTI40 was the same as control values. These findings indicate that the alveolar fluid content of a type I cell-specific protein can be used as a sensitive and specific biochemical marker of type I cell injury.

Animals↗
PubMed1994

Activation of G proteins may inhibit or stimulate surfactant secretion in rat alveolar type II cells.

To investigate how G proteins regulate surfactant secretion, we subjected rat alveolar type II cells to conditions known to activate or to inactivate G proteins. AlF-4, which activates G proteins, inhibited secretion in intact cells. Guanosine-5'-O-(3-thiotriphosphate), which activates G proteins in permeabilized cells, stimulated secretion at basal cytosolic [Ca2+], but inhibited secretion at higher [Ca2+]. In contrast, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), which inactivates G proteins, stimulated secretion at each [Ca2+] tested. Because treatment with GDP beta S stimulated secretion at basal cytosolic [Ca2+], surfactant secretion appears to be subject to G protein-regulated tonic inhibition. Pertussis toxin (PTX) inhibited terbutaline- and ionomycin-stimulated secretion in intact cells, but did not inhibit secretion stimulated by either forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate. Inhibition by PTX of terbutaline-stimulated, but not 8-bromoadenosine 3',5'-cyclic monophosphate- or forskolin-stimulated secretion, suggests that PTX-sensitive G proteins regulate beta-adrenergic-stimulated surfactant secretion proximal to second messenger generation. Inhibition of ionomycin-stimulated secretion, however, suggests that PTX-sensitive G proteins may also regulate non-receptor-mediated secretory events.

Aluminum Chloride↗
PubMed1993

Low density lipoprotein- and high density lipoprotein-mediated signal transduction and exocytosis in alveolar type II cells.

Low density lipoproteins (LDL) and high density lipoproteins (HDL) from serum stimulate signal-transduction pathways and exocytosis in rat alveolar type II cells. Both LDL and HDL stimulated primary cultures of type II cells to secrete phosphatidylcholine (PtdCho), the major phospholipid component of pulmonary surfactant. The effects on secretion were preceded temporally by stimulation of inositol phospholipid catabolism, calcium mobilization, and translocation of protein kinase C from cytosolic to membrane compartments. Heparin, which blocks the binding of ligands to the LDL receptor, completely inhibited the effects of LDL on signal transduction and PtdCho secretion but did not inhibit the effects of HDL. Unilamellar PtdCho liposomes the size of native LDL had no effect on type II cells; however, PtdCho complexes containing either apolipoproteins E or A-I stimulated both signal transduction and PtdCho secretion. LDL receptors were present in type II cell membranes by immunoblotting. In contrast to findings with hepatic membranes, type II cells exhibited two major bands of 130 kDa and 120 kDa and a minor band at 230 kDa that also was present under reducing conditions. These results are consistent with our hypothesis that the LDL-receptor pathway functions in vivo to deliver cholesterol to type II cells and that this process is coupled to surfactant assembly and secretion via signal-transduction pathway(s). HDL elicits similar responses independent of the LDL receptor, suggesting that type II cells may use the selective uptake pathway to obtain cholesterol or that HDL triggers signal transduction by mechanisms unrelated to lipid delivery.

Adenosine Triphosphate↗
PubMed1992

Stretch-dependent regulation of atrial peptide synthesis and secretion in cultured atrial cardiocytes.

We have developed a novel system to study stretch-dependent secretion of atrial natriuretic peptide (ANP) using cultured neonatal rat atriocytes in vitro. Application of tension (i.e., 2 sequential stretches) to cells grown on a flexible culture surface effected a dose-dependent increase in immunoreactive (ir) ANP release into the medium. Analysis of atriocyte cytoplasmic RNA 24 h poststretch revealed an increase in ANP mRNA levels of about ninefold relative to the unstretched controls. Medium ATP levels, measured as an index of cellular damage, were similar in control and stretched cells. Furthermore, cooling the cultures to 0 degrees C suppressed both basal as well as stretch-stimulated release. These findings argue against cellular damage and nonspecific release of irANP as an explanation for the increase in medium immunoreactivity. Stretch was incapable of amplifying the secretory response to prostaglandin F2 alpha, suggesting possible overlap in the pathways whereby these stimuli effect release of the peptide. The calcium channel blocker verapamil had no effect on stretch-dependent irANP release, whereas calmidzolium, a calmodulin inhibitor, suppressed basal as well as stretch-dependent secretion, implying a potentially important relationship between intracellular calcium metabolism and irANP release.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Regulation of ATP-dependent surfactant secretion and activation of second-messenger systems in alveolar type II cells.

