Overexpression of Pulmonary Surfactant Apoprotein A mRNA in Alveolar Type II Cells and Nonciliated Bronchiolar (Clara) Epithelial Cells in Streptozotocin-induced Diabetic Rats Demonstrated by In Situ Hybridization Kazuhiro Sugahara, Ken-ichi Iyama, Kimihiko Sano, and Tohru Morioka Departments of Anesthesiology and Developmental Biology, Kumamoto University Medical School, Kumamoto, and Department of Pediatrics, Kobe University School of Medicine, Kobe, Japan
Pulmonary surfactant is critical for gas exchange and is composed of both phospholipids and specific surfactant-associated proteins. The most abundant surfactant protein is termed surfactant apoprotein A (SP-A). This protein is thought to be important in the formation of tubular myelin, in absorption of surfactant to the air-liquid interface, in recycling of surfactant in alveolar type II cells, and in the regulation of secretion. We have examined the expression and localization of SP-A mRNA in streptozotocin-induced diabetic rats by in situ hybridization using a specific rat cDNA probe. Diabetes was induced by intraperitoneal injection of 60 mg/kg streptozotocin. After 10 wk, lungs were excised and examined by in situ hybridization and by light and electron microscopy. The ultrastructural examination demonstrated the marked changes of endoplasmic reticulum of alveolar type II cells, as reported previously. Immunohistostaining of SP-A in diabetic lungs was weak in alveolar type II cells. However, by autoradiographs of in situ hybridization, compared with the control lungs, a larger number of silver grains for the SP-A mRNA were shown in alveolar type II cells and also in some bronchiolar epithelial (Clara) cells from the diabetic lungs. Alveolar type II cells having high contents of silver grains were also increased in number. These results were confirmed by measurement of the SP-A content and by Northern blot analysis. The present study demonstrates an overexpression of SP-A mRNA despite the ultrastructural changes in the endoplasmic reticulum of alveolar type II cells in the diabetic lungs, which will provide new information on the regulatory mechanism of SP-A gene expression. In situ hybridization may be a useful approach to evaluating the surfactant apoprotein gene expression in respiratory disorders.
A number of studies have reported that diabetes mellitus adversely affects mechanical and biochemical functions of the lungs. We have demonstrated that arterial oxygen tensions and pulmonary diffusing capacities for carbon monoxide in diabetic patients are often lower than those in a normal corresponding age group (1). In addition, two types of cells in the lungs, i.e., alveolar type II cells and Clara cells, are selectively affected in experimentally induced diabetes of adult rats (2, 3). We have also demonstrated that isolated alveolar type II cells have high-affinity insulin receptors (4). Furthermore, maternal diabetes predisposes newborn infants to re(Received in original form March 25, 1991 and in final form August 20, 1991) Address correspondence to: Kazuhiro Sugahara, M.D., Ph.D., Department of Anesthesiology, Kumamoto University Medical School, Honjo I-I-I, Kumamoto 860, Japan. Abbreviations: adult respiratory distress syndrome, ARDS; bovine serum albumin, BSA; phosphate-buffered saline, PBS; surfactant apoprotein A, SP-A. Am. J. Respir. Cell Mol. BioI. \bl. 6. pp. 307-314, 1992
spiratory distress syndrome (RDS), which is thought to be caused by an immaturity in the synthesis and secretion of surfactant by alveolar type II cells (5). Pulmonary surfactant is a complex mixture of lipids and proteins, which stabilizes alveoli by lowering the surface tension at the air-liquid interface (6). Pulmonary surfactant contains at least three lung-specific proteins that are thought to be important for surfactant structure and functions (7). Pulmonary surfactant apoprotein A (SP-A) is the major protein component of the surfactant complex, and the cDNAs for human, rat, and canine SP-A have been sequenced (7). SP-A has been shown to play an important role in the formation of tubular myelin and in enhancing the adsorption of surface-active phospholipids to an air-liquid interface (7). Recent studies indicate that synthesis of SP-A and its mRNA are affected by various hormones, including glucocorticoid and insulin (7). However, less information is available about the SP-A gene expression in abnormal physiologic states, such as adult respiratory distress syndrome (ARDS) and diabetes mellitus, which have been reported to affect the pulmonary surfactant (8).
