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J M Shannon

Publications and source records attributed to J M Shannon.

At least 37 records · Page 2Linked to original sources
PubMed1997

Developmental changes of fetal rat lung Na-K-ATPase after maternal treatment with dexamethasone.

Late in gestation, the prenatal fetal alveolar epithelium switches from fluid secretion to resorption of salt and water via apical sodium channels and basal Na-K-ATPase. The amounts of lung sodium pump activity protein and mRNA increase in the lung just before birth. Because maternal glucocorticoids (GC) may promote maturation of the alveolar epithelium and augment fetal surfactant apoprotein levels, we hypothesized that GC increase the fetal lung Na-K-ATPase alpha- and beta-subunit gene expression in development. Timed-pregnant Sprague-Dawley rats were injected daily with intraperitoneal dexamethasone (1 mg/kg) or saline for 1, 3, or 5 days before death at fetal day (FD) 17 or 19. Maternal GC treatment altered the fetal lung wet to dry weight, decreasing it at FD17 and increasing it at FD19. Northern analysis of total lung RNA for the alpha1- and beta1-pump subunits demonstrated differential regulation of the mRNA in response to GC. At FD17, beta1-mRNA increased after 1 (FD16) or 3 days (FD14-FD16) of GC treatment, whereas alpha1-mRNA was not altered. There were accompanying increases in beta1-, but not alpha1-, protein. At FD19, GC treatment for 5 days (FD14-FD18) increased beta1- and decreased alpha1-mRNA levels, but treatment for 1 (FD18) or 3 days (FD16-FD18) had no effect. In all groups, the alpha1-Na-K-ATPase protein was predominantly on the basolateral surface of airspace epithelium by immunofluorescence. In summary, maternal dexamethasone differentially affected the fetal lung mRNA levels of the two sodium pump subunits in a complex manner, with increased beta1-mRNA levels dependent on duration of treatment and fetal age.

Animals↗
PubMed1997

Heparin-binding EGF-like growth factor is a mitogen for rat alveolar type II cells.

Alveolar type II cells proliferate and differentiate into type I epithelial cells to restore the alveolar epithelium after lung injury. Since mitogens that bind the epidermal growth factor (EGF), EGF, receptor and transforming growth factor alpha (TGF alpha) have been shown to stimulate type II cell proliferation, studies were undertaken to determine whether the recently described protein, heparin-binding EGF-like growth factor (HB-EGF), was a mitogen for rat alveolar type II cells in primary culture. In addition, since HB-EGF is produced by macrophages, it was of interest to determine whether mitogenic activity for type II cells present in macrophage conditioned medium was due to HB-EGF. Rat and human recombinant HB-EGF stimulated thymidine incorporation into rat type II cells in a concentration-dependent manner up to 10-50 ng/ml then became inhibitory. The nuclear labeling index of type II cells increased from 2% to 16% with 10 ng/ml HB-EGF. However, HB-EGF induced only a small increase in cell number after 48 h and did not support low-density proliferation of alveolar type II cells. Conditioned medium from the human monocytic cell line, U937, stimulated type II cell DNA synthesis, and stimulatory activity could be partially purified by S-sepharose and heparin-sepharose chromatography. The growth-promoting activity from U937 cells that bound to heparin-sepharose was inhibited by a neutralizing antibody to human HB-EGF. Immunoblot analysis of active fractions also verified the presence of HB-EGF. However, the neutralizing antibody to rat HB-EGF did not inhibit mitogenic activity for type II cells found in rat bronchoalveolar lavage fluid. HB-EGF mRNA was found to be expressed in human alveolar macrophages to similar levels as differentiated U937 cells but was not detected in rat alveolar macrophages by Northern analysis of total mRNA. There was no difference in the level of HB-EGF mRNA expression in human alveolar macrophages from patients with interstitial lung disease compared with macrophages from normal subjects. The results demonstrate that HB-EGF is a mitogen for rat alveolar type II cells but appears to show species-specific differences with regard to its production by macrophages. Leslie, C. C., K. McCormick-Shannon, J. M. Shannon, B. Garrick, D. Damm, J. A. Abraham, and R. J. Mason. 1997. Heparin-binding EGF-like growth factor is a mitogen for rat alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 16:379-387.

Adult↗
PubMed1997

Prevention of bleomycin-induced lung injury in rats by keratinocyte growth factor.