Several different classes of agonists are known to stimulate exocytosis in type II cells. These agonists cause increases in second messengers, such as adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+, and/or stimulate protein kinase C. We studied generation of cAMP and phosphoinositide (PI) turnover in monolayer cultures of type II cells and measured [Ca2+]i in single cultured cells. ATP [10(-4) M], which stimulates secretion of phosphatidylcholine (PC) and increases cellular cAMP, also stimulated PI turnover and increased [Ca2+]i. 12-O-tetradecanoylphorbol-13-acetate (TPA), which stimulates PC secretion and activates protein kinase C, did not increase [Ca2+]i. Pretreatment of type II cells with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the PC secretion induced by ATP and TPA and blocked the increase in PI turnover caused by ATP. ATP-dependent surfactant secretion and stimulation of PI turnover could also be inhibited by pretreatment of the cells with pertussis toxin. We used the fluorescent probe indo-1 to measure [Ca2+]i in single cultured type II cells. ATP produced rapid transient increases in [Ca2+]i, which could be prevented by pretreatment of the cells with either TPA or W-7. Our data suggest that pertussis toxin-sensitive G protein(s) are involved in ATP-dependent activation of PI turnover and in secretion of surfactant in type II cells. Activation of protein kinase C blocks the ATP-stimulated increase in [Ca2+]i. Finally, calmodulin may be involved in the regulation of ATP-dependent increase in [Ca2+]i, the activation of PI turnover, and the secretion of surfactant in type II cells.

Adenosine Triphosphate↗
PubMed1990

Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells.

Deep inflation of the lung stimulates surfactant secretion by unknown mechanisms. The hypothesis that mechanical distension directly stimulates type II cells to secrete surfactant was tested by stretching type II cells cultured on silastic membranes. The intracellular Ca2+ concentration was measured in single cells, before and after stretching. A single stretch of alveolar type II cells caused a transient (less than 60 seconds) increase in cytosolic Ca2+ followed by a sustained (15 to 30 minutes) stimulation of surfactant secretion. Both Ca2+ mobilization and exocytosis exhibited dose-dependence to the magnitude of the stretch-stimulus. Thus, mechanical factors can trigger complex cellular events in nonneuron, nonmuscle cells and may be involved in regulating normal lung functions.

Animals↗
PubMed1990

Isolation and culture of alveolar type II cells.

The alveolar type II cell performs many important functions within the lung, including regulation of surfactant metabolism, ion transport, and alveolar repair. Because type II cells comprise only 15% of all lung cells, it is difficult to attribute specific functions to type II cells from studies of whole lungs or mixed cell cultures. At the present time, there is no passaged line that exhibits the full range of known type II cell functions. For these reasons, investigators have used isolated type II cells to study alveolar cell biology, biochemistry, and molecular biology. This review addresses many of the issues involved in isolating and culturing type II cells, including the choice of a method of isolating cells, the importance of using specific markers of the differentiated type II cell phenotype, and the problems of maintaining these differentiated phenotypic characteristics in tissue culture.

Animals↗
PubMed1990

Expression of cell-specific markers for alveolar epithelium in fetal rat lung.

In adults, the alveolar epithelium is composed of types I and II cells which differ structurally and functionally although they appear to belong to the same cell lineage. Using cell-specific markers (type I cells, monoclonal antibody; type II cells, Maclura pomifera lectin [MPA]), we have determined when and in what pattern their binding sites occur during development of the fetal rat lung. Rather than first appearing on days 19 to 20, when morphogenesis of type I cells occurs and lamellar bodies provide positive identification of type II cells, the markers appeared on day 15 (for type I cell marker) and day 16 (for type II cell marker). The type I cell marker was widespread by day 17 and was sufficiently abundant to be detected on a Western blot. MPA binding appeared more gradually and was often found on isolated cells. On serial sections of day 20 lung, the markers appeared to be localized to the same cells. The early appearance of cell-specific markers suggests an early onset of the developmental program that leads to full differentiation of types I and II cells. Co-expression of both cell-specific markers suggest that fetal cell lineage may differ from the scheme proposed by others that type II cells serve as type I cell precursors during development.