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Therefore, in the present study, we have examined the expression and localization of SP-A mRNA in streptozotocininduced diabetic rats by in situ hybridization using a specific rat cDNA probe. We found that there is a markedly increased expression of SP-A rnRNA in both alveolar type II cells and nonciliated bronchiolar epithelial (Clara) cells from the lungs of diabetic rats. This study has been previously published in abstract form (9).
Materials and Methods Animals Adult, specific pathogen-free, male F344 rats (100 to 150 g) were obtained from Charles River Laboratories (Shizuoka, Japan). After a 24-h fast, experimental diabetes was induced by .a single intraperitoneal injection of 60 mg/kg rat body weight streptozotocin in citrate buffer (0.1 M, pH 4.5). Control rats received an equal volume of citrate buffer without streptozotocin. Diabetes was confirmed by glycosuria, elevated blood sugar (> 300 mg/dl) , insulin deficiency, and impaired rate of growth. Glycosuria was checked with Urostik (Miles Sankyo Co., Tokyo, Japan), and blood glucose and serum insulin level were measured with a refractance photometer (Miles Sankyo Co.) and commercial radioimmunoassay (Shionogi Co., Tokyo, Japan), respectively. The animals were fed ad libitum and were killed 10 wk after drug treatment. Development of streptozotocin-induced diabetes and the histologic changes were similar to those of the previous study of alloxan-induced diabetes (2). Preparation of Tissue Sections The rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally), and the trachea was cannulated after tracheostomy. The lungs were fixed by intratracheal instillation of 4 % paraformaldehyde in 0.01 M phosphatebuffered saline (PBS) (pH 7.4). After 5 min of fixation in situ, the lungs were excised and cut for in situ hybridization and for light and electron microscopy. In addition, although diabetic rats weighed less, the lungs of another three small control rats that matched the diabetic rats in body weight were prepared in the same manner. Preparation of cDNA Probe SP-A is encoded by two rnRNA species of 0.9 and 1.6 kb, which have identical coding regions and differ only in the length of the 3' untranslated sequences (10). The rat SP-A cDNA probe used for in situ hybridization was a 0.9-kb insert that encodes the entire protein sequence of SP-A. The isolation and characterization of this cDNA have been described by Sano and associates (10). The insert (double-stranded probe) was labeled with PH]deoxythymidine triphosphate (dTTP) using a conventional nick translation with minor modifications (11, 12). The reacti~:}ll mixture. contained 100 p.Ci of lyophilized PH]dTTP, DIck translation buffer (50 mM Tris-HCl [pH 7.9], 5 mM MgCI2 , 10 rnM ,B-mercaptoethanol, 50 p.g/rnlbovine serum albumin [BSA]) (Sigma Chemical Co., S1. Louis, MO), cold nucleotide-mix (30 p.Meach of deoxyadenosine triphosphate [dATP]), deoxyguanosine triphosphate [dGTP], deoxycytidine triphosphate [dCTP]), 1.0p.g/rnl DNase I (Takara Shuzo Co., Kyoto, Japan), 15 U DNA polymerase (Takara Shuzo Co.), and 40 p.g/rnl DNA fragments.
The mixture was incubated at 14°C for 2 h, fractionated, and precipitated by chromatography on a Nensorb'" 20 cartridge (Dupont Co., Wilmington, DE). The final sample usually had a specific activity of 2 to 2.5 X 107 cpm/ug DNA.
