Expression of myosin isoforms in smooth muscle cells in the corpus cavernosum penis

Michael E. DiSanto1, Ze Wang2, Chandrakala Menon1, Yongmu Zheng1, Thomas Chacko1, Joseph Hypolite1, Gregory Broderick1, Alan J. Wein1, and Samuel Chacko1,2

1 Division of Urology and 2 Department of Pathobiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses ~4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17 isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (~10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches ~23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.

erectile function; light chain phosphorylation; quantitative reverse transcription polymerase chain reaction

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

IN BOTH HUMANS AND ANIMALS, erection (tumescence) and detumescence are regulated by contraction and relaxation of smooth muscle located in the trabeculae of the corpus cavernosum. These processes are regulated by the flow of blood into and out of the cavernous bodies (2, 6). The male penis remains in the flaccid state the majority of the time. Thus the corpus cavernosum smooth muscle (CCSM), unlike most other smooth muscles, spends the majority of its time in the contracted state and must relax rapidly in order for an erection to occur. Although the signaling pathways regulating the CCSM have been investigated in some detail (e.g., membrane channels, cyclic nucleotides, and nitric oxide), how alterations in these pathways effect relaxation and contraction of the smooth muscle cells have not been thoroughly investigated. To understand which changes may be involved in erectile dysfunction, one must first understand the normal composition and function of the proteins that form the contractile apparatus that is necessary for force generation and maintenance in the corporal smooth muscle.

The myosin molecule contains two long polypeptide chains of ~200 kDa, called heavy chains. The COOH-terminal region forms the alpha -helical rod portion of the myosin molecule, which is proposed to be involved in filament assembly (15), whereas the NH2-terminal region of both heavy chains are folded into globular structures to form two enzymatically active heads. In addition, each head also has two smaller polypeptide light chains at the base of the heads known as LC20 (20 kDa, phosphorylatable) and LC17 (17 kDa, essential). The major mechanism for regulation of contraction in smooth muscle is phosphorylation of the LC20 (1); in addition, the type of smooth muscle myosin isoform may contribute to the contractility.

Both the myosin heavy chain (MHC) and myosin light chain (MLC) LC17 have been demonstrated to exist as different isoforms. Splicing at the 3' end of the MHC pre-mRNA produces two isoforms, SM1 (204 kDa) and SM2 (200 kDa), which differ only at the COOH terminus (4). Alternative splicing also occurs at the 5' end of the MHC pre-mRNA transcript, which results in a 21-nt insertion that encodes a seven-amino acid sequence in the NH2-terminal head region of the myosin near the ATP binding site (17), which is shown as the nucleotide pocket in the crystalline structure of myosin subfragment-1 (24). The mRNA possessing this 21-nt insertion is known as SMB, whereas mRNA lacking this insertion is known as SMA. In addition, alternative splicing in the 3' end of the LC17 pre-mRNA generates two light chains known as LC17a and LC17b (27), consisting of the same number of amino acid residues but differing in five of the last nine amino acids and thus separable into two bands, an acidic (LC17a) and a basic (LC17b) form, on isoelectric focusing (IEF) (11).

Existence of smooth muscle myosin isoforms that are produced by alternative splicing of a single gene and variations in the relative proportion of these isoforms have been demonstrated in smooth muscle tissues from various sources (26, 32, 33). These isoforms have been shown to affect a variety of functions including in vitro motility, shortening velocity, and actin-activated Mg2+-ATPase activity (8, 17, 25).

We report that CCSM possesses an overall myosin isoform composition intermediate between aorta (smooth muscle that is considered tonic) and bladder smooth muscle (phasic). We also show that the basal level of myosin phosphorylation in the CCSM is relatively low (~10%), despite the high resting tone, and reaches only ~23% in response to maximal stimulation. The presence of myosin isoforms, with or without the seven-amino acid insert, which, respectively, have high and low actin-activated ATPase activities (8, 17) in the CCSM is likely to affect the kinetics of cross-bridge cycling. ATPase activity of the myosin, in combination with other regulatory systems such as thin-filament regulation mediated by caldesmon or calponin, may account for the high resting tone in the flaccid state and the ability of the CCSM to relax rapidly during penile erection

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Tissue preparation. Corporal tissue was obtained from normal male adult New Zealand White rabbits as previously described (23). The cleaned tissue was immediately frozen and stored in liquid nitrogen until needed. Tissue stored >3 wk was not used for this study.