Animals↗
PubMed1989

Pulmonary surfactant.

Pulmonary surfactant is a chemically heterogeneous material that provides a stable, low surface tension within the lung, thereby preventing alveolar collapse at low transpulmonary pressures. Both the lipid and the protein components of surfactant are important for establishing and maintaining a low surface tension. The composition of surfactant, the biogenesis of surfactant components, and the biologic roles of surfactant components are discussed in this brief review. Surfactant functions in adult lung disease and surfactant replacement therapy are also briefly considered.

Animals↗
PubMed1988

Synthesis of surfactant components by cultured type II cells from human lung.

We examined the effect of monolayer culture on surfactant phospholipids and proteins of type II cells isolated from human adult and fetal lung. Type II cells were prepared from cultured explants of fetal lung (16-24 weeks gestation) and from adult surgical specimens. Cells were maintained for up to 6 days on plastic tissue culture dishes. Although incorporation of [methyl-3H]choline into phosphatidylcholine (PC) by fetal cells was similar on day 1 and day 5 of culture, saturation of PC fell from 35 to 26%. In addition, there was decreased distribution of labeled acetate into PC, whereas distribution into other phospholipids increased or did not change. The decrease in saturation of newly synthesized PC was not altered by triiodothyronine (T3) and dexamethasone treatment or by culture as mixed type II cell/fibroblast monolayers. The content of surfactant protein SP-A (28-36 kDa) in fetal cells, as measured by ELISA and immunofluorescence microscopy, rose during the first day and then fell to undetectable levels by the fifth. Synthesis of SP-A, as measured by [35S]methionine labeling and immunoprecipitation, was detectable on day 1 but not thereafter. Levels of mRNAs for SP-A and for the two lipophilic surfactant proteins SP-B (18 kDa) and SP-C (5 kDa) fell with half-times of maximally 24 h. In contrast, total protein synthesis measured by [35S]methionine incorporation increased and then plateaued. In adult cells, the content of SP-A and its mRNA decreased during culture, with time-courses similar to those for fetal cells. We conclude that in monolayer culture on plastic culture dishes, human type II cells lose their ability to synthesize both phospholipids and proteins of surfactant. The control of type II cell differentiation under these conditions appears to be at a pretranslational level.

Adult↗
PubMed1988

Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells.

The alveolar surface of the lung is lined by two classes of epithelial cells, type I and type II cells. Type I cells cover more than 97% of the alveolar surface. Although this cell type is felt to be essential for normal gas exchange, neither unique identifying characteristics nor functions have been described for the type I cell. We have produced monoclonal antibodies to (a) component(s) of molecular weight 40,000 and 42,000 of the apical surface of rat alveolar type I cells. The antibodies are specific to the lung in Western blots of organ homogenates. In immunocytochemical studies of frozen lung at the level of both light and electron microscopy, the monoclonal antibodies appear to react specifically with the apical plasma membrane of type I cells. Airway, vascular, interstitial cells, type II cells and macrophages are not immunoreactive. Western blots of isolated type I cells (approx. 70% pure) also demonstrate immunoreactivity at molecular weights of 40,000 and 42,000. When the lung is injured, type I cells may be damaged and sloughed from the alveolar surface. Alveolar repair occurs when the second type of alveolar cell, the type II cell, divides. Cell progeny may retain type II cell morphology or may differentiate into type I cells. Western blots of freshly isolated type II cells (approx. 85% pure) do not display immunoreactivity with our monoclonal antibodies. However, type II cells maintained in culture acquire immunoreactivity to monoclonal antibodies, demonstrating that type II cells in vitro have the capacity to develop a characteristic associated with type I cells in situ. The availability of markers for a specific membrane component of type I cells should facilitate the study of many questions on alveolar functions, development and response to injury.

Animals↗
PubMed1988

Positive correlation between cytosolic free calcium and surfactant secretion in cultured rat alveolar type II cells.

