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Considerable progress has been made in defining the phenotype of contractile cells of the lung during development, in the adult and during the remodeling process. The high degree of phenotypic heterogeneity of subgroups of these cells, such as SMCs, is appreciated. Recent studies also have explored the relationship between phenotype and cell function, though this remains an important area for research in the coming years. Similarly, though our understanding of the regulation of cell phenotype is expanding rapidly, much remains to be done, particularly at the level of gene regulation. New transgenic models, coupled with gene-promotor analyses in transgenic animals and in cultured cells should allow rapid progress. Studies of the regulation of specific contractile and cytoskeletal proteins at the gene level by specific cytokines and extracellular-matrix elements will be particularly important.

The lung contains a full complement of vascular and visceral smooth-muscle cells (SMCs) [2, 16, 18]. Additionally, in the normal alveolar wall, there are contractile interstitial cells that contribute to normal function [10, 12] as well as to alveolar remodeling seen in such disorders as pulmonary hypertension and interstitial fibrosis [11, 21]. This review will present three topics: (1) the differentiation of these cells during normal lung development, (2) the phenotypic changes that occur during pulmonary interstitial remodeling, including the appearance of alveolar and interstitial myofibroblasts during pulmonary fibrosis, and (3) the regulation of cell phenotype by transforming growth factor-β (TGF-β). Other chapters in this volume also are particularly relevant to what is covered here (Chaps. 1, 5, 6, 15, 18).

The myofibroblast is an ultrastructurally and metabolically distinctive connective tissue cell identified as a key participant in tissue remodeling in human granulation tissue, organ fibrosis, and the fibroblastic host response to malignant neoplasms. In this study of myofibroblasts in human lung diffuse alveolar damage (DAD), we identified 36 autopsy cases in which DAD could be histologically documented. DAD is known to progress from initial injury through an exudative, proliferative, and terminal fibrotic phase. In the exudative phase (16 cases), myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA) are found in the septa and less frequently in hyaline membranes. In the proliferative phase (18 cases), many myofibroblasts in septa, hyaline membranes, and intra-alveolar fibroplasia express alpha-SMA. The alpha-SMA phenotype should be used in additional studies of myofibroblast differentiation, replication, and apoptosis. A better understanding of the biology of this cell type should offer new therapy for patients with DAD.

Epidermal growth factor (EGF) is a potent mitogen for human mesothelial cells, and autophosphorylation of the EGF receptor (EGF-R) occurs in these cell types after exposure to asbestos, a carcinogen associated with the development of mesothelioma. Here, the intensity and distribution of EGF-R protein was documented by immunocytochemistry in a human mesothelial cell line (MET5A) exposed to various concentrations of crocidolite asbestos and man-made vitreous fibers (MMVF-10). Whereas cells in contact with or phagocytizing shorter asbestos fibers ( or =60 microm) showed intense staining for EGF-R. In contrast, human A549 lung carcinoma cells showed neither elongation nor increased accumulation of EGF-R protein in response to long fibers. Patterns of aggregation and increases in EGF-R protein in mesothelial cells phagocytizing long asbestos fibers were distinct from diffuse staining of phosphotyrosine residues observed in asbestos-exposed cultures. These studies indicate that aggregation of EGF-R by long fibers may initiate cell signaling cascades important in asbestos-induced mitogenesis and carcinogenesis.

Alpha-cristobalite causes pulmonary interstitial disease in humans and experimental animals. Aerosol exposure of rats to cristobalite for 8 days results in early and sustained alveolar type II cell hyperplasia in areas of inflammation characterized by the presence of macrophages and polymorphonuclear leukocytes. Irregular interstitial fibrosis and coalescence of alveoli are apparent by day 120. The inflammatory response is characterized by increased lavage cell recoveries, principally macrophages and neutrophils. Lavage recoveries of protein, nonpolar lipid, phospholipid, and saturated phosphatidylcholine also are increased. The recovery ratio for two important surfactant phospholipids, phosphatidylcholine and phosphatidylglycerol, is decreased at all points following exposure. Our morphologic analyses, together with results correlating lavage cell and lipid recoveries, point to the potential importance of macrophages and neutrophils in the epithelial cell response to cristobalite exposure.