In Situ Hybridization Portions of the right lobe of each fixed lung were cut into 5-mm cubes and fixed in 4 % paraformaldehyde in 0.01 M PBS at room temperature for 1 h and washed 3 times in PBS for 5 min each. These tissue blocks were then dehydrated in a graded series of ethanol and embedded in paraffin. Consecutive sections were cut at 5 p.mand were then processed for in situ hybridization. Hybridization procedures used in this study were practically the same as those described previously 01, 12). Briefly, deparaffinized sections mounted on subbed microscope slides were treated with pronase E (0.25 mglrnl in 50 rnM Tris-HCl [pH 7.6] and 5 mM disodium EDTA [Sigma]) for 10 min and acetylated with a freshly diluted acetic anhydride (0.25 % in 0.1 M triethanolamine buffer [pH 8.0]) for 10 min. The treated sections were processed for in situ hybridization at 45° C for 18 h in a mixture containing the tritiated cDNA probe 0 p.gIrnl), yeast tRNA (500 p.g/rnl), salmon sperm DNA (80 p.g/rnl), 50% formamide, 10 mM Tris-HCl (pH 7.0), 0.15 M NaCI, 1 mM EDTA (pH 7.0), ix Denhardt's mixture, and 10% dextran sulfate. . After hybridization and removal of the cover glass by Immersing the slides in 2x SSC (IX SSC = 0.15 M NaCl, 0.D15 M trisodium citrate [pH 7.0]) for 1 h at room temperature, sections were washed 3 times in 2 X SSC for 10 min each at room temperature, once in 0.5 X sse for 10 min at 45° C, and 3 times in O.lx SSC for 10 min each at 45° C. Afterwards, the slides were dehydrated in ethanol, dried in air, immersed in Kodak NTB-2 nuclear track emulsion, and exposed for 12 to 14 days at 4° C. The exposed slides were developed in Kodak D-19 developer for 3 min at 18° C. The sections were stained with hematoxylin. In addition, sections digested with RNase (2 mg/rnl, 1 h at room temperature) before in situ hybridization with the cDNA probe showed no labeling, implying that the hybridization with the probe was dependent on the presence of RNA in tissue sections. For the quantitative analysis of labeling intensity, the number of silver grains per cell was counted at 250 X oil immersion for 100 cells in five random fields, and the number of alveolar cells having a high density of silver grains (signalto-noise ratio> 10) per unit area was also counted at 500x enlarged photomicrographs in 30 random fields of the sections of each of three different experiments, which were performed by two independent individuals. Results are expressed as means ± SE and were analyzed by Student's t test for independent means. P < 0.05 was considered to be statistically significant. Immunohistochemistry After deparaffinization by heat and in xylene, and a rinse in ethanol (12), 5-p.m sections were treated with 1% hydrogen peroxide in methanol for 30 min to minimize endogenous peroxidatic activity in the tissue and washed in PBS. The slides were then placed in a moist chamber, and sections
Sugahara, Iyama, Sano et al.: Pulmonary Surfactant Apoprotein A mRNA in Diabetic Rats
were covered with 5 % normal goat serum in PBS for 30 min. The excess serum was removed by blotting, and sections were covered with the primary antibody solution and incubated overnight at 4° C. The primary antibody was a rabbit polyclonal antibody directed against rat SP-A (13), and it was used at a 1:200 dilution in PBS containing 1% normal goat serum. After PBS washing, sections were then covered with the biotinylated second antibody, goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at a 1:50 dilution for 1 h, rinsed in PBS, covered with Vectastain ABC reagent (Vector) at a 1:50 dilution for 1 h at room temperature, and rinsed in PBS again. Antigenic sites on sections were demonstrated by reacting the sections with a mixture of 0.05 % 3,3'diaminobenzidine tetrahydrochloride (Dojindo Co., Kumamoto, Japan) in 0.05 M Tris-HCl buffer (pH 7.6), and 0.01% hydrogen peroxide for 7 min. The sections were then counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, and mounted in Entellan Neu (Merck, Darmstadt, Germany). Negative controls for immunostaining were performed by replacing the primary antibody with the normal rabbit IgG. In addition, some of the sections were treated with 0.1 to 1.0% saponin or 0.1% trypsin before being covered with 5 % normal goat serum to increase the antibody's permeability or to unmask an epitope of antigen. Light and Electron Microscopy Some of the excised lung tissues were