To determine whether increases in the cytosolic free Ca2+ concentration ([Ca2+]i) accompany agonist-stimulated surfactant secretion by cultured alveolar type II cells, we measured the [Ca2+]i of quin2-loaded cells isolated from adult rats before and after cells were stimulated with ionomycin, terbutaline or tetradecanoylphorbol acetate (TPA). To determine whether increases in [Ca2+]i are necessary for stimulated surfactant secretion to occur, we measured secretion in cells after [Ca2+]i had been reduced by loading cells with quin2 in medium containing low [Ca2+]. Ionomycin increased [Ca2+]i and stimulated surfactant secretion in a dose-dependent manner. Reductions in [Ca2+]i correlated with reductions in secretion stimulated by ionomycin, terbutaline or TPA. Ionomycin-stimulated secretion was most sensitive to reductions in [Ca2+]i; terbutaline-stimulated secretion was more sensitive than TPA-stimulated secretion. When [Ca2+]i was less than 65 nM, all stimulated secretion was blocked. Restoration of [Ca2+]i to greater than 100 nM restored ionomycin-stimulated secretion. We conclude that ionomycin increases [Ca2+]i and stimulates surfactant secretion in cultured alveolar type II cells, and that increased [Ca2+]i appears to be necessary for ionomycin-stimulated secretion to occur. Terbutaline-stimulated surfactant secretion seems to be more easily inhibited by a reduction in [Ca2+]i than does TPA-stimulated secretion.

Aminoquinolines↗
PubMed1987

Pulmonary surfactant and its components inhibit secretion of phosphatidylcholine from cultured rat alveolar type II cells.

Pulmonary surfactant is synthesized and secreted by alveolar type II cells. Radioactive phosphatidylcholine has been used as a marker for surfactant secretion. We report findings that suggest that surfactant inhibits secretion of 3H-labeled phosphatidylcholine by cultured rat type II cells. The lipid components and the surfactant protein group of Mr 26,000-36,000 (SP 26-36) inhibit secretion to different extents. Surfactant lipids do not completely inhibit release; in concentrations of 100 micrograms/ml, lipids inhibit stimulated secretion by 40%. SP 26-36 inhibits release with an EC50 of 0.1 microgram/ml. At concentrations of 1.0 microgram/ml, SP 26-36 inhibits basal secretion and reduces to basal levels secretion stimulated by terbutaline, phorbol 12-myristate 13-acetate, and the ionophore A23187. The inhibitory effect of SP 26-36 can be blocked by washing type II cells after adding SP 26-36, by heating the proteins to 100 degrees C for 10 min, by adding antiserum specific to SP 26-36, or by incubating cells in the presence of 0.2 mM EGTA. SP 26-36 isolated from canine and human sources also inhibits phosphatidylcholine release from rat type II cells. Neither type I collagen nor serum apolipoprotein A-1 inhibits secretion. These findings are compatible with the hypothesis that surfactant secretion is under feedback regulatory control.

Animals↗
PubMed1986

The role of calcium in the secretion of surfactant by rat alveolar type II cells.

Beta adrenergic agonists, tetradecanoylphorbol acetate, and the ionophore A23187 all stimulate surfactant secretion in type II cells isolated from rats. We found that combinations of these agonists cause augmented secretion, suggesting that the agonists may effect different steps in the secretory process. Previous studies have shown that cAMP is likely to be an intracellular 'second messenger' in type II cells. A23187, which has been reported to increase cAMP in some cell systems, did not increase the cAMP content of type II cells. We investigated the possible role of Ca2+ as another 'second messenger' by studying cellular 45Ca fluxes and the effect of extracellular calcium depletion on secretion. Depletion of extracellular calcium for as long as 3 h did not alter stimulated secretion, although basal secretion was increased. Secretagogues did not stimulate 45Ca influx from extracellular sources. A23187 and, to a lesser extent, terbutaline caused an acceleration of 45Ca efflux from type II cells. The addition of terbutaline or tetradecanoylphorbol acetate to A23187 further accelerated 45Ca efflux, suggesting that these agonists may act on separate calcium pools or by different mechanisms on the same calcium pool. Although secretion from type II cells is not inhibited by extracellular calcium depletion, the studies on 45Ca efflux suggest that Ca2+ plays a role in the regulation of surfactant secretion from isolated type II cells.

Animals↗