Intratracheal instillation of bleomycin produces pulmonary fibrosis in rats. Alveolar type II cell proliferation is thought to minimize the fibrotic response after lung injury. Because keratinocyte growth factor (KGF) stimulates type II cell proliferation in the rat, we designed experiments to evaluate whether intratracheal KGF before or after intratracheal bleomycin would prevent pulmonary fibrosis. Intratracheal bleomycin without KGF resulted in moderate to severe lung injury and subsequent fibrosis. Conversely, intratracheal KGF pretreatment at 48 or 72 hr before bleomycin resulted in minimal to no visible lung injury. Rats pretreated with phosphate buffered saline before bleomycin had significantly more neutrophils and protein in bronchoalveolar lavage fluid at 4 and 6 days and higher hydroxyproline levels after bleomycin as compared to KGF-pretreated rats. Pretreatment with KGF at 48 hr protected against bleomycin-induced alterations in pulmonary physiology and increased surfactant protein C-positive (SP-C)-positive cells and SP-A, SP-B, SP-C, and SP-D mRNA levels after bleomycin instillation when compared to saline pretreated rats on day 1 or day 7. KGF posttreatment protocols did not prevent bleomycin lung injury and fibrosis. We conclude that KGF pretreatment attenuates bleomycin lung injury and increases type II cell proliferation and surfactant protein gene expression after bleomycin instillation in the rat.

Animals↗
PubMed1996

Acidic fibroblast growth factor and keratinocyte growth factor stimulate fetal rat pulmonary epithelial growth.

We have shown that pulmonary epithelial growth and differentiation can occur if pulmonary mesenchyme is replaced with a mixture of growth factors [total growth medium (TGM)] that consists of adult rat bronchoalveolar lavage fluid, insulin, epidermal growth factor (EGF), cholera toxin (CT), acidic fibroblast growth factor (aFGF), and fetal bovine serum. In the present study, we have defined the importance of specific components of TGM. Day 14 fetal rat distal lung epithelium, devoid of mesenchyme, was enrobed in growth factor-depleted Matrigel and cultured for 5 days in various soluble factors. We found that deleting aFGF or CT from TGM significantly reduced DNA synthesis. Epithelial proliferation was not significantly different when keratinocyte growth factor (KGF) replaced aFGF in TGM. KGF, however, required the presence of a basal medium containing CT, insulin, and serum for optimal proliferation. We then added specific growth factors to the basal medium and showed that aFGF and KGF were more potent mitogens than EGF, transforming growth factor-alpha, and hepatocyte growth factor. Additionally, basal medium + KGF also allowed progression to a distal alveolar phenotype. We conclude that aFGF and KGF may be important mediators in epithelial-mesenchymal interactions.

Animals↗
PubMed1996

Keratinocyte growth factor reduces lung damage due to acid instillation in rats.

Acid aspiration is a serious complication of anesthesia and other forms of unconsciousness that can result in the adult respiratory distress syndrome (ARDS), which continues to have a very high mortality despite our current therapeutic interventions. This type of injury damages the alveolar epithelium, principally alveolar type I cells, and requires proliferation of alveolar type II cells to restore gas exchange units. Since keratinocyte growth factor (KGF) has been shown to be a potent mitogen for alveolar type II cells, we evaluated whether intrabronchial administration of KGF would minimize lung injury due to the unilateral instillation of 0.1 N hydrochloric acid (HCl). Rats were pretreated or post-treated by intrabronchial instillation of KGF (5 mg/kg) into the left lung before HCl instillation. All rats receiving KGF at 48 or 72 h before HCl instillation survived for the 7-day observation period, whereas the mortality rate for those receiving HCl alone or saline followed by HCl was 31% and 33%, respectively. Pretreatment with KGF at 72 h but not at 24 or 48 h considerably ameliorated morphologic damage produced by HCl. Inflammatory cells in bronchoalveolar lavage were markedly decreased 3 and 7 days after HCl instillation by the 72-h KGF pretreatment. Pretreatment with KGF at 72 h also attenuated the reduction of total lung capacity, decreased the alpha 1(I) procollagen mRNA levels, and diminished hydroxyproline accumulation due to HCl instillation. Saline pretreatment at 72 h had no significant effect on the HCl injury and subsequent physiologic abnormalities. Our attempts to improve outcome with post-treatment instillation of KGF were unsuccessful. We conclude that KGF pretreatment reduces lung injury due to acid instillation and can prevent subsequent pulmonary fibrosis.