fixed in 10% neutral formaldehyde solution and embedded in paraffin. The thin sections were stained with hematoxylin-eosin and examined microscopically. Other lung tissues were fixed in 2 % glutaraldehyde, postfixed in osmium tetroxide, and embedded in Epon 812 as described in detail elsewhere (2). The ultrathin sections were stained with uranyl acetate and lead citrate, and examined on a Hitachi H-300 electron microscope. Measurement of SP-A To confirm our result obtained by immunohistochemistry, we also performed another experiment for measurement of SP-A in the lung tissues of control and diabetic rats. The rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), and the lungs were removed from the chest cavity immediately and separated into the right and left lung. The former was placed in PBS containing 1% (vol/vol) Triton X-IOO (1% Triton X-IOO/PBS) for determination of SP-A and was homogenized with Polytron and stored at -80° C until the assays were performed. The latter was examined morphologically to confirm the diabetic changes. The amount of SP-A was measured by a double-sandwich enzyme-linked immunosorbent assay using a rabbit anti-rat SP-A polyclonal antibody, as described previously (14). Anti-SP-A IgG fraction (100 I-'g/mI in 0.1 M NaHC03) was incubated overnight at room temperature in wells of untreated 96-well microtiter plates (Dynatech, Chantilly, VA). The wells were then incubated in PBS containing 1% (vol/vol) Triton X-IOO and 3 % (wt/vol) BSA (3 % BSA/1% Triton X-IOO/BSA). After two washes with 1% Triton X-IOO/PBS, 1001-'1 of affinity-purified rat SP-A standard or various dilutions of samples were added to each well and allowed to incubate at 37° C for 90 min. After the wells were
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washed 3 times with 3 % BSA/l % Triton X-IOOIBSA, 1001-'1 ofbiotinylated anti-SP-A IgG fraction was added to each well and allowed to incubate at 37° C for 90 min. Anti-SP-A IgG was biotinylated using a biotinylation kit from Amersham (Arlington Heights, IL). After four washes with 1% Triton X-IOO/PBS, 1001-'1 of diluted horseradish peroxidase-conjugated streptavidin (Amersham) was added to each well and allowed to incubate at 37° C for 30 min. After three washes with 1% Triton X-IOO/PBS, 1001-'1 of substrate solution (14) was added to each well. The reaction was stopped by the addition of 50 1-'1 of 1.5 mM sodium azide in 0.1 M citric acid. The absorbance at 410 nm was recorded with a Microplate Autoreader (Bio-Rad Laboratories, Richmond, C· .). RNA Analysis To confirm our result obtained by in situ hybridization, we also performed Northern blot analysis to measure relative amounts of SP-A mRNA in the lung tissues of control and diabetic rats. Total RNAs were extracted from lung tissues with 4 M guanidinium isothiocyanate containing 0.1 M {j-mercaptoethanol and 25 mM sodium acetate. RNA was purified by centrifuging the lysate through a cushion of 5.7 M cesium chloride at 80,000 X g for 16 h at 22° C. The amount of RNA was quantitated by measurement of OD at 260 nm. For Northern blot analysis, total RNA (20 I-'g) was sizefractionated by electrophoresis on a 1% agarose gel containing 3 % formaldehyde and 0.5 /-tg/mI of ethidium bromide and transferred to a nylon membrane sheet (Gene Screen; NEN Research Products, Boston, MA) by capillary action in iox SSe. After baking at 80° C for 3 h, the membrane was prehybridized at 42 ° C overnight in 5 x SSC, 3 x Denhardt's solution, 0.2 mg/mI salmon sperm DNA, and 45% formamide and then hybridized in the same solution that additionally contained 1.0 x 1{)6 cpm/mI of a 32P-labeled cDNA probe for rat SP-A. The probe was labeled using a random primer labeling kit (Amersham) with [a-32P]dCTP. After a 24-h hybridization period, the membrane was washed twice in 2 x SSC at room temperature for 5 min, twice in 2x SSC containing 0.1% sodium dodecyl sulfate at 65° C for 30 min, and finally twice in O.1x SSC at room temperature for 30 min. The membrane was then exposed to a Kodak X-Omat AR film with an intensifying screen at -70° C for 24 h. To quantify the relative amounts of the SP-A mRNA, the film was scanned at 550 nm using a dual-wavelength TLC Scanner (CS-930; Shimadzu, Kyoto, Japan) and the area corresponding to each dot was computed. The area of each dot in a single sample was well correlated with the amount of total RNA, and the area of each dot was calculated.
Results Diabetic Rats Changes in blood glucose, serum insulin, and body weight of control and streptozotocin-induced diabetic rats are shown in Table 1. Diabetic rats were hyperglycemic, insulin deficient, and gained weight poorly, as we have previously reported (2), with an average blood glucose of 553 ± 51 mg/dl, serum insulin of 4.0 ± 0.8 /-tV/mI and body weight of 140 ± 10 g at surgery compared with 198 ± 48 mg/dl, 35.0 ± 2.3 /-tV/mI, and 311 ± 15 g, respectively, for the con-
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TABLE I
Comparison of body weight, blood glucose, and serum insulin of control and streptozotocin-induced diabetic rats*
Control rats (n = 5) Diabetic rats (n = 5)
Body Weight
Blood Glucose
Serum Insulin
(g)
(mgldl)
(p'ulml)
311 ± 15 140 ± 10
198 ± 48 553 ± 51
35.0 ± 2.3 4.0 ± 0.8
* Values are mean ± SE. trol rats (P < 0.01, n = 5). Rats matched in weight (to diabetic rats) had an average body weight of 152 ± 2 g (n = 3). Ultrastructural Changes There was no evidence of respiratory infection in control or diabetic rats, either grossly or microscopically. There were two major changes observed in the ultrastructure of the lungs in diabetic rats. There was a marked dilatation of endoplasmic reticulum in alveolar type II cells and in nonciliated bronchial epithelial (Clara) cells in rats killed 10 wk after injection of streptozotocin. It was likely that there were some accumulations of membrane-like materials within dilated endoplasmic reticulum in alveolar type II cells. There was a decrease in number of lamellar bodies in alveolar type II cells (data not shown) (2). The myelin structures of these lamellar bodies still remained, but some of them were irregular or deformed. In Situ Hybridization of SP-A mRNA By autoradiographs of in situ hybridization, silver grains for the SP-A mRNA were clearly shown in alveolar type II cells
and some bronchiolar epithelial cells from the diabetic lungs, but more grains were apparent over cells from the diabetic lungs than those from the control lungs (Figures 1 and 2). Some but not all of the positive bronchiolar epithelial cells had the appearance of Clara cells by light and electron microscopy. Clara cells are characterized by a nonciliated dome-shaped apex that protrudes into the bronchiolar lumen and a large number of dense ovoid secretory granules in their cytoplasm. No silver grains were seen over alveolar type I cells, alveolar macrophages, or cells of the interstitium or vascular endothelium, which indicated that SP-A mRNA localized only in SP-A-producing cells. Compared with the control cells, the densities of silver grains over alveolar type II cells and Clara cells were markedly higher in the diabetic lungs (Figures I and 2). Furthermore, the cells having a high density of silver grains were also increased in number in the diabetic lungs. Therefore, we examined the count of silver grains per alveolar cell, and the number of alveolar cells having positive silver grains in a certain field in sections from the control and diabetic lungs. In one of five hybridization experiments, the silver grain counts per cell in the control and diabetic lungs were 46.0 ± 2.0 and 90.0 ± 5.0, respectively (n = 100, P < 0.01; Figure 3). The number of alveolar cells having SP-A mRNA per mm- were 229 ± 14 and 511 ± 19, respectively (n = 30, P < 0.01). Similar results were obtained in four other separate experiments. Three rats matched in weight (to diabetic rats) showed similar results to control rats (data not shown). As a negative control, sections digested with RNase before in situ hybridization with the cDNA probe showed no labeling in any cells of both control and diabetic lungs (Figure 1), confirming the specificity of the in situ hybridization reaction.
Figure 1. Autoradiographs of in situ hybridization of pulmonary surfactant apoprotein A (SP-A) mRNA in paraffin sections of alveolar epithelium from a control rat (C), a diabetic rat (DM), and in an RNase-treated paraffin section of alveolar epithelium from a diabetic rat (RNase). Note a number of silver grains over cells in DM, a lower number of grains over cells in C, and no specific silver grains over cells from an RNase-treated section. Bars = 20 IJ.ID.
Sugahara, Iyama, Sano et al.: Pulmonary Surfactant Apoprotein A mRNA in Diabetic Rats
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Figure 2. Autoradiographs of in situ hybridization of SP-A mRNA in paraffin sections of bronchiolar epithelium from a control rat (C) and a diabetic rat (DM). Note a number of silver grains over cells in DM, a lower number of grains over cells in C. Bars = 20 Jlm.
Immunohistochemistry The polyclonal antibody to the rat SP-A, which has been reported in a previous study (13), was used to stain the lung tissues from the control and diabetic rats by immunohistochemistry. Imrnunostaining of normal lungs (Figure 4) demonstrated a strong positive reaction in the alveolar wall cells as well as focal inner surface of the alveoli. The location and distribution of the SP-A-positive cells and their morphologic characteristics were consistent with those of alveolar type II cells. Some positive cells in the alveolar space appeared to be alveolar macrophages. In airways, SP-A-positive cells in the bronchiolar epithelium were also observed. These cells were nonciliated bronchiolar epithelial (Clara) cells. In the diabetic lungs (Figure 4), on the other hand, SP-A-positive reactions localized in the alveolar wall cells, focal inner surface of alveoli, and some bronchiolar epithelium in patterns similar to those of the control lungs. However, the staining reaction in diabetic lungs was less intense over the entire sec-
Figure 3. Quantitation of SP-A mRNA in control (C) and diabetic lungs (DM). Sections of the control lungs (solid bars) and the diabetic lungs (hatched bars) were hybridized using the cDNA probe for SP-AmRNA. The numbers of silver grains per cell (A) and alveolar cells having positive silver grains per unit area (B) were counted in fivedifferent views of the sections in one of five hybridization experiments. Similar results were obtained in four other separate experiments. Values are expressed as mean ± SE.
tion. Because it was possible that this weak staining could be due to a masking by some materials, we also treated some sections in organic solvents or detergents, such as 0.1 to 1.0% saponin or 0.1% trypsin, to increase the antibody's penetration or to unmask the epitope of SP-A. But weak staining in diabetic lungs still remained. No specific staining was shown in the control sections treated with normal (nonspecific) rabbit IgG (data not shown).
Content of SP-A in Lung Tissues To confirm our result obtained by immunohistochemistry, we also measured the content of SP-A in the lung tissues of control and diabetic rats. The content of SP-A in lung tissues of control and diabetic rats is shown in Table 2. Diabetic rats had less SP-A in lung tissues than did control rats. Although diabetic rats gained weight poorly, SP-A per lung weight in diabetic rats was significantly reduced compared with that of the control rats (P < 0.05).
B
A
Cell number! mm
Grains! cell
100
2
600
80
60 40
20
o
C
OM
o
C
OM
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Figure 4. Immunohistostaining using anti-rat SP-A polyclonal antibody in paraffin sections of alveolar epithelium (upper panels) and bronchiolar epithelium (lower panels) from a control rat (C) and a diabetic rat (DM). Note the less intense staining of SP-A in the diabetic rat compared to the control lungs. Bars = 20 p.m.
Relative Amount of the SP-A mRNA To confirm our result obtained by in situ hybridization, we also performed Northern blot analysis using the 1.6-kb insert as a probe. Northern blot of RNA extracted from the lung tissues of control and diabetic rats is shown in Figure 5. The relative abundance of the SP-A mRNA in the diabetic lungs was higher than that in the control lungs, and the calculated dot areas in the control and diabetic lung tissues were 0.22 ± 0.01 g (n = 3) and 0.28 ± 0.01 g (n = 3), respectively (P < 0.05).
Discussion Alveolar type II epithelial cells are essential for the normal function of the lungs. These cells synthesize and secrete pulmonary surfactant that reduces the surface tension at the airliquid interface, promotes gas exchange, and prevents alveolar collapse. The importance of pulmonary surfactant to normal lung physiology is emphasized by the fact that alterations in the surfactant are associated with clinical lung impairments, such as ARDS as well as infant respiratory distress syndrome (6, 8).
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TABLE 2
The content of SP-A in the lungs of control and streptozotocin-induced diabetic rats* SP-A in Homogenate
Control rats (n = 3) Diabetic rats (10 wk, n
=
3)
Body Weight
Right Lung Weight
SP-A in Homogenate
Right Lung Weight
(g)
(g)
(p.glml)
(p.glmllg)
336 ± 13 154 ± 9t
1.78 ± 0.10 1.66 ± 0.28
193.5 ± 32.5 126.5 ± 11.3
117.2 ± 8.0 70.9 ± 4.9t
* Values are mean ± SE. < 0.05 compared with control.
t P
Pulmonary surfactant is composed primarily of phospholipid and lung-specific proteins. SP-A is the most abundant and non-serum protein in pulmonary surfactant with a reduced molecular weight between 26,000 and 36,000, and is thought to have a regulatory function not only in surfactant metabolism but also in the host defense of the lung (7). In the present study, we demonstrated that expression of SP-A mRNA was markedly increased in lungs from streptozotocin-induced diabetic rats by in situ hybridization using a specific cDNA probe. Alveolar type II epithelial cells and the nonciliated bronchiolar epithelial (Clara) cells in the diabetic lungs were more intensely labeled with the cDNA probe for SP-A mRNA, compared with the control (shown in Figures 1 and 2). Those cells with a high content of silver grains were also increased in number. Although the technique of tissue in situ hybridization has been used to localize the mRNAs for the surfactant proteins SP-A and SP-B (15), to our knowledge, this is the first report demonstrating the overexpression of SP-AmRNA in the diabetic lung by in situ hybridization. Several studies have demonstrated that surfactant apoproteins are present not only in alveolar type II cells but also in nonciliated bronchiolar epithelial (Clara) cells (15, 16). In this study, we provide additional support for the synthesis of surfactant apoproteins in bronchiolar epithelial cells by demonstrating the presence of mRNA for the SP-A in these cells in adult rat lungs, similar to that in rabbit and human lungs (15). The increased SP-A mRNA expression in the diabetic lungs was also confirmed by Northern blot analysis, and it showed an approximately 1.3-fold increase in the diabetic lungs compared with control lungs. This increase was consistent with that observed by in situ hybridization. It is likely,
Figure 5. Northern blot of RNA isolated from control lungs and diabetic lungs. Total RNA was extracted from rat lungs in each group. A total of 20 !Jog of RNA was electrophoresed, transferred to a nylon membrane sheet, and hybridized with the radiolabeled SP-A cDNA probe. The intensity of signal in the diabetic lungs (lane B) was higher than that seen in the control lungs (lane A).
_1.6 kb
A B
however, that in situ hybridization has demonstrated the more specific localization and expression of SP-A mRNA in the lungs. Isolating alveolar type II cells from the diabetic lungs remains to be done for Northern blot analysis. The precise reason or mechanism for the increased SP-A mRNA expression in the diabetic lung is unclear. The principal factors regulating SP-A gene expression in vivo have not yet been established, but the rapidly expanding literature on this subject suggests that a number of hormones and growth factors including insulin (7, 17) are able to modulate this expression either transcriptionally or post-transcriptionally. Snyder and Mendelson (17) showed that insulin decreases SP-Aexpression levels in fetal lung explants. In the present study, serum insulin levels were very low in diabetic rats. Therefore, this may be one reason for the overexpression of the SP-A mRNA in the diabetic lungs. Furthermore, overexpression of SP-A mRNA was similarly observed not only in alveolar type II cells but also in bronchiolar epithelial (Clara) cells from diabetic lungs, which indicates that diabetes itself or other factors associated with diabetes, such as acidosis, may cause an abnormality in the regulation of SP-A gene expression. However, there have been few studies to evaluate the roles of insulin or other factors in the regulation of SP-A levels in vivo. Further study, especially about the expression of SP-A mRNA in the insulin-treated diabetic lungs, remains to be done. In the present study, immunohistostaining of the SP-A by rabbit anti-rat SP-A polyclonal antibody showed that the staining reaction of the diabetic lungs was less intense than those of the control lungs, Although one of the reasons for weak staining was likely to be associated with maskings of the antigen site, the staining of the tissues treated by organic solvents or detergents was almost similar to those before treatment, which suggests that the production of SP-A protein may be decreased in the diabetic lungs. In this connection, it is of interest that our study (2) including this experiment (data not shown) and another study (3) demonstrated the ultrastructural alterations of only two types of cells, that is, alveolar type II cells and Clara cells, in experimentally induced diabetic rats. Those changes were marked dilatations of the endoplasmic reticulum, which plays a central part in the biosynthesis of the macrcmolecules in cells. Therefore, those morphologic changes in the diabetic lungs might be attributed to the disturbance of SP-A metabolism, and dysfunction of endoplasmic reticulum might also alter the efficiencyof SP-A translation. Furthermore, to verify our results obtained by immunohistochemistry, we also measured the amount of SP-A in the lung tissues of control and diabetic rats by an enzyme-linked immunosorbent assay, as
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reported previously (14), and we found a decreased SP-A content in the diabetic lungs compared with the control lungs (Table 2). Our combined morphologic results of in situ hybridization and immunohistostaining, and measurement of SP-A and Northern blot analysis, indicate that levels of SP-A mRNA in the diabetic lungs actually increased, although SP-A protein decreased. However, the exact reason for the discrepancy between SP-A mRNA levels and SP-A protein is also unknown. Such changes might be due to changes in the transcriptional rate or changes in the stability or translational efficiencyof the SP-A mRNA. Recently, similar observations between some other proteins and their mRNA levels have been reported in other tissues (18), including tissues in a diabetic state (19). Further investigation will be required to characterize the SP-A gene regulation in the lung and to clarify this discrepancy. In summary, we have demonstrated overexpression of SP-A mRNA in alveolar type II cells and Clara cells of the lungs from streptozotocin-induced rats, despite their ultrastructural alterations. This study indicates that studies of lungs from diabetic animals provide useful information on the regulatory mechanism of surfactant protein gene expression, and in situ hybridization may be a useful way of evaluating this information in respiratory disorders. Acknowledgments: We gratefully thank Drs. Y. Kuroki and T. Akino (Sapporo University Medical School, Japan) for providing anti-rat polyclonal antibody for SP-A, and Dr. M. Hayashi (UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ) for his helpful advice and discussions. We are also especially appreciative of Dr. Robert J. Mason (Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO) for his time and valuable criticism of the manuscript. This work was supported in part by the Grants-in-Aid for Scientific Research No. 03807102 to Dr. Sugahara and No. 03670176 to Dr. Iyama from the Ministry of Education, Science and Culture, Japan.
References 1. Sugahara, K., and T. Morioka. 1983. Studies of the lungs in diabetes mellitus. Kokyu To Junkan 31: 1287-1294. 2. Sugahara, K., K. Ushijima, T. Morioka, and G. Usuku. 1981. Studies of
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