RNA extraction and RT-PCR. RNA was isolated and reverse transcribed, and PCR was carried out as previously described (38). An upstream primer and a downstream primer that could anneal with the 3'-untranslated region of rabbit smooth muscle alpha -actin cDNA, as described previously, were also included in the PCR reaction as an internal standard (10). Aliquots (20 µl) of the PCR products were electrophoresed on a 2% agarose gel and visualized after ethidium bromide staining. Band intensities were quantitated by reflectance scanning of the corresponding photograph of the gel using a Bio-Rad (Hercules, CA) GS-700 imaging densitometer and subsequent analyses using the Bio-Rad Molecular Analyst program. The agarose gels were all photographed under the same conditions. Band intensities were always in the linear range of the light emission by the ethidium bromide based on previously constructed standard plots.

Sequence determination. PCR products were electrophoresed on a 2% Tris-acetate agarose gel, and the bands of interest were excised and eluted from the gel. The PCR products of the MHC isoforms were directly sequenced according to standard methods (31). The corresponding DNA products of the light chain were subcloned into the PCR 2.1 vector (Invitrogen; San Diego, CA) and then were sequenced in both directions using T7 and M13 sequencing primers. The sequences obtained were aligned with previously published sequences using the Lasergene software program (Madison, WI) to confirm their identity.

Quantitative competitive RT-PCR. To generate a competitive internal standard for quantitative determination of rabbit smooth muscle MHC transcript, a 258-nt cDNA fragment of the smooth muscle MHC was isolated using an RT-PCR upstream primer, 5'-TCAGCAACGAGCTGGCCA-3', and downstream primer, 5'-GCATCTCCTTCAGCTTCT-3'. The RT-PCR product contains the DNA sequence encoding the region between residues 5402 and 5659 for rabbit smooth muscle MHC SM1 and SM2 or SMA and SMB (3), as demonstrated by DNA sequencing. The RT-PCR product was subcloned into the pBluescript SK vector (Stratagene; La Jolla, CA), and a BamH I site in the middle of the DNA fragment was created using a site-directed mutagenesis system (37). A 90-nt DNA fragment covering part of the intron 7 of mouse caldesmon genomic DNA was also isolated using a PCR cloning approach and was confirmed by DNA sequencing (data not shown). Next, both ends of this caldesmon intron fragment were linked to a BamH I site and then inserted into the BamH I site of the RT-PCR product (see Fig. 3A). Competitor cRNA was produced using the linearized recombinant pBluescript plasmid and an in vitro transcription technique (38). The cRNA contained the target sequence present in SM1 and SM2 transcripts or SMA and SMB transcripts but differed from it in the presence of the 90-nt caldesmon intron fragment (see Fig. 3A). Total RNA was extracted from rabbit bladder, aorta, or corpora as described above. For each sample, seven to nine aliquots of rabbit total RNA (0.1 µg) were co-reverse transcribed with various concentrations (determined by ultraviolet spectrophotometry) of the competitor cRNA (29). The reverse transcription reactions were terminated by incubating the samples at 90°C for 5 min. The cDNAs were immediately subjected to competitive PCR, in which the smooth muscle MHC DNA fragments were coamplified with the competitive template (see Fig. 3B). The PCR conditions were 20 µl RT product, 1× PCR buffer (500 mM KCl, 100 mM Tris · HCl, pH 8.3, 15 mM MgCl2, and 0.01% gelatin), 1.25 mM dNTP, 0.1 µM each of upstream (5'-TGGCCACAGAGCGCAG-3') and downstream (5'-TCAGCTTCTTGTCCCTCT-3') primers, and 2.5 units of Taq DNA polymerase (Perkin-Elmer, Norwalk, CT). Cycle conditions were 50 s at 94°C, 30 s at 60°C, and 60 s at 72°C, with a 7-min final extension at 72°C after 25 cycles. Aliquots (20 µl) of PCR product were electrophoresed on a 2% agarose gel, and band intensities were quantitated as described above. The exact number of smooth muscle MHC copies was determined by graphical analysis (see Fig. 3C).