Administration, Inhalation↗
PubMed1995

Keratinocyte growth factor increases mRNAs for SP-A and SP-B in adult rat alveolar type II cells in culture.

The production of pulmonary surfactant, a complex of phospholipids and lung-specific surfactant proteins, is a primary function of alveolar type II cells. Although previous studies have demonstrated a role for cell-extracellular matrix interactions and normal cell shape in the maintenance of differentiated function in primary cultures of adult rat type II cells, a positive role for growth factors in surfactant protein gene expression in isolated normal adult type II cells has not been reported. In the present study, we have examined the effects of a panel of hormones, growth factors, and cytokines on the expression of mRNAs for surfactant proteins A, B, and C (SP-A, SP-B, and SP-C). Our results show that keratinocyte growth factor (KGF) induced a two- to threefold increase in steady-state levels of mRNAs for SP-A and SP-B, but had no effect on or decreased SP-C mRNA. The increase in SP-A mRNA was accompanied by an increase in SP-A protein. The effects of KGF were both dose and time dependent, and they could be neutralized by a monoclonal antibody against KGF. The effects of KGF were mimicked by acidic fibroblast growth factor, which will bind the KGF receptor. We conclude that KGF can support differentiation of alveolar type II cells as well as act as a mitogen, thus suggesting an important role for KGF in maintenance of the alveolar epithelium.

Animals↗
PubMed1995

Reversible transdifferentiation of alveolar epithelial cells.

Alveolar epithelial type II (AT2) cells have been thought to be the progenitors of terminally differentiated type I (AT1) cells in the adult animal in vivo. In this study, we used an AT1 cell-specific monoclonal antibody (mAb VIII B2) to investigate expression of the AT1 cell phenotype accompanying reversible changes in expression of the AT2 cell phenotype. AT2 cells were isolated and cultured either on attached collagen gels or on gels detached 1 or 4 days after plating and maintained thereafter as floating gels. Monolayers on both attached and floating gels were harvested on days 4 and 8 and analyzed by electron microscopy for changes in morphology and binding of mAb VIII B2. Results indicate that: (1) alveolar epithelial cells (AEC) on attached gels develop characteristics of the AT1 cell phenotype, (2) AEC on gels detached on day 1 maintain features of the AT2 cell phenotype (and do not react with mAb VIII B2), and (3) the expression of AT1 cell phenotypic traits seen by day 4 on attached gels is reversed after detachment. We conclude that commitment to the AT1 and AT2 cell lineages requires continuous regulatory input to maintain the differentiated states, and that transdifferentiation between AT2 and AT1 cells may be reversible.

Animals↗
PubMed1995

Proliferation and differentiation of fetal rat pulmonary epithelium in the absence of mesenchyme.

Previous studies have shown that pulmonary mesenchyme is required to maintain epithelial viability and to support branching morphogenesis and cytodifferentiation. We have examined whether pulmonary mesenchyme can be replaced by a medium containing a combination of soluble factors. Day 13-14 fetal rat distal lung epithelium was enzymatically separated from its mesenchyme, enrobed in EHS tumor matrix, and cultured for 5 d in medium containing concentrated bronchoalveolar lavage, EGF, acidic fibroblast growth factor, cholera toxin, insulin, and FBS (TGM), or in control medium containing only FBS. After 5 d in culture, marked growth and morphological changes occurred in epithelial rudiments cultured in TGM, whereas no changes were seen in controls. [3H]Thymidine incorporation and nuclear labeling indices during the last 24 h of culture confirmed that epithelial rudiments cultured in TGM had significant proliferative capacities. Evaluation of surfactant protein gene expression by Northern analysis, in situ hybridization, and immunocytochemistry demonstrated that distal lung epithelial differentiation progressed in TGM. Ultrastructural analysis demonstrated that fetal distal lung epithelium cultured in TGM contained lamellar bodies and deposited a basal lamina. These results are the first demonstration that sustained proliferation and differentiation of glandular stage distal pulmonary epithelium can proceed in the absence of mesenchyme.

Animals↗
PubMed1994

Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme.

