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Correspondence to: Thomas J. Eddinger, Marquette University Dept. of Biology, Wehr Sciences Building, Room 109, PO Box 1881, Milwaukee, WI 53201-1881. E-mail: thomas.eddinger@marquette.edu
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Summary |
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To evaluate the distribution of smooth muscle myosin heavy chain isoforms (SMB, with head insert), we examined frozen sections from the various regions of swine stomachs using isoform-specific antibodies. We previously reported variable SMB myosin heavy chain (MHC) expression in stomach cells that correlates with unloaded shortening velocities. This is consistent with the generalization of tonic fundic muscle having low expression and phasic antral muscle having high expression of the SMB MHC isoform. Using immunohistochemistry (IHC), we show a progression of the SMB MHC from very low immunoreactivity in the fundus to very intense immunoreactivity in the antrum. In the body, the average level of SMB MHC immunoreactivity lies between that of the antrum and fundus. Intercellular heterogeneity was observed in all stomach regions to a similar extent. However, the intercellular range in SMB MHC immunoreactivity decreases from fundus to antrum. All stomach regions show isolated pockets or clusters of cells with similar SMB MHC immunoreactivity. There is a non-uniform intracellular immunoreactivity in SMB MHC, with many cells showing greater-intensity staining of SMB MHC in their cell peripheries. This information may prove useful in helping to elucidate possible unique physiological roles of SMB MHC. (J Histochem Cytochem 50:385393, 2002)
Key Words: smooth muscle, myosin isoform, SMB, heterogeneity, immunohistochemistry, tissue, swine
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Introduction |
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MYOSIN, a hexamer composed of two myosin heavy chains (MHCs) and two sets of myosin light chains (MLCs), has several isoforms resulting from differences in each subunit. Alternate splicing of a single MHC gene results in four different MHC isoforms. Alternate splicing of the 3' end of the MHC gene codes for SM1 and SM2, which differ by 43 amino acids including a 34-amino-acid size difference in the COOH terminal of the myosin molecule (
Isoform content may be particularly important to smooth muscle physiology because isoform composition is the principle determinant of contractile characteristics in other contractile cells (
The stomach is traditionally divided into three regions: the fundus, which comes directly off the esophagus; the body, which lies between the fundus and antrum; and the antrum, which encompasses the distal third of the stomach and feeds into the duodenum (Fig 1A). Histologically, all three stomach regions are composed of two separate discernible muscle layers, the muscularis mucosa and the more massive muscularis externa (Fig 1C) (
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This study was designed to define the histological distribution of SMB MHCs in the stomach regions previously examined by our lab. By examining single cells, it was technically impossible to determine a tissue's histological distribution of SMB MHC. Results from this study confirm the trend in SMB MHC immunoreactivity from low to high as one traverses along the greater curvature of the stomach from fundus to antrum. In addition, we report intercellular and intracellular heterogeneity in SMB MHC immunoreactivity. Cells with high SMB MHC immunoreactivity are equally likely to be next to a cell of similar SMB MHC immunoreactivity, or not, throughout all regions examined. Finally, we report intracellular heterogeneity as a tendency for SMB MHCs to be localized to the perimeter of a cell and otherwise randomly distributed within the cell.
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Materials and Methods |
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Tissues were isolated from pigs killed in a local slaughterhouse. The stomachs were removed, cleaned of blood, adipose, and loose connective tissue, and dissected into fundus (region 2), body (regions 45), and antrum (region 8) (Fig 1A). These portions were frozen in liquid nitrogen-cooled 2-methylbutane, and stored at -20C until serially sectioned (approximately 4 µm).
The polyclonal SMB MHC antibody (gift of Dr. A.S. Rovner; University of Vermont) was generated in rabbit against the following peptide: glutamine-glycine-proline-serine-phenylalanine-alanine-tyrosine-glycine-glutamic acid-leucine-glutamic acid-cysteine. The working dilution for the SMB MHC antibody was 1:500 in 0.1% BSA. The specificity of this antibody has been characterized extensively (
Sections from all tissue regions were labeled with the SMB MHC antibody following the methods of
PBSTween [(in g/liter) NaCl 8.0, KH2PO4 0.2, Na2HPO4 1.15, KCl 0.2; 0.1% Tween-20, pH 7.4] was used to dilute all solutions and to rinse the tissues between all steps except after BSA blocking, where no rinsing was performed. After the staining procedure, sections were mounted in media containing several flakes of p-phenylenediamine (anti-fading compound), 75% glycerol [(in mM) KCl 75.0, Tris base 10.0, EGTA 2.0, MgCl2 1.0, pH 8.5], covered with a coverglass, and sealed with clear nailpolish before being stored at 4C until being examined. Negative controls were done for each experiment by replacing primary antibodies with PBSTween or by using pre-immune serum (Fig 1B and Fig 2).
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During each experiment, sections from all three stomach regions were stained at the same time and under the same conditions. Similarly, to preserve the integrity of the experiment, strict standards were employed for all tissue visualization using an Olympus IX70 microscope with fluorescent capabilities. Micrograph exposure times were held constant between images in a set of slides to standardize fluorescent intensities between micrographs. Neutral density filters were used as needed to limit brightness during DAPI visualization. By strictly adhering to these criteria, the only significant variable affecting micrograph light intensity was the actual labeling of SMB MHCs. Cell images were recorded using a Princeton Instruments (Princeton, NJ) digital camera linked to a PC loaded with IP Lab v 3.0 for Windows (Scanalytics; Fairfax, VA).
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Results |
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Negative controls did not produce artifacts that could be mistaken for the varying trends in SMB MHC distribution; only slight background staining was observed in some stomach preparations (Fig 1B and Fig 2). Negative controls using no primary antibody (Fig 1B and Fig 2A) or pre-immune serum from the rabbit that produced the antibody (Fig 2B) showed little to no immunoreactivity compared to the fluorescent signal observed using the SMB MHC serum (Fig 2C). Some immunoreactivity was observable in the stomach mucosa: However, this staining was of a much smaller magnitude than in muscle cells in the muscularis mucosa and externa (data not shown). It is currently unclear if this is nonspecific binding, SMCs from the capillary and lymphatic systems, or if other cell types present can express SMB MHC.
Major differences in SMB MHC immunoreactivity are observable between the stomach regions. Although no absolute quantitation was attempted, stomach antrum showed greater reactivity of SMB MHC than stomach fundus or body (Fig 3, antrum vs body and fundus). SMB MHC immunoreactivity in the body appeared to range between fundic and antral extremes (Fig 3, body).
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An intercellular mosaic pattern of SMB MHC immunoreactivity was observed (Fig 4A1 and 4A2). No significant homogeneity was observable even between adjacent smooth muscle cells, and cells showing high levels of SMB MHC immunoreactivity were equally likely to neighbor a cell of similar immunoreactivity, or not (Fig 3F3L). This range in heterogeneity is most clearly observable in transverse sections of smooth muscle, where the limits of each cell are most discernible, and brightly illuminated cells appear randomly distributed throughout the tissue (Fig 3H3L and Fig 4A1) but is also observable in longitudinal sections (Fig 4A2 and 4B). This range in heterogeneity, although present in all regions of the stomach, stands out most dramatically in the body and fundus. The range in heterogeneity of SMB MHC in the antrum appeared to be narrower because all cells in this region show relatively high levels of SMB MHC immunoreactivity. Although the distribution of SMB MHC-dense cells appeared to be random, small pockets of cells with similarly high or low levels of SMB MHC immunoreactivity were observed.
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Intracellular heterogeneity, similar to intercellular heterogeneity, is present in cells from all tissue regions. A mosaic or "checkerboard" immunoreactivity of SMB MHC is discernible in transverse and longitudinal tissue sections. Although most intracellular heterogeneity appears to be randomly distributed, as reported for intercellular distributions, a tendency for high SMB MHC immunoreactivity to be localized in the periphery of muscle cells was frequently observed (Fig 4C).
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Discussion |
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In this study we focused on the SMB MHC isoform to extend our previous studies on its distribution in the stomach. SMB MHC has been identified as a myosin isoform with unique contractile properties. At the protein level, SMB MHC is necessary and sufficient for myosin to propel actin two to three times more rapidly in in vitro motility assays and to cause a twofold increase in ATP hydrolysis over the SMA MHC isoforms (
Although these correlations have been made at the protein, cell, and tissue levels, the magnitude of changes due to SMB MHC is not consistent among studies.
It has been suggested that differences in contractile force and shortening velocity may result, at least in part, from variants in isoforms from other parts of the myosin molecule, such as LC17a/b or SM1/2. Correlations between LC17a and an increased rate of contraction have been reported in tissue (
Although this study is the first to show the histological distribution of SMB MHC in the various regions of the stomach, heterogeneity has been previously suggested by other studies.
This study shows that SMB MHC immunoreactivity increases as one moves along the greater curvature of the stomach from fundus to antrum, consistent with
Small pockets or clusters of cells with high levels of SMB immunoreactivity were observed in longitudinal sections (Fig 4B) and in transverse sections (Fig 3G3L) in all three regions of the stomach. A possible physiological role for this heterogeneity might be to increase the rate of SM cell activation. SM cells expressing greater amounts of the SMB MHC isoform can shorten more rapidly than cells with low levels of this isoform (
It is possible that this electromechanical model could present a way for a contractile stimulus to spread more rapidly than by relying on chemical diffusion or enteric input alone. With this in mind, a limited number of cells expressing high levels of SMB MHC could recruit many other cells with which they associate. A greater demand for such stimulation could be thought to exist in thicker, more muscular tissues, such as the antrum, in which diffusion would be a less desirable way to transmit contractile stimulus. Further research in this area is needed to test this hypothesis.
The observation of heterogeneous distribution of SMB MHCs in the stomach may require reinterpretation of previous data reporting the ratio of SMA/SMB MHCs in the stomach as one value rather than a series of values from tonic and phasic regions of this organ. Traditionally, the stomach has been considered a homogeneous tissue, and this oversimplified assumption has been shown not to be the case. Future research can take SMB MHC heterogeneity in the stomach into account by recognizing the organ's full isoform complexity.
Heterogeneity in protein isoform expression is known to differ in certain diseased states, such as arteriolosclerosis or hypertension in vascular SM (
This study has mapped out the histological distribution of SMB MHCs in three regions of the swine stomach. SMB MHC shows the least immunoreactivity in tonic fundic cells and increases as one moves along the greater curvature of the stomach to the body and antrum. Random intercellular heterogeneity with areas of cells expressing similar amounts of SMB MHCs was observed in all three stomach regions. A greater range in heterogeneity was observed in the fundus, while a smaller range in cells expressing high levels of SMB MHC was observed in the antrum. Intracellular heterogeneity was also observed, with a tendency for SMB MHC to be located around the periphery of a cell. This study is important in providing basic information regarding the distribution of the SMB MHC isoform, and allows hypotheses to be made and tested regarding the physiological significance of heterogeneous expression of SMB MHC.
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Acknowledgments |
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Supported in part by National Institutes of Health Grants HL-62237 and AR-45294.
We thank A. S. Rovner for the gift of the SMB antibody, Hansen Meat Service for providing the tissue, D. A. Riley for advice on immunohistochemistry, and Elizabeth Beaulieu for generating Fig 2.
Received for publication April 30, 2001; accepted October 10, 2001.
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