Introduction

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Smooth muscle performs several functions in the developing and adult organism, including the synthesis of extracellular matrix and contraction (1, 2). Differentiating smooth muscle is characterized by the sequential appearance of cytoskeletal and contractile proteins that typify smooth-muscle cells in the adult (1). A high degree of plasticity is observed in smooth-muscle cells in the adult in diseases that affect both vascular and visceral smooth muscle as well as during normal remodeling (1, 2, 4). These changes reflect backward and forward transitions between states of relative dedifferentiation and differentiation and/ or expansion of resident smooth-muscle cell subpopulations.

Myosin, the major motor protein for all muscle tissues, is a hexamer composed of two heavy chains and two pairs of light chains (2, 7). Smooth-muscle myosin is a well-characterized marker of the smooth-muscle cell phenotype, which is exclusively expressed in smooth muscle (2, 8). The known mammalian smooth-muscle myosin heavy-chain (SMMHC) isoforms are the products of alternative splicing of a single gene (9). Alternative splicing in the region encoding the tail of the heavy chain produces the SM-1 and SM-2 isoforms, and splicing in the region encoding the 25/50-kD junction produces the SM-A and SM-B isoforms. The use of both splice sites enables the production of at least four heavy-chain isoforms that have been described to date: SM-1A, SM-1B, SM-2A, and SM-2B (11). The B-isoforms, which contain an additional seven amino acids located at the 25/50-kD junction, are known to possess unique functional properties (10, 13). Rovner and colleagues (13), for example, have shown that the seven-amino acid insert in SM-B is necessary and sufficient to double the isoform's Mg²⁺-adenosine triphosphatase (ATPase) activity as well as the rate at which it can support the movement of actin filaments. The proportions of SM-A to SM-B have been demonstrated to differ in tonic versus phasic smooth muscle (10), suggesting that differential expression of the A and B isoforms is of functional importance. Finally, recent studies of molluscan myosins have demonstrated that sequence variations at the 25/50-kD "loop" serve to modulate ATP turnover rate (14).

The changes that occur in smooth muscle cell replication and contractility in diseases that affect the vasculature and visceral tissues such as the pulmonary airways have been extensively characterized (1, 2, 6, 7). Much less is known about the regulation of smooth-muscle-specific genes and the changes in gene expression that occur in the contractile protein phenotype during development and airway remodeling (2, 8). This is particularly true for critical contractile proteins such as myosin.

We hypothesized that there would be a differential distribution of the SM-B isoform in the airways and vasculature, commensurate with the differential functional requirements of those tissues. Accordingly, we used antibodies to SM-B, SM-1, and SM-2, and an antibody that reacts with all SMMHC to determine the myosin heavy-chain composition of lung airway and vascular smooth muscle. We found a tissue distribution in adult lung that indicates the presence of SM-B in fast, phasic muscles such as the trachea and in the smooth muscle of the airways. SM-B is rarely present in smooth muscle of large pulmonary blood vessels of the adult but is present in smaller vessels, including those perfusing the trachea and the gas exchange regions of the distal lung. During lung development, SM-B first appears in smooth muscle of the large airways the first day after birth and remains restricted to the airways through the first 2 wk of life. Reactivity to large vessels is faint in the large pulmonary vessels and rare in the adult. The information provided by this study is essential toward understanding myosin heavy-chain distribution in adult and developing lung smooth muscles, and toward ultimately understanding the molecular basis for contractility in smooth muscle of the lung and how it is affected in pulmonary vascular and airway disease.

	Materials and Methods

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Animals

Guaranteed timed-pregnant Fischer 344 rats were obtained from the National Cancer Institute (Frederick, MD) at Day 14 of gestation and maintained in an Association for Assessment and Accreditation of Laboratory Animal Care-approved barrier facility with food and water ad libitum. Animals were studied at Days 15 through 21 of gestation and at Days 1, 3, 8, and 15 of postnatal life and adulthood (approximately 200 g). Multiple sections from three different litters from different mothers were examined to control for different litter sizes. Multiple sections from a minimum of four adult animals also were studied. No differences were observed between animals of the same age in terms of patterns of SM-B antibody reactivity.

Adult animals were killed by sodium pentobarbital overdose according to protocols approved by the University Institutional Animal Care and Use Committee.

Preparation of Tissues and Tissue Extracts

For studies of fetal animals, uteri were removed from anesthetized pregnant animals and dissected rapidly in phosphate-buffered saline (PBS) under a dissecting microscope. The entire thoracic region of the fetus was fixed en bath in 100% ethanol for immunohistochemistry. For lung tissues from neonates, the lungs of 1-, 3-, 7-, 15-, and 28-d-old animals were dissected free of other tissues, perfused with PBS via the heart, gently inflated with 100% ethanol via the trachea by means of a syringe, and then submersed in ethanol. Lungs from older animals were perfused in situ with PBS via the right ventricle, the trachea was cannulated, and the tissue was fixed with ethanol at a pressure head eqivalent to 20 cm water. The trachea was then tied off and the lungs were fixed by immersion.

For biochemical studies of tissue extracts, trachea and lungs were dissected and immediately frozen in liquid nitrogen. The frozen tissue was then pulverized with a mortar and pestle under liquid nitrogen before the tissue was homogenized using a Polytron (Brinkman Instruments, Westbury, NY) at a tissue/buffer ratio of 300 to 500 mg frozen tissue per milliliter in extraction buffer consisting of 0.1 M sodium pyrophosphate, 10% glycerol, 0.01 M disodium ethylenediaminetetraacetic acid, 0.005 M ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid, pH 8.8, to which 50 µg/ml (final concentration) leupeptin, 0.1 µM phenylmethylsulfonyl fluoride, 500 ng/ml pepstatin, and 5% -mercaptoethanol were added immediately before use. Tissues were then mixed with 2× or 4× sodium dodecyl sulfate electrophoresis sample buffer (15). Electrophoresis was in 3.8% acrylamide, 0.05% bis-acrylamide gels to separate SM-1 and SM-2 isoforms, using a Bio-Rad Protean II apparatus. Molecular-weight standards were purchased from Amersham (Arlington Heights, IL).

Antibodies

The following antibodies were used for immunohistochemistry, as described elsewhere (3, 16): anti-SM1 (1:200); anti-SM2 (1:100); anti-smooth-muscle -actin (1:200); and anti-desmin (1:100). The rabbit anti-SMMHC antibody that was generated against bovine aorta, and which recognizes all smooth-muscle heavy-chain isoforms, was kindly provided by Dr. Robert Adelstein (National Institutes of Health, Bethesda, MD) and used at 1:400 for immunohistochemistry and 1:8,000 for Western blotting (see below). The SM-B antibody was generated against the following peptide: glutamine-glycine-proline-serine-phenylalanine-alanine-tyrosine-glycine-glutamic acid-leucine-glutamic acid-cysteine. The peptide was coupled to keyhole limpet hemocyanin at the cysteine residue and injected into rabbits for antibody production (HTI Bioservices, Inc., Ramona, CA). The antibody was initially characterized by examining pre- and postimmune sera reactivity in Western blots (1:4,000) and tissue sections (1:400) (see RESULTS). To further demonstrate monospecificity against the SM-B isoform, we examined reactivity against baculovirus-expressed SM-B intermediate heavy meromyosin (IHMM) and IHMM lacking the SM-B insert (Figure 1; 10, 13). Construction of the SMMHC complementary DNAs with and without SM-B insert and their expression in baculovirus were as described (13).

View larger version (57K): [in this window] [in a new window]   Figure 1.   The SM-B antibody specifically recognizes the SM-B isoform. (A) Western blot of baculovirus-expressed SM-B intermediate length heavy chain HMM (IHMM; 28) probed by ECL with SM-B antibody (left) and then reprobed with anti-SMMHC antibody (right). SM-B antibody reacts only with IHMM containing the seven-amino acid difference peptide (+ lanes, left) whereas anti-aorta antibody reacts with IHMM with and without the insert (+ and  lanes, right). (B) ECL of tissue extracts probed with SM-B antibody. From left: bladder (200 µg); molecular-weight standard which is biotinylated and therefore reactive with streptavidin-peroxidase; trachea (200 µg). SM-B antibody reacted with both SM-1 (upper band) and SM-2 (lower band) isoforms in both bladder and trachea. (C) Coomassie-stained polyacrylamide gel showing the protein distribution of SM-1 and SM-2 isoforms in rat tracheal muscle. Molecular-weight markers were myosin (220 kD), phosphorylase B (97 kD), and bovine serum albumin (66 kD).

Immunoperoxidase Staining and Western Blotting

Staining of tissue sections was performed as described previously (3). Acrylamide gels were blotted to nitrocellulose and antibody detection was performed as previously described (3), with the exception that enhanced chemiluminescence (ECL) was used for antibody detection (Amersham). The same results were obtained when blots were stained with diaminobenzidene tetrahydrochloride (3; data not shown).

	Results

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Antibody Specificity

The SM-B antibody was generated in rabbit against the seven-amino acid peptide present in the SM-B isoform. This antibody was found to identify specifically baculovirus-expressed heavy meromyosin containing the seven amino acids, and does not cross-react with the SM-A isoform, which lacks this seven-amino acid region (Figure 1A). Using this antibody in Western blots of tracheal smooth-muscle extracts, the SM-B isoform was identified in association with both SM-1 and SM-2 tail isoforms (Figure 1B).

Adult Lung

Figures 2-4 show the overall pattern of antibody staining seen in adult lung tissues. The SM-B isoform is expressed in airway smooth muscle and in small blood vessels (Figures 2B, 3A, and 4B). Large vessels containing elastic laminae typically do not express the SM-B isoform (Figures 2B and 3A); however, both the SM-1 and SM-2 isoforms are expressed (Figures 2D and 2E, respectively). The general smooth-muscle myosin antibody (anti-SMMHC) reacts with all airway smooth muscle and all vascular smooth muscle (Figures 2C, 3B, and 4C).

View larger version (143K): [in this window] [in a new window]   Figure 2.   The SM-B isoform is expressed in airway smooth muscle and small blood vessels. (A) Preimmune serum from the same animal that produced the SM-B antibody. (B and F ) SM-B antibody. (C) Anti-SMMHC antibody. (D) SM-1 antibody. (E) SM-2 antibody. There is no reactivity with preimmune serum. SM-B antibody reacts with smooth muscle of airway (a) and small but not large blood vessels (v). Original magnifications: A-E, ×100, bar = 5 µm; F , ×400, bar = 1 µm.

View larger version (109K): [in this window] [in a new window]   Figure 3.   SM-B expression in lung vascular smooth muscle is heterogeneous. (A) SM-B antibody reacts with airway (a), but its expression is variable in blood vessels (v). Large arrow indicates a vessel with little SM-B antibody reactivity. Small arrow indicates a vessel with a heterogeneous pattern of reactivity. A serial section shows that all smooth muscle stains with anti-SMMHC (B). All original magnifications, ×100, bar = 5 µm.

View larger version (143K): [in this window] [in a new window]   Figure 4.   The SM-B isoform is expressed in septal-tip cells and peripheral blood vessels of the adult peripheral lung. (A) Preimmune serum shows no reactivity. SM-B antibody reacts with septal-tip cells (s) and small blood vessels (v) of peripheral lung (B, E, G, and H). Anti-SMMHC antibody reacts with all smooth-muscle tissues (C and F ), as does the antibody against SM-1 (D). Original magnifications: A-D, ×100, bar = 5 µm; E-H, ×400, bar = 1 µm.

The expression of the SM-B isoform, with regard to blood vessels in the lung, is heterogeneous (Figures 3A, 4B, 4E, and 4G through 4H). Whereas large, muscular vessels in the lung infrequently contain detectable amounts of this isoform, its expression in smaller blood vessels (0.1 to 200 µm) is variable. Note, for example, the almost complete absence of SM-B reactivity in the vessel directly above the airway (Figure 3B, large arrow), whereas the somewhat smaller vessel below the airway and another vessel to the left of the airway (small arrow) clearly show a heterogeneous pattern of reactivity with the SM-B antibody.

SM-B isoform expression extends to the septal tips of the peripheral lung and includes blood vessels adjacent to the tips (Figure 4B). Under higher magnification, expression of SM-B in these structures is seen to be heterogeneous, with some unreactive cells (Figures 4E, 4G, and 4H). Reactivity with the anti-SM-1 and anti-SMMHC antibodies is also observed in the septal tips and adjacent vessels (Figures 4C, 4D, and 4H). Expression of the SM-2 isoform was not observed in the septal tips or the peripheral vasculature (not shown; 3).

The smooth muscle of the trachea, as well as many vessels investing the connective tissue adjacent to the trachealis, expresses the SM-B isoform (Figure 5A). Under closer examination, this expression is also shown to be somewhat heterogeneous in terms of levels of SM-B antibody reactivity (Figure 5B, arrows). Both SM-1 and SM-2 isoforms are also expressed in the trachea (Figures 5C and 5D), as are other smooth-muscle markers such as -smooth muscle actin and desmin (Figures 5E and 5F).

View larger version (145K): [in this window] [in a new window]   Figure 5.   Rat tracheal smooth muscle contains the SM-B isoform. Shown is antibody reactivity against SM-B (A and B); SM-1 (C); SM-2 (D); smooth muscle -actin (E); and desmin (F ). Large arrow in B identifies a highly reactive cell; small arrow indicates a cell with much less reactivity. Original magnification for A and C-F, ×100, bar = 5 µm; and for B, ×400, bar = 1 µm.

Developing Lung

We have previously demonstrated that SM-1 and SM-2 myosin heavy-chain isoforms are detectable at relatively late stages of lung development (3). The SM-B isoform also appears rather late in development, at around the time of birth (Figure 6). Reactivity to the SM-B antibody is not observed at fetal Days 17 or 20 (Figures 6B and 6D) or at intervening developmental time points (not shown). Very faint reactivity is observed in smooth muscle of the largest airways and in larger blood vessels at Day 21 of fetal development, but this varies by individual animal (not shown). Reactivity is clearly present, however, in the largest airways of all 1-d-postnatal rats (Figures 6E and 6F). The expression of SM-B in the airway smooth muscle is heterogeneous, with expression restricted to the larger airways through the first 2 wk of neonatal development, thereafter spreading distally. Expression of the SM-B isoform in large, elastic vessels also is apparent on Day 1 of postnatal development (Figures 6E and 6F), although its expression is heterogeneous in that not all vessels are positive. Again, reactivity spreads distally as development proceeds to give the adult pattern. The adult pattern of SM-B reactivity is observed by Day 21 (not shown).

View larger version (120K): [in this window] [in a new window]   Figure 6.   Expression of the SM-B isoform during rat lung development. Preimmune serum does not react with any tissue elements at fetal Day 17 (A) or fetal Day 20 (C). SM-B antibody also does not react at these ages (B and D, respectively). SM-B antibody reactivity against airway (a) smooth muscle, and very faint reactivity in large vessels, at postnatal Day 1 (E and F ). Reactivity with SM-B antibody at Days 3 (G) and 15 (H). The dark borders around certain tissue structures are artifacts of tissue processing, as also indicated by their black appearance on the initial slide, rather than the characteristic brown, intracellular material indicating positive antibody reactivity. Original magnifications, ×100, bar = 5 µm.

	Discussion

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We show here for the first time the differential distribution of a myosin isoform, SM-B, that has been shown in in vitro ATPase and motility assays to differ in contractile properties as compared with the SM-A isoform (10, 13). The differential expression of these isoforms may provide an additional potential explanation of the molecular basis for differential contractile properties of pulmonary vascular and airway smooth muscle and perhaps a means of modulating smooth-muscle contractile properties in response to physiologic stresses (4, 16, 17).

Four SMMHC isoforms (SM-1A, SM-1B, SM-2A, and SM-2B), produced by alternative splicing from a single gene, have now been identified (11). Studies of lung vascular and airway smooth muscle have demonstrated changes in the proportions of SM-1 and SM-2 tail isoforms during development and remodeling (3, 18), but correlations with muscle performance have been difficult to demonstrate (7, 18). This may be the result of the inability at the time to distinguish between the A and B head isoforms. The subsequent observations that the SM-B and SM-A isoforms have different functional properties and tissue distributions (10, 11, 13, 17), may provide an important link between smooth-muscle isoform content and contractile performance.

Our results clearly demonstrate the differential expression of the SM-B isoform in lung. The presence of this isoform in the adult lung in airways, septal tips, and small blood vessels suggests that the smooth muscle in these structures has faster, phasic contractile properties that may be essential for airway responsiveness and for regulation of pulmonary blood pressure and alveolocapillary perfusion. The heterogeneity of SM-B isoform expression observed in the large versus small blood vessels of the adult lung further implies that SM-B versus SM-A content is of physiologic significance.

The expression of the SM-B isoform in blood vessels is not merely a function of size, because some small vessels do not express it whereas similarly sized vessels in the same section clearly do. Rather, its expression seems to correlate with a type of vessel: It is almost always observed in thin-walled vessels and is typically not observed in multilayered, muscular vessels containing elastic laminae in the adult. The heterogeneity of expression observed in individual vessels will require further detailed examination (5).

SM-B reactivity is often heterogeneous within a given vessel or airway element. This is in keeping with the finding of others regarding the phenotypic heterogeneity of vascular smooth-muscle cells (1, 2), including in lung (5, 22), and extends our knowledge with regard to similar levels of heterogeneity in airway smooth muscle. It will be important in these regards to determine how the presence of SM-B relates to that of other markers of heterogeneity such as meta-vinculin (5). The heterogeneity of smooth-muscle phenotype, especially regarding such key contractile proteins as myosin, would provide a molecular basis for functional heterogeneity as well as for maintaining tissue function during remodeling (5).

Our results also clearly indicate that SM-B can occur in both the SM-1 and SM-2 isoform subfamilies. The proportion of SM-B in these two subfamilies must vary in lung because there are cases in which SM-B does not appear to be present in some tissues where both SM-1 and SM-2 are present (e.g., large blood vessels; Figure 2) but is clearly associated with both SM-1 and SM-2 in others (e.g., trachea; Figures 1B and 6). We have found the same to be true in development of vascular and other visceral muscles of the rat (R.B.L. and S.L.W., unpublished results).

RNA analyses have demonstrated the presence of both SM-B and SM-A transcripts in fetal lung at the time we first see protein expressed (23). The expression of SM-B in large blood vessels of the lung in neonatal rats, while absent in adult large vessels, suggests that these vessels in the neonatal lung may have different contractile properties from that of the adult. This pattern of expression is also observed in the aorta, where neonatal aorta expresses SM-B messenger RNA that diminishes during development until it is absent in adult aorta (23).

The SM-B isoform first appears in the developing lung in large airways and blood vessels. Expression in the airways later expands to include all airways and septal-tip cells. The pattern of expression of SM-B in the lung vasculature changes such that SM-B expression diminishes during development in the large vessels and increases in smaller vessels, until finally only the smaller blood vessels express SM-B. This shift in isoform expression is intriguing, and suggests that the phenotype of neonatal smooth-muscle cells in the large vessels of the lung is different from that of adult. Preliminary studies of the effects of remodeling in the lung on SM-B isoform distribution in both airways and blood vessels, for example, during the evolution of pulmonary hypertension, suggest changes that may be important in helping to define the role of isoform switching with respect to contractile performance (24, 25). In this regard it is tempting to speculate that changes in SM-B may prove to be partly responsible for the alterations in airway contractile function that accompany asthma (4, 16).

Despite the results of in vitro studies (10, 13) the relationship between SM-B expression and muscle performance remains to be determined. In support of such a role, Sjuve and associates (26) have described a positive correlation between maximal shortening velocity and SM-B content in studies of hypertrophied versus normal rat bladder. At the same time, Haase and Morano (27) have reported 8-fold higher amounts of SM-B in rat bladder than in quiescent rat myometrium, even though shortening velocities are approximately equal. In a related study, Calovini and coworkers (28) reported SM-B is decreased in pregnant rat uterus and shortening velocity is increased (28, 29). Finally, the studies of Siegman and colleagues (30) also suggest there may be no relationship between SM-B and velocity of shortening. The differences in contractile behavior observed by Rovner and coworkers (13) are also insufficient to explain the 7-fold differences in speed of contraction that have been observed for different smooth-muscle tissues (7, 13). Thus, differences involving additional isoforms of myosin and/or other contractile proteins (such as myosin light chains [4, 7, 31, 32]) as well as other factors (such as internal load [33]) are also likely at play. In this regard, it would appear likely that there may be subgroups of contractile protein isoforms that characterize smooth muscles that are functionally different from one another (7).

Our present results illustrate how demonstrating a correlation between SM-B content and muscle tissue contractile performance may be difficult. Clearly, the distribution of SM-B within a vessel class, and perhaps even the viscera, can be heterogeneous. Different input pathways to smooth-muscle cells of differing SM-B content in terms of signals causing contraction might be an important additional means of altering tissue contractile properties in vivo. Which of the signal pathways is active in vivo or during experimental manipulation thus might affect parameters such as shortening velocity. One way to approach this problem would be to develop transgenic animals in which the desired SMMHC isoform is selectively expressed as, for example, has been done in studies of cardiac muscle (34).

In conclusion, we report the differential distribution in rat lung of the SM-B isoform of the SMMHC. It will be important in the future to determine the degree to which the distribution of this isoform in different smooth muscles of the lung (and other organs) reflects their differential contractile properties. Additionally, changes in the expression of SM-B may help to explain some of the alterations that occur in contractile properties in airway diseases such as asthma, as well as the changes in vascular smooth-muscle performance that accompany such disorders as pulmonary hypertension.