Antibody production. A polyclonal antibody to the seven-amino acid peptide of the same sequence as the NH2-terminal 25,000/50,000 seven-amino acid (QGPSLAY) insert of rabbit smooth muscle MHC (SMB) was produced in mice as follows. An extra cysteine residue was added to the NH2 terminus of the seven-amino acid synthetic peptide to enable cross-linking of this peptide with a carrier protein (keyhole limpet hemocyanin; KLH). Cross-linking was accomplished using an Imject maleimide-activated KLH kit (Pierce, Rockford, IL), and conjugated peptide was separated from unconjugated peptide by gel filtration. The fractions of the conjugated peak exhibiting the highest absorbance at 280 nm were pooled and used for immunization of BALB/c mice. IgG was purified from the mouse serum using Ultralink Affinity Pak immobilized protein A columns (Pierce). This IgG fraction was used for the Western blotting and immunofluorescence studies described below.

Immunohistochemistry. Corporal tissue was removed as described above, covered with OCT compound (Sakura Finetek, Torrance, CA), and frozen in liquid nitrogen. All subsequent solutions were prepared in PBS. Frozen tissue sections (6 µm) made using a cryostat microtome (Reichert; Scientific Instruments, Buffalo, NY) were incubated in 3.7% formaldehyde-0.2% Triton X-100 and then were rinsed three times with PBS. BSA (1%) was used to block nonspecific binding, and then the sections were incubated overnight with anti-smooth muscle myosin antibody (Sigma, St. Louis, MO) at a dilution of 1:500 or with antibody to the seven-amino acid insert that we produced as described in this paper at a dilution of 1:200. Next, the sections were rinsed several times in PBS and then treated with 5 µg/ml of anti-mouse IgG-Texas red containing 1% BSA for 2-3 h, rinsed several times with PBS, and mounted with a drop of mounting medium (Aqua-Mount; Lerner Labs, Pittsburgh, PA). Sections were viewed under a fluorescence microscope (Nikon FX-A) equipped for phase-contrast and epifluorescence illumination at a magnification of ×31.2.

Hematoxylin and eosin staining of tissue sections. Briefly, the sections were placed in deionized water for 5 min and then in acetone for 5 min. After the sections were rinsed with deionized water, they were then treated with about five drops of Mayer's hematoxylin (Bio Genex, San Ramon, CA) for 2 min, rinsed under tap water for 2 min, and treated with five drops of 0.05% wt/vol aqueous eosin B (Fisher, Philadelphia, PA) for 30 s. After staining was completed, the sections were washed with deionized water for several minutes, mounted with Aqua-Mount, and viewed under a light microscope.

Gel electrophoresis and quantitation of proteins. Corporal tissue was ground in a mortar cooled in liquid nitrogen, and a fine powder was made without allowing the tissue to thaw. The powder was then mixed with buffer (0.05 M Tris, pH 6.8, 20% glycerol) at a ratio of 100 mg powder/ml buffer and then was homogenized on ice with an Ultra-Turrax T8 mini-homogenizer (IKA Works, Wilmington, NC) at top speed. The mixture was then brought to 1% SDS, 25 mM dithiothreitol, and 0.003% bromphenol blue and boiled for 5 min. The extracted muscle proteins present in the supernatant were obtained by spinning at 13,000 g for 10 min. The MHCs SM1 and SM2 were separated by electrophoresis of the supernatant on slowly running, highly porous 4.5% SDS-polyacrylamide slab gels (1-mm thick) as previously described (35). Coomassie brilliant blue-stained proteins were quantified by scanning densitometry of the gel. The relative ratio of SM2 to SM1 heavy chains was estimated from the areas under the peaks of SM2 and SM1.

Western Blot analysis. SM1 and SM2 were separated by 4.5% SDS-PAGE as described above and then were transferred to Immobilon-P membrane (Millipore, Bedford, MA). Transfer was carried out for 1 h at 100 V using a Bio-Rad Mini Trans-Blot electrophoretic cell and buffer of the following composition (25 mM Tris, 192 mM glycine, 0.005% SDS, and 0.056% beta -mercaptoethanol. The membrane was then blocked with 10% Sea Block (Pierce) for 1 h and subsequently was incubated with a 1:200 dilution of primary antibody (overnight at 4°C) and then a 1:1,000 secondary peroxidase conjugated antibody (goat anti-mouse IgG; Sigma) for 1 h at room temperature. All antibody solutions were diluted in 10% Sea Block, and thorough washing with PBS containing 0.05% Tween 20 was performed between incubations. Proteins recognized by the antibody were then visualized by using 3,3'-diaminobenzidine (Sigma).