Normal branching morphogenesis and cytodifferention of the lung require a specific interaction between the epithelial rudiment and pulmonary mesenchyme. Previous studies in the mouse have shown glandular stage pulmonary mesenchyme can elicit a lung-like pattern of branching morphogenesis when grafted onto tracheal epithelium of the same age that has been denuded of its own mesenchyme. We have examined whether are not this pattern of branching is accompanied by changes in epithelial cytodifferentiation. Purified pulmonary mesenchyme was isolated from the distal tips of day 13-14 fetal rat lungs and grafted onto a stretch of tracheal epithelium from which the mesenchyme had been removed. The grafts were cultured on semisolid 0.5% agarose in Waymouth's medium containing 10% serum for 5 days and then for an additional 2 days in the same medium containing 10(-6) M cortisol. Unoperated or operated-ungrafted tracheae and intact lung explants served as controls. Grafting distal tip pulmonary mesenchyme onto the tracheal epithelium induced a pattern of branching identical to that seen in control lung explant cultures, while ungrafted control tracheal cultures formed cystic structures. Light microscopy of the induced tracheal epithelium showed that the constituent cells exhibited a morphology virtually identical to cells in control lung explants. Ultrastructural analysis demonstrated that the induced tracheal epithelial cells contained lamellar inclusion bodies, and the lumina of the induced acinar structures contained tubular myelin figures. Reverse transcription polymerase chain reaction analysis of the induced tracheae revealed the presence of mRNAs for surfactant protein A (SP-A) and SP-B, as well as SP-C, which is a specific marker for type II cell differentiation in the adult rat. Control tracheal cultures contained mRNAs only for SP-A and SP-B. Immunocytochemistry demonstrated that many cells within the induced tracheal epithelium were positive for SP-A and SP-C proteins. Further studies demonstrated that the ability of mesenchyme to induce tracheal epithelium varied within the pulmonary tree, and that the competence of the tracheal epithelium to respond to the influence of distal tip mesenchyme was temporally restricted. Our results demonstrate that pulmonary mesenchyme can act instructively on the tracheal epithelium to induce a program of type II cell differentiation.

Animals↗
PubMed1994

Regulation of surfactant protein D expression by glucocorticoids in vitro and in vivo.

SP-D is a recently described lung-associated protein that is produced by alveolar type II cells and may function in pulmonary host defenses. Since little is known regarding the hormonal regulation of SP-D, and since the other surfactant proteins (SP-A, SP-B, and SP-C) are known to be regulated by glucocorticoids, we sought to determine the effects of glucocorticoids on SP-D mRNA and protein expression, both in vitro and in vivo, in the fetal rat lung. In vitro experiments were performed on lung explants from fetuses on gestational day 15 or 18. Explants were cultured in serum-free conditions with or without hydrocortisone for 3 days. SP-D mRNA expression was evaluated by Northern blot analysis. SP-D protein expression was analyzed using a polyclonal antibody against SP-D and standard immunohistochemical techniques. The expression of SP-D mRNA increased in fetal day 15 explants but remained unchanged in fetal day 18 explants cultured without the addition of hydrocortisone, compared with in vivo controls. The addition of hydrocortisone resulted in increases in SP-D mRNA expression at both gestational ages. This pattern of SP-D mRNA expression was compared with the expression of the other surfactant proteins and found to be most similar to that of SP-B. In vivo experiments were performed using maternal administration of dexamethasone (1 mg/kg) or an equal volume of saline on fetal days 15, 16, and 17 or on fetal day 17 with sacrifice on fetal day 18.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Hepatocyte growth factor is a growth factor for rat alveolar type II cells.

Proliferation of alveolar type II cells is thought to be critical for restoration of gas exchange units after diffuse alveolar damage. However, the factors that regulate type II cell proliferation are not well understood. Hepatocyte growth factor (HGF) is a potentially important mitogen because it causes epithelial cells but not fibroblasts to proliferate and is found in the lung. We used rat alveolar type II cells in primary culture to demonstrate that HGF stimulates DNA synthesis in a concentration-dependent manner. The half maximal effect on stimulation of thymidine incorporation was less than 1 ng/ml. By autoradiography, HGF increased nuclear labeling from 1.3% of type II cells with medium alone to 9.4% with 5 ng/ml HGF. During this time, HGF modestly increased cell number in comparison to control media. However, in an assay of colony formation in low-density cultures, HGF did not consistently increase colony formation by alveolar type II cells and was less effective than acidic fibroblast growth factor or bronchoalveolar lavage fluid in this assay. The receptor for HGF (c-met proto-oncogene) was expressed in rat type II cells and whole lung but not in macrophages. In contrast, the mRNA for HGF was detected in rat macrophages and lung but not in type II cells. However, HGF message was not detected in human alveolar macrophages under conditions in which the HGF message was detected in rat alveolar macrophages and in human fibroblasts. Hence, HGF is a potential paracrine growth factor for alveolar type II cells, but there may be important species differences in the relative level of expression.