We measured the rate of protein synthesis and total RNA content in the right ventricle (RV) at day 2 and day 4 after pulmonary artery constriction to determine the contributions of changes in capacity and efficiency of in vivo protein synthesis to pressure overload (PO) cardiac hypertrophy. A significant increase in the proportion of RV weight to total heart weight was observed at day 2 and day 4 when compared with untreated controls. The rate of protein synthesis was significantly higher at day 2 post-PO (0.31 +/- 0.06 day-1 or 30 +/- 5 mg.g RV-1.day-1, means +/- SD, P less than 0.05) as well as at day 4 (0.25 +/- 0.05 day-1 or 28 +/- 9 mg.g RV-1.day-1, P less than 0.05) than for untreated rabbits (0.15 +/- 0.03 day-1 or 17 +/- 4 mg.g RV-1.day-1). RNA content was significantly higher at day 2 (1.47 +/- 0.17 mg/g RV, P less than 0.05) than in controls (1.16 +/- 0.14 mg/g RV), whereas there was a slight but nonsignificant increase at day 4 (1.36 +/- 0.21 mg/g RV, P less than 0.1). The efficiency of protein synthesis (synthesis/RNA) per gram RV was significantly increased both at day 2 (20.5 +/- 2.2 g protein.g RNA-1.day-1, P less than 0.05) and day 4 (19.8 +/- 3.5 g protein.g RNA-1.day-1, P less than 0.05) compared with control (14.6 +/- 2.3 g protein.g RNA-1.day-1). The increase in efficiency appeared to be caused by pressure overload itself based on a comparison of 0-4 day data vs. data obtained from sham animals (P less than 0.05).

Collagen content, the ratio of collagen types I and III and elastin content were measured in 5-6- and 10-12-week-old rabbits with and without right-ventricular pressure overload. Significant and equivalent hypertrophy occurred in both age groups. A 2-day pressure overload caused a fall in collagen concentration below control levels in right-ventricular tissue from the older animals, but no change in the younger ones. A 2-week pressure overload in the older animals resulted in a rise in collagen concentration, a decreased ratio of type III to type I plus III [III/(I + III)] collagens, a fall in desmosine concentration and a fall in the desmosine/hydroxyproline ratio in the right ventricle. None of these changes occurred in the younger age group. We hypothesize that the changes in connective-tissue proteins after overload in the older group may contribute to previously observed changes in mechanical performance. The divergent connective-tissue responses in the two groups suggest the importance of age in determining outcome, as well as the possibility of separate regulatory mechanisms for contractile and for architectural elements of the heart.

An initial examination was made of the hypothesis that one action of cigarette smoke components on pulmonary alveolar macrophage function involves the inhibition of contractile protein adenosine triphosphatase activity. Pulmonary alveolar macrophage calcium-dependent adenosine triphosphatase activity, magnesium-dependent adenosine triphosphatase activity, sodium-potassium-dependent adenosine triphosphatase activity, phagocytosis, and cell adhesiveness were measured in the presence of cigarette smoke, acrolein, ouabain, and ethacrynic acid. Calcium-dependent adenosine triphosphatase activity, magnesium-dependent adenosine triphosphatase activity, phagocytosis, and adhesiveness were inhibited by smoke and ethacrynic acid, but not by ouabain. Acrolein, a component of smoke, inhibited phagocytosis, adhesiveness, and calcium-dependent adenosine triphosphatase activity, indicating that another component of smoke must be effective at inhibiting magnesium-dependent adenosine triphosphatase activity. Sodium-potassium-dependent adenosine triphosphatase activity was inhibited by ouabain and ethacrynic acid, but not by smoke or acrolein. Finally, sulfhydryl reagents at least partially protected the macrophages against the inhibitory actions of each of the agents. The results are in accord with recently obtained experimental evidence that calcium-dependent adenosine triphosphatase and, perhaps, magnesium-dependent adenosine triphosphatase play a role in phagocytosis. The data also suggest that smoke components affect a number of macrophage activities, including adhesion and phagocytosis, by altering the cell's contractile apparatus.