Two-dimensional gel electrophoresis. Corporal smooth muscle tissue or bladder smooth muscle tissue that had been previously frozen in liquid nitrogen was ground to a fine powder in a liquid nitrogen-cooled mortar using a pestle. Endogenous kinase and phosphatase were inactivated by adding the frozen powder to a mixture of dry ice and acetone and by allowing them to thaw (5). The sample was then spun for 5 min at 6,000 g, and the acetone was removed. The pellet was mixed with IEF sample buffer (50 µl/10 mg of tissue) and then homogenized with a mini-electric homogenizer. After centrifugation, 50 µl of this supernatant were applied to mini-IEF cylindrical gels (1 × 65 mm) and electrophoresed. IEF gels were subjected to PAGE (7 cm × 10 cm × 1 mm gels) with slight modification (35) of the procedure published by O'Farrell (28). The spots corresponding to LC17a and LC17b, as well as unphosphorylated and monophosphorylated LC20, were quantitated by scanning densitometry and a program (Bio-Rad) for analysis of two-dimensional gels. The identity of these spots was confirmed by Western blotting of the two-dimensional slab gels and by staining with monoclonal antibody (Sigma) to LC20 (not shown). Pure myosin phosphorylated with 32P was added to the frozen powder to ensure the absence of dephosphorylation of myosin by endogenous phosphatase during myosin isolation and electrophoresis and to confirm that the acidic spot in the Coomassie blue-stained two-dimensional gels was indeed phosphorylated 20-kDa LC. Free [gamma -32P]ATP added to the tissue during homogenization was not incorporated into the MLC, ruling out additional phosphorylation by endogenous kinase during the preparative procedure and gel electrophoresis.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Expression of mRNA transcripts for MHC isoforms. Total RNA from corporal smooth muscle was reverse transcribed and the cDNA was assayed by PCR using upstream (P1) and downstream (P2) primers that span the region coding the 25- to 50-kDa junction (Fig. 1) of the rabbit smooth muscle MHC (ATP binding region). As shown in Fig. 2A, the PCR products corresponding to the mRNA transcripts with or without the 21-nt insert in the 5' region of the cDNA migrate in a 2% agarose gel as 358- and 337-bp bands, respectively. The presence of the 21-nt insertion was confirmed by direct sequence analysis. CCSM expresses a greater amount of cDNA without the insert (SMA cDNA) than that with the insert (SMB cDNA), as shown in Fig. 2A (lane 3). Reflectance densitometric scanning of photographs of the agarose gels stained with ethidium bromide revealed a mean value of 31% SMB cDNA for the corporal sample (n = 5). For comparison, the cDNAs from PCR of total RNAs from bladder and aorta smooth muscles, which contain predominantly SMB (lane 2) and SMA types (lane 4), respectively, are shown. No discernible bands were observed in a negative control that lacked RNA (lane 5). Hence, although the CCSM expresses predominantly the SMA mRNA, which is typical of the tonic type muscles (for example aorta), it also expresses significant amounts of the SMB mRNA.


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Fig. 1.   Nucleotide sequences of primers used in RT-PCR of smooth muscle myosin isoforms. A: RT-PCR primers were designed for the following regions in rabbit smooth muscle myosin heavy chain (MHC) mRNA: ATP binding (P1 and P2), SM1/SM2 (P3 and P4), and the COOH-terminal region of rabbit 17-kDa myosin light chain (MLC) mRNA (P5 and P6). MHC primers were designed according to the rabbit uterus smooth muscle MHC full-length cDNA sequence (3). MLC primers were designed based on the mouse sequence (Halstones and Gunning, GenBank accession no. UO4443). B and C: schematic drawings of the entire MHC and MLC molecules. The targeting region of cDNA is shown in parenthesis, and the corresponding region of the myosin molecule is indicated by the solid line above it.


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Fig. 2.   Analysis of MHC and 17-kDa MLC mRNA transcripts using RT-PCR. A: P1 and P2 primers were used to amplify the NH2-terminal region containing the MHC SMA/SMB alternative splice site. B: P3 and P4 primers were used to amplify the COOH-terminal region containing the MHC SM1/SM2 alternative splice site. C: P5 and P6 primers were used to amplify the region containing the LC17a/LC17b alternative splice site. A-C: lane 1, 100-bp DNA ladder used as standard; lane 2, rabbit urinary bladder; lane 3, rabbit corpus cavernosum; lane 4, rabbit aorta; lane 5, negative control lacking RNA. Rabbit uterine smooth muscle alpha -actin was amplified in each reaction as an internal standard as described in METHODS.

To determine the expression of SM1 and SM2 heavy chains that are different at the COOH-terminal tail region of the myosin, a set of upstream and downstream oligonucleotide primers (P3 and P4) that span the 3'-terminal alternative splicing region of the cDNAs corresponding to both SM1 and SM2 (Fig. 2B) were used. As shown in Fig. 2B, two PCR products of 206 and 245 bp, representing SM1 cDNA fragments and SM2 cDNA fragments (confirmed by direct sequence analysis), respectively, were obtained for the corporal tissue (lane 3). Densitometric scanning revealed a SM2/SM1 cDNA ratio of ~1.2:1 (n = 5). For comparison, the cDNAs from rabbit bladder myosin (lane 2) and rabbit aortic myosin (lane 4) are shown in Fig. 2B. Bladder muscle has been shown to exhibit an SM2/SM1 ratio of ~1.7:1 (38), and rabbit aorta myosin contains an SM2/SM1 ratio of ~1:1 (8). Hence the relative ratio of SM2/SM1 in the corpora is more similar to the aortic muscle than that of the smooth muscle in the bladder wall.

Quantitative competitive RT-PCR. Although the RT-PCR results (shown above) yield information about the relative ratios of the mRNAs that encode the MHC isoforms, it does not provide quantitative information. To determine the absolute concentrations of the mRNAs that encode the various MHC isoforms in the tissues, quantitative competitive RT-PCR was performed on rabbit bladder, aorta, and corpus cavernosum, as described in METHODS. As shown schematically in Fig. 3A, a mutant cRNA that contained a 90-nt region of intron 7 of mouse caldesmon was constructed. A representative gel and the corresponding data analysis from quantitative competitive RT-PCR obtained for one of the corporal samples are shown in Fig. 3, B and C (see legend of Fig. 3 for details).


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Fig. 3.   Quantitative competitive RT-PCR (QC RT-PCR). A: a mutant cRNA that contained the sequence [nucleotides 5402-5659 for rabbit smooth muscle MHC (SMMHC)] present in SM1 and SM2 transcripts or SMA and SMB transcripts, but differing from them by introduction of a 90-nt caldesmon intron fragment, was transcribed from the mutant SMMHC. Known amounts of mutant cRNA were mixed with total RNA and used to perform QC RT-PCR using appropriate primers. B: representative ethidium bromide-stained agarose gel from a QC RT-PCR performed on corpus cavernosum. It can be seen that the competimer fragment runs above the fragment produced from the SMMHC cDNA. C: bands in the gel in B were scanned using a densitometer and the data plotted to determine the exact number of SMMHC copies, which is the same as the mutant SMMHC (mSMMHC) copy number when the amounts of the two are equal.

The results from quantitative competitive RT-PCR analyses are summarized in Fig. 4. The corpus cavernosum contained relatively the same number of mRNA copies of the smooth muscle MHC (SMMHC) gene per 100 ng of total RNA (~1.7 × 106) as the aorta, whereas the bladder possessed roughly 1.6-fold more copies (Fig. 4A). Using this quantitative information, along with the information we obtained for the relative ratios of the MHC mRNA transcripts from Fig. 2, A and B, we were able to determine the absolute concentrations of MHC alternatively spliced mRNAs in these three tissues. Corpus cavernosum and aorta possessed roughly the same amount of SM1 and SM2 mRNA, whereas the bladder possessed a nearly equivalent amount of SM1 mRNA but approximately twofold more SM2 mRNA than either of the former two tissues (Fig. 4C). With regard to myosin isoforms encoded by mRNA alternatively spliced at the 5' end, the aorta consisted of predominantly the SMA transcript (lacking the 21-nt insert), whereas the bladder contained predominantly SMB mRNA (containing the 21-nt insert). Similar to the bladder, the corpus cavernosum contained the mRNA coding for SMB, but the absolute concentration was ~4.5-fold lower than the bladder (Fig. 4B).


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Fig. 4.   Quantitative determination of myosin isoform ratios in rabbit tissues. With the use of information gathered from gels as in Fig. 3B, it was determined that the corpus cavernosum and aorta smooth muscles contain relatively the same number of mRNA copies of the SMMHC gene per 100 ng of total RNA (~1.7 × 106), whereas the bladder possessed roughly 1.6-fold more copies (A). Using this quantitative information along with the relative ratios of the individual isoforms that we had determined (in Fig. 2), we were able to calculate the absolute concentrations of alternatively spliced MHC isoforms in these 3 tissues. Corpus cavernosum and aorta possessed roughly the same amount of SM1 and SM2, whereas bladder possessed a nearly equivalent amount of SM1 but ~2-fold more SM2 than either of the former 2 samples (C). In regard to the NH2-terminal alternatively spliced myosin isoforms, the aorta consisted of predominantly the SMA isoform, whereas the bladder contained predominantly SMB. Similar to the bladder, the corpus cavernosum contained some of the SMB isoform but at a relative concentration ~4.5-fold lower than the bladder.