Animals↗
PubMed1993

Proliferation of rat alveolar epithelial cells in low density primary culture.

Alveolar type II cells proliferate to restore the alveolar epithelium after lung injury and differentiate into type I epithelial cells. A variety of factors promote rat type II cell DNA synthesis in vitro; however, only low levels of proliferation occur when type II cells are cultured at high density. We plated type II cells at low density to determine if those growth factors that stimulate thymidine incorporation also stimulate low density proliferation. Type II cells were plated at 1 x 10(3) cells/cm2 in Dulbecco's modified Eagle's medium containing 2% fetal bovine serum, cholera toxin, insulin, epidermal growth factor, acidic fibroblast growth factor (aFGF), and concentrated bronchoalveolar lavage fluid from normal rats. By 7 days, numerous colonies had grown out that exhibited an epithelial morphology and stained positively for cytokeratin. The cell number at day 7 in the presence of the combined factors was 5.9 x 10(3) (+/- 0.6 x 10(3)) cells/cm2 (n = 4). There was no colony formation in the absence of fetal bovine serum. The addition of linoleic acid to serum-free medium containing all the growth supplements was found to partially restore colony formation. When aFGF or lavage fluid was omitted from the culture medium, colony formation was dramatically reduced. The colonies lacked characteristics of differentiated type II cells, which was anticipated since these cells were cultured on tissue culture plastic. To see if these cells could express differentiated functions, we maintained the colonies under growth conditions, removed them from the plastic substratum, and then replated them on EHS matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Modulation of alveolar type II cell differentiated function in vitro.

We have investigated whether the loss of differentiated function observed in adult rat alveolar type II cells cultured on a substratum that promotes cell spreading and flattening represents a reversible phenotypic change. Cells were cultured for 4 and 8 days in association with fetal rat lung fibroblast feeder layers on either attached collagen gels, which promote the loss of differentiated function, or on floating collagen gels, which support differentiation. A fifth group of cultures were maintained as attached gels for 4 days, then detached and cultured as floating gels for the remaining 4 days. Expression of mRNAs for surfactant proteins A, B, and C, patterns of phospholipid biosynthesis, rates and patterns of protein synthesis, and cell morphology were evaluated as markers of differentiation. Without exception, detaching the gels after 4 days in culture resulted in significant recovery of differentiated characteristics, demonstrating that type II cells modulate differentiated function in response to the culture environment. The results are discussed in relation to the importance of normal cell architecture to normal cell function and to the possible in vitro progression of type II cells to type I cells.

Acetates↗
PubMed1992

Surfactant protein C is expressed in alveolar type II cells but not in Clara cells of rat lung.

The expression of the surfactant-associated proteins in bronchiolar cells remains to be defined. We used in situ hybridization to identify sites of message expression of the surfactant-associated proteins A, B, and C (SP-A, SP-B, and SP-C) in adult and fetal rat lung. The expression of these messages by in situ hybridization was also compared with the localization of SP-A by immunocytochemistry. The localization of SP-A was used to identify type II cells and nonciliated bronchiolar epithelial (Clara) cells in these sections. The cRNA antisense probes for SP-A, SP-B, and SP-C appeared to hybridize over type II cells. Sense probes showed no localization or apparent specific hybridization. Messages for both SP-A and SP-B were also found in nonciliated bronchiolar epithelial (Clara) cells. However, no message for SP-C was observed in these cells. Clara cells from terminal to large bronchioles lacked detectable mRNA for SP-C. Expression of surfactant protein mRNAs was not detectable in type I cells, alveolar macrophages, interstitial cells, or vascular cells. Similarly, in fetal rat lung the messages for SP-A and SP-B but not SP-C were detected in bronchiolar cells. We conclude that rat Clara cells do not express SP-C mRNA, and thus SP-C can be regarded as a specific marker for rat type II cells.

Animals↗
PubMed1991

Effects of maternal dexamethasone on expression of SP-A, SP-B, and SP-C in the fetal rat lung.

Prenatal administration of glucocorticoids has been shown to enhance surfactant production in the fetus. Since the surfactant proteins play an important role in surfactant function and secretion, we wished to determine the effects of maternal glucocorticoid administration on their fetal expression and appearance. Daily dexamethasone (DEX) (1 mg/kg/day) or 0.9% saline was administered to timed-pregnant rats on gestational days 14 through 16 or on day 16 with sacrifice on day 17 (term day 22), and on gestational days 14 through 18, or days 16 through 18, or day 18 with sacrifice on day 19. SP-A content was determined in lung homogenates from treated and control male and female fetal rats by an enzyme-linked in lung homogenates from treated and control male and female fetal rats by an enzyme-linked immunosorbent assay. The abundance of mRNAs for SP-A, SP-B, and SP-C per fixed amount of total cellular RNA was also determined in lungs from treated and control male and female fetal rats by Northern blot analysis. In litters sacrificed on day 17, DEX administered on days 14 through 16 and on day 16 resulted in significant increases in SP-A content. Expression of SP-A mRNA, which was not detectable in control fetuses on day 17, became clearly apparent after either 1 or 3 d of DEX treatment. The abundance of mRNAs for SP-B and SP-C also increased in day-17 fetuses after either 1 or 3 d of DEX treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

In vivo regulation of surfactant proteins by glucocorticoids.

Surfactant proteins have key roles in regulating surfactant secretion, in recycling, and in the assembly of the surfactant monolayer but little is known about their regulation in vivo. Surfactant proteins SP-A, SP-B, and SP-C have been shown to be upregulated by glucocorticoids in vitro, but the role of glucocorticoids in the physiologic regulation of surfactant protein synthesis remains unknown. We have studied the effects of exogenously administered glucocorticoids on the regulation of steady-state surfactant protein mRNA accumulation. We have also studied the effects of adrenalectomy on the accumulation of the surfactant protein mRNAs. Surfactant protein genes appear to have quantitatively different responses to exogenously administered glucocorticoids, with SP-C mRNA increasing at the lowest dose, SP-A and SP-B mRNA increasing in response to similar glucocorticoids doses but with SP-B yielding the highest maximum response. Adrenalectomy, however, does not alter surfactant protein mRNA levels. These observations support a minor role for glucocorticoids in maintaining the steady-state accumulation of surfactant protein mRNA. Adrenalectomy decreases total pulmonary SP-A when compared to sham-operated animals in the absence of changes in its mRNA. Therefore, glucocorticoids may have translational or post-translational effects that regulate total pulmonary SP-A accumulation, but the effects appear to be minor. These findings support a potential role for the adrenal in the pulmonary response to stress and demonstrate for the first time differential accumulation of the surfactant protein mRNAs to glucocorticoids in vivo.

Adrenalectomy↗
PubMed1990

Expression and characterization of rat surfactant protein A synthesized in Chinese hamster ovary cells.

Rat surfactant protein A (SP-A) was expressed in a Chinese hamster ovary (CHO-K1) cell line and characterized for biologic activity using assays for receptor binding and modulation of phospholipid secretion from isolated type II cells. The CHO-K1 cell line was cotransfected with separate plasmids encoding for the rat SP-A, dihydrofolate reductase and neomycin phosphotransferase, respectively. Antibiotic (Geneticin-G418)-resistant transformants were screened by ELISA for the secretion of recombinant SP-A into the media. Northern analysis of the transfected cell lines demonstrated the expression of both 1.6 kb and 0.9 kb mRNA species for SP-A, consistent with the proposed differential polyadenylation of the primary transcript. Amplification with methotrexate resulted in a dose-dependent increase in mRNA for SP-A and a 20-fold increase in the production of recombinant SP-A relative to untreated cells. Maximum production of SP-A was 370 micrograms of SP-A/l of media in a 4-day incubation. Recombinant SP-A was purified from the serum-free media of large scale cultures of transfected, amplified CHO cells by affinity chromatography on mannose-Sepharose. The recombinant SP-A migrated similarly to native SP-A by NaDodSO4-PAGE analysis under reducing and nonreducing conditions and under reducing conditions after digestion with N-glycanase. Recombinant SP-A effectively competed with 125I-native SP-A for binding to the high affinity receptor for SP-A on isolated plasma membranes from rat alveolar type II cells. The recombinant SP-A was as effective as native SP-A in the inhibition of secretion of phospholipid from isolated type II cells. We conclude that recombinant rat SP-A produced in Chinese hamster ovary cells is physically and functionally similar to native rat SP-A.

Animals↗