Quantitatively accurate studies of macromolecule and lipid synthesis in lung and other tissues by using radioactive substrates require detailed knowledge of the specific radioactivity of the appropriate pool of precursor molecules serving the synthetic pathway. A brief summary is provided of how considerations of precursor availability, metabolism, and compartmentation, as well as product remodeling, may affect the accuracy with which rates of protein, DNA, RNA, and lipid synthesis can be measured. Where possible, the application of this material to studies of lung metabolism is discussed, along with approaches that may minimize experimental uncertainties.

Rates of protein synthesis and degradation were measured in the isolated rat epitrochlearis muscle by radiotracer techniques, by using the specific radioactivity of tRNA-bound amino acid as precursor for protein synthesis. The tissue maintained linear rates of protein synthesis for 3 h of incubation in the presence of amino acids and glucose and in the absence of insulin. Under these conditions, however, the muscles were in negative nitrogen balance, with rates of protein degradation exceeding rates of protein synthesis. Under steady-state conditions of labelling, the specific radioactivities of tRNA-bound leucine, phenylalanine and valine were significantly less than their respective values in the incubation medium, at concentrations in the medium varying from 1 to 10 times those in normal rat serum. Insulin caused a dose- and time-dependent increase in tRNA-based protein synthesis rates, more than doubling rates at 5 and 50 ng of insulin/ml. At the lower, physiological, concentration of insulin, the stimulation of protein synthesis was not observed until the third hour of incubation with the hormone, whereas the rate of protein synthesis at the higher concentration was elevated during the second hour. There were no delays in the stimulation by insulin of glucose conversion into glycogen. The delayed stimulatory effects of insulin on the rate of protein synthesis brought the tissue to a nitrogen balance near zero. The presence of the hormone also prevented the increase in the rate of protein degradation seen in the third hour of incubation in the absence of the hormone. These studies demonstrate the viability of the incubated rat epitrochlearis muscle with respect to protein metabolism and sensitivity to the protein anabolic effects of physiological concentrations of insulin, and indicate that the preparation is a suitable experimental model for the study of the control of protein metabolism in fast-twitch skeletal muscle.

Levels of type III procollagen N-terminal peptide were immunoassayed in sera and bronchoalveolar lavage fluids from healthy volunteers and patients with idiopathic pulmonary fibrosis or sarcoidosis. Peptide levels normalized to protein were higher than corresponding serum levels in all groups, suggesting that the lung is a producer of this peptide. Levels were significantly elevated in both patient groups, suggesting increased rates of type III collagen synthesis. Values were highest in idiopathic pulmonary fibrosis samples. Correlation of peptide levels with clinical severity of disease and lavage cell profiles was poor, perhaps because the latter reflects aspects of the disease process other than that stage in which type III collagen synthesis is altered. Lavage analyses of procollagen peptides may provide an important index of altered collagen synthesis in interstitial lung disease.

The synthesis rates of different myosin isoenzymes in a single muscle, and of the same isoenzymes in different muscles (soleus, masseter and plantaris), were measured. The rate of total protein synthesis was significantly higher in the soleus [greater than 95% slow myosin (SM)] than in the plantaris [greater than 95% fast myosin (FM)]. Two fast isoenzymes, FM2 and FM3, were synthesized at different rates in the masseter, and SM was synthesized at a faster rate than FM. Intermediate myosin had a synthesis rate similar to that of FM. There was a small but significant difference between the synthesis rates of the SM isoenzymes of the soleus and masseter muscles. FM3 was synthesized faster in the masseter than in the plantaris, whereas FM2 was synthesized faster in the plantaris than in the masseter.

Cardiac hypertrophy, induced by pressure overload, leads to a depression in the rate of force development, velocity of shortening, tension-dependent heat generation, and myosin ATPase activity, whereas cardiac hypertrophy, induced by thyroxine administration, leads to an increase in these parameters. These changes have been attributed, in part, to structural changes in myosin. In this study, we have investigated changes in the relative content of myosin isozymes and differences in primary structure of the isozymes in pressure-overloaded and thyrotoxic cardiac hypertrophy in the rabbit. Three myosin isozymic forms (V1 = fastest, V2 = intermediate, V3 = slowest mobility) were observed in pyrophosphate polyacrylamide gels from normal hearts with the V3 component being the predominant species. In the pressure-overloaded model, the V1 and V2 components disappeared or were present in reduced amounts leaving the V3 more predominant. The most striking difference was the isozymic profile produced in thyrotoxic hearts where the V1 became the predominant component and V2 and V3 the minor components. alpha-Chymotryptic digestion of myosin heavy chains produced characteristic, reproducible peptide patterns for each of the animal models, as did fluorographic analyses of alpha-chymotryptic digests of 14C-iodoacetamide (IAA)-labeled SH1 peptides of myosin. Our results suggest that altered proportions of myosin isozymes may be responsible for altered cardiac performance.

Subsegmental bronchoalveolar lavage of human subjects provides an accessible sample of cells and secretions from the lower respiratory tract for clinical and research study. Technical factors that may modify lavage results have received little attention. The effects of lavage volume on the patterns of recovered cells and biochemical components were studied by separately analyzing each of 4 sequential 60-ml syringes of saline instilled into a subsegment of the right middle lobe. Fourteen healthy normal subjects volunteered for lavage. Recovery of cells from both nonsmokers and smokers increased and then remained essentially constant in subsequent syringes, suggesting elution of adherent macrophages from the alveolar surface. Protein, carbohydrate, lipid, and potassium appeared in decreasing concentrations in sequential syringes, but different constituents followed separate patterns. Simple mixing models do not explain the observed results. Measured values for the components in a "small" 120-ml lavage may be substantially different from the values in a "large" 240-ml lavage. Further study of the kinetics of recovery of selected lavage components may provide better understanding of the origins and functions of these substances in the lung.

The in vivo synthesis rates of myosin isozyme heavy chains beta and alpha were measured in right ventricular (RV) muscle at 2 and 4 days following pulmonary artery constriction in rabbits, together with measurements of their relative mRNA levels. The synthesis rate of beta-myosin heavy chains was elevated in 2-day (0.27 +/- 0.06 day-1 or 2.5 +/- 0.7 mg/g RV/day, mean +/- SD) and in 4-day (0.25 +/- 0.08 day-1 or 2.8 +/- 1.0 mg/g RV/day) pressure overload, when compared to untreated rabbits (0.15 +/- 0.04 day-1 or 1.5 +/- 0.4 mg/g RV/day). However, the synthesis rates of alpha-myosin heavy chains in the same hearts were not altered significantly. There was a differential increase in the fractional synthesis rate of beta vs. alpha heavy chains in 2-day and 4-day pressure overload and in 2-day shams, suggesting switching toward beta heavy chain synthesis had occurred at these time points. beta heavy chain synthesis, as a proportion of total (alpha + beta) heavy chain synthesis, was significantly higher in 4-day pressure overload (78 +/- 9%) than in 4-day sham rabbits (63 +/- 6%). This increase in relative beta-synthesis was associated with a significant increase in the relative proportion of beta heavy chain mRNA level (76 +/- 13% vs. 56 +/- 7%). Furthermore, relative beta-synthesis and the beta-mRNA levels correlated linearly with each other in all experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The success or failure of pulmonary defense mechanisms largely determines the appearance of clinical lung disease. The lung is protected by interlucking systems of nonspecific and specific defenses. Inhaled substrances can be isolated by mechanical barriers or can be physically removed from the lung either by transport up the bronchial mucociliary escalator or by transport through interstitial and lymphatic channels leading to lymph nodes. Substances can be locally detoxified within the lung by interaction with secretory proteins, such as antibodies, or by neutralization and dissolution within phagocytic cells. The pulmonary alveolar macrophage is the central figure in the protection of the respiratory membrane, operating in all 3 of the nonspecific modes of defense and augmented by specific immunologic mechanisms as well. Alterations in macrophage function and physiology may be crucial in determining the effectiveness of pulmonary defense. Recent advances in the cell biology of the alveolar macrophage have led to a greater understanding of its complex funcition. The multiple origins of macrophages from local and circulating cell pools and the variability in their fate and lifespan reflect the multi-faceted role of this cell type. The importance of the interactions between macrophages, orther lung cells, and other defense mechanisms has become increasingly clear. As well as functioning as resident defender of the alveolus, the macrophage is an important effector of the pulmonary immune response and plays a key role in the pathogenesis of a wide variety of inflammatory, destructive, and fibrotic lung diseases. Humoral and cell-mediated immune responses amplify and direct lung defenses against infection and may also participate in protection against other agents. Immunoglobulin A and G, microbial neutralizing and opsonizing anti-bodies, and macrophage-stimulating T lymphocytes are the major immunospecific forms of lung defense. Infectious agents, cigarette smoke, air pollutants, industrial dusts, and a spectrum of coexistent disease states may impair pulmonary defense mechanisms and increase susceptibility to asute and chronic respiratory diseases. A thorough understanding of the ways in which the lung protects itself against the daily assault of infectious, toxic, and immunogenic materials should lead to a beter understanding of pathogenesis and consequences of lung disease and to better clinical care of the patient with respiratory disease.

The patterns of myosin isoenzymes in fast- and slow-twitch muscles of the rat hindlimb were studied, by pyrophosphate/polyacrylamide-gel electrophoresis, with hypertrophy (induced by synergist removal) and with spontaneous running exercise of 4 and 11 weeks duration. At 11 weeks, changes with hypertrophy in the slow-twitch soleus, composed of greater than 95% SM2 (slow myosin 2) in normal muscles, were minor, and consisted of an increase in the SM1 and SM1′, and a loss of intermediate myosin (IM), an isoenzyme characteristic of Type IIa fibres [Fitzsimons & Hoh (1983) J. Physiol. (London) 343, 539-550]. The changes were dramatic, however, in the fast-twitch plantaris muscle. There was a 3-fold increase in the proportion of SM. In addition, IM became the predominant isoenzyme in the profile of hypertrophied plantaris by 4 weeks. These increases were balanced by decreases in the proportion of FM2 (fast myosin 2), with FM1 completely absent from the profile at 11 weeks. The changes in the plantaris with exercise were similar in direction but not as extensive as those with hypertrophy, and FM1 remained present at control levels throughout the study. When hypertrophy and exercise were combined, the increase in slow myosin was equal to the sum of the increases with each treatment alone. Changes at 4 weeks were intermediate between those of control and 11-week muscles. Peptide mapping of individual myosin isoenzymes showed that the heavy chains of IM were different from either fast or slow heavy chains. Furthermore, IM was found to be composed of a mixture of fast and slow light chains. These changes suggest that a transformation of myosin from fast to slow isoforms was in progress in the plantaris in response to hypertrophy, via a Type-IIa-myosin (IM) intermediate stage, a phenomenon similar to that occurring in chronically stimulated fast muscles during fast-to-slow transformation [Brown, Salmons & Whalen (1983) J. Biol. Chem. 258, 14686-14692].

A large proportion of parenchymal lung is composed of cells with an abundance of actin-containing cytoplasmic microfilaments. This may explain the substantial contractile capability of the tissue, which it appears cannot be ascribed totally to vascular or airway smooth muscle. Parenchymal contractility is increased in bleomycin-induced fibrosis, together with an increase in microfilaments and actin- and myosin-directed immunofluorescence. The present biochemical studies indicate that at least 10% of detergent-extractable protein from peripheral lung is actin. Approximately 71% of this actin is polymerized and 29% is monomeric, on the basis of differential ultracentrifugation. Isoelectric focusing and tryptic peptide analysis show that nonmuscle actin types predominate in the parenchyma, of which approximately 78% is beta-nonmuscle actin and 19% is gamma-nonmuscle actin, together with approximately 3% gamma-smooth muscle actin. Total actin as a percentage of extractable protein was not increased significantly in lungs of rats 4 or 8 wk after bleomycin instillation. Thus, actin per total lung is increased only in proportion to increased total lung weight. There is, in addition, no detectable shift in the beta/gamma actin ratio in the fibrotic lung or increase in percentage of smooth muscle actin. There is, however, a significant 16% decrease in monomeric G-actin and a commensurate significant increase in the percentage of polymerized or F-actin. Therefore, increased contractility and actin-specific immunofluorescence characteristic of fibrotic lung does not appear to be due to increases in total actin but rather to increases in its degree of polymerization, as is found in a variety of remodeling tissues. The consequence of these changes in contractile protein organization to lung function requires further investigation.

An effort to optimize conditions for studying protein biosynthesis by the pulmonary alveolar macrophage in vitro has led to a comparative analysis of the activity of suspended and adherent cells. A number of differences were observed. (1) Suspended cells synthesized protein for only a limited period of time, after which they responded only partially to incubation in fresh medium. This was true even under reincubation conditions in which the cells were allowed to adhere to a surface. Adherent cells, however, synthesized protein during a longer period of time and were fully capable of responding to new medium within the time periods examined. (2) Analyses of the radioactive proteins synthesized using a dual-isotope technique suggested that, during a period of 2 hours, suspended cells synthesized relatively smaller quantities of high molecular weight proteins than adherent cells. (3) The administration of a phagocytic load (zymosan; particle to cell ratio, 10:1) inhibited by 20 per cent the incorporation of isotopic amino acid into protein during a period of 3 hours. The same phagocytic load, however, stimulated incorporation by 20 per cent in adherent cells. (4) The rate of particle uptake measured using oil red O-albumin complexes decreased by approximately 50 per cent in suspended cells preincubated for 2 hours, but was maintained in similarly preincubated adherent cells. It was concluded that pulmonary alveolar macrophages incubated adhered to a surface are more appropriate for metabolic studies than are pulmonary alveolar macrophages incubated in suspension.

A study was made of the effects of kaolinite, an aluminum silicate found in cigarette smoke and in alveolar macrophages of cigarette smokers, on the in vitro function of rabbit alveolar macrophages. Macrophages lavaged by standard procedures were incubated as adherent monolayers in the presence or absence of kaolinite, and amino acid incorporation into protein and transport subsequently measured. In the presence of dialyzed serum, kaolinite slightly inhibited incorporation into protein during the first 2 to 3 hr of incubation, after which incorporation ceased and a large percentage of newly synthesized protein was released (50% effect at approximately 0.5 mg/ml kaolinite). A dual-isotope experiment indicated that any change in the synthesis of protein which may have occurred was not selective for any protein or group of proteins. Kaolinite also stimulated noncompetitive amino acid accumulation after 2 to 3 hr in the presence of serum. The effects of kaolinite were immediate when incubations were conducted in the absence of serum. Control experiments showed all of the effects of the aluminum silicate to be on the cells and not on the incubation medium. These results suggest that kaolinite is cytotoxic and exerts its effects by a mechanism similar to that proposed for magnesium silicates and silica, in which the naked silicate is immediately cytotoxic, but if coated with serum protein must first be uncoated by lysozomal enzymes before destroying the cells.

The contractile capability of lung parenchymal strips isolated from normal rats was compared with that of strips isolated from rats with pulmonary fibrosis induced by intratracheal instillation of bleomycin sulfate. Subsequently, the population of contractile cells, both muscle and nonmuscle, was analyzed in each strip by histochemical, immunocytochemical, and ultrastructural means. The force (g force/g tissue wet weight) generated by fibrotic strips was approximately double that of the control strips in response to acetylcholine, epinephrine, and potassium depolarizing solution. The content of smooth muscle in fibrotic and control strips was not significantly different. Immunofluorescent examination indicated increased contractile proteins (actin and myosin) in the thickened alveolar walls of the fibrotic strips in areas devoid of histologically demonstrable smooth muscle. Ultrastructural examination of the fibrotic interstitium revealed an increased population of filament-laden cells that appeared related to the contractile interstitial cell, or myofibroblast. The results indicate that parenchymal strips isolated from fibrotic rat lung can generate increased force in vitro. These responses may be related to increased nonmuscle contractile cells in the fibrotic interstitium.

A detailed study was carried out to measure the relative contents of V1 and V3 myosin isozymes in different regions of rabbit ventricle as a function of age, to assess animal-to-animal variability, and to compare different experimental approaches aimed at minimizing the effects of such variability. In addition, comparisons were made in normal developing hearts between ventricular isozyme composition and myofibrillar myosin calcium-stimulated adenosine triphosphatase. V1 isozyme predominated relative to V3 isozyme in the hearts of 2-week-old rabbits, decreasing to become a minor component in 10-week-old animals. Despite this trend, there was considerable variability in relative isozyme content of whole ventricular tissue among different rabbits of the same age. This variability was reduced in comparisons of littermates and by use of cardiac biopsies to measure changes in isozyme content in the same animal over time. Within different regions of a given heart, there also were small but significant differences in the percent V1 isozyme. The percent V1 was greatest for right ventricular papillary, followed by right ventricular free wall and then the left ventricle (free wall plus septum). There also were differences in the percent V1 within those regions, as exemplified by the significantly higher values for ventricular epicardium vs. endocardium. There was a linear correlation between the myofibrillar myosin calcium-stimulated adenosine triphosphatase and percent V1 of total isozyme for both right and left ventricles in normal and developing hearts. The regression lines for calcium-stimulated adenosine triphosphatase vs. percent V1 had a steeper slope in the left than in the right ventricle.

The insulin sensitivity of protein synthesis and glucose incorporation into glycogen by the soleus and epitrochlearis muscles from fed rats and 24 h-starved rats was determined in vitro during the first and second hours of incubation after isolation of the muscles. Rates of protein synthesis by both muscles from fed rats in the first hour of incubation were 2-fold higher than in the second hour and were not increased by insulin. Rates of protein synthesis during the first hour in the presence of 6000 microunits of insulin/ml were increased in soleus, but not in epitrochlearis, muscles from starved rats. Rates of protein synthesis in both muscles from fed and starved rats were increased significantly by insulin during the second hour. High concentrations of insulin caused a marked stimulation of the rates of glucose incorporation by both muscles from fed and starved rats in both the first and second hours of incubation. The insulin sensitivity of glucose incorporation during the second hour, defined as the concentration of insulin causing half-maximal stimulation, was increased 10-fold for both muscle types from starved rats (soleus, 65 microunits/ml; epitrochlearis, 45 microunits/ml) relative to muscles from fed rats (soleus, 600 microunits/ml; epitrochlearis, 500 microunits/m). The insulin sensitivity of protein synthesis in the second hour was greater for soleus muscles from starved rats (65 microunits/ml) than from fed rats (500 microunits/ml). In contrast, the insulin sensitivity of protein synthesis in epitrochlearis muscles from starved rats was significantly decreased (225 microunits/ml) compared with fed rats (25 microunits/ml Maximal rates achieved by high concentrations of insulin were not different from those in the same muscle from fed rats. It is suggested that protein synthesis, in distinction to glucose utilization, may be resistant to insulin stimulation during periods of acute starvation in muscles with fibre compositions similar to the epitrochlearis, but not in muscles with fibre compositions similar to the soleus. Partial reversal of the resistance observed in vitro for epitrochlearis muscles from starved rats may be due to the loss of factors which suppress the effect of insulin in vivo.

A detailed understanding of alveolar epithelial cell transitions during remodeling after lung injury requires the identification of specific markers. We have developed a panel of monoclonal antibodies against species of the intermediate filament protein, keratin. These individual species are recognized markers of the state of differentiation of various epithelial cells. These and complementary protein analytic methods have been applied to studies of isolated, enriched Type II pneumocyte preparations as well as to normal and injured lung tissues. Monoclonal antibody 24A3, initially raised against Morris hepatoma 7777 keratins, decorated a filament network in isolated cultured rat Type II pneumocytes by indirect immunofluorescence; it reacts by 2-dimensional polyacrylamide gel immunoblot procedures with an acidic, 46,000-dalton keratin. Monoclonal antikeratin antibodies AE1 and AE3, raised against human epidermal keratins, reacted poorly with isolated Type II cells; however, AE3 reacted by immunoblot technique with the 55,000-dalton keratin subclass. The bronchial epithelium reacted intensely with 24A3 as well as with a mix of AE1 plus AE3 in ethanol-fixed, paraffin-embedded sections of normal and injured rat lung. Alveolar regions of normal lung reacted poorly with all 3 antibodies, however, as visualized by light microscopy. At the same time, very large, presumptive epithelial cells in the alveolar regions stained intensely with 24A3 3 days after intratracheal instillation of bleomycin, whereas thin cells lining the alveoli in injured regions were intensely reactive 14 days after bleomycin treatment. These elongated cells may represent Type II pneumocytes in the process of converting to Type I cells.