Expression of MHC isoforms at the protein level. To determine whether the expression patterns of SM1 and SM2 at the mRNA level determined by RT-PCR are also seen at the protein level, tissue homogenates from rabbit corpus cavernosum were electrophoresed on 4.5% SDS-PAGE gels (Fig. 5). The SM1 (204 kDa) and SM2 (200 kDa) MHCs were separated on this highly porous gel, and the protein bands stained with Coomassie blue were quantitated by densitometry. Two separate regions of the bands in each lane for all sample loadings (lanes 2 and 3) were estimated and the ratios averaged. The SM2/SM1 ratio was determined to be ~1.1:1 (n = 5). Thus the ratios of SM2/SM1 at the mRNA and protein levels are similar.


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Fig. 5.   SDS-PAGE analysis of total protein extracts from corpus cavernosum smooth muscle. Total protein extracted from rabbit corporal tissue was loaded onto a 4.5% SDS-polyacrylamide gel at 2 different protein concentrations (lanes 2 and 3), and SM1 and SM2 were separated by electrophoresis as outlined in METHODS. Proteins were visualized by Coomassie brilliant blue staining. Relative protein levels of SM1 and SM2 were determined by densitometric scanning of the stained gels. Rabbit skeletal muscle MHC, with a calibrated molecular mass of 200 kDa, was run as a control in lane 1. SM1 protein (204 kDa) runs slightly higher than the SM2 (200 kDa) protein.

The SM1 and SM2 heavy chain isoforms with or without the NH2-terminal insertion are not separable by gel electrophoresis; thus it is difficult to determine the relative ratio of SM1 and SM2 MHC with or without the insert. To determine whether CCSM contains both inserted and noninserted SM1 and SM2 MHC, we generated an antibody specific for the seven-amino acid insert as outlined in METHODS, and the isoforms were distinguished by Western blotting using this antibody. Bladder, aorta, and corporal samples were separated on a 4.5% gel as described above and then transferred to membrane by Western blotting. The antibody against the seven-amino acid peptide reacts with both the SM1 and SM2 bands of the myosin isolated from the bladder (Fig. 6, top, lane 3). On the contrary, myosin from aortic smooth muscle consists of predominantly the noninserted SMA isoform, since the antibody does not recognize either the SM1 or SM2 MHC isoform in the adult rabbit aorta (lane 1). These results would be predicted since, in the adult rabbit urinary bladder, all of the MHC mRNA contain the 21-nt insertion, coding for the seven-amino acid peptide, whereas mRNA containing this insertion is not detectable in the aorta (see Fig. 2A). Because the normal SM2/SM1 isoform composition in the bladder is ~2.8:1 (38), the antibody reaction is more dominant in the lower band (which represents SM2) than the upper SM1 band (Fig. 6, top, lane 3).


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Fig. 6.   Western blot analysis of smooth muscle myosin SM1/SM2 using antibody against the NH2-terminal 7-amino acid insertion. Top: crude extracts were prepared from smooth muscle tissues as described in METHODS, SM1/SM2 were separated by a highly porous 4.5% SDS-polyacrylamide gel, and the proteins were blotted to Immobilon-P membrane. Lane 1, rabbit thoracic aorta; lane 2, rabbit corpus cavernosum; lane 3, rabbit urinary bladder body. Western blot analysis was performed using the antibody specific for the NH2-terminal 7-amino acid insertion. Relative position of a 200-kDa marker protein (rabbit skeletal myosin) is indicated at right. Bottom: gel identical to that described in top was prepared and then stained with Coomassie blue as outlined in METHODS.

Although the CCSM consists of slightly more SM2 than SM1 at the protein level (Fig. 5), both the SM1 and SM2 bands of the CCSM myosin (Fig. 6, top, lane 2) react with the antibody to relatively the same extent. The ratio of SM2/SM1 containing the insert was ~1:1 (n = 3) as determined by densiometric scanning. An identical gel stained with Coomassie blue is shown in Fig. 6, bottom.

Localization of the SMB isoform in the CCSM. To conclusively demonstrate that the CCSM does contain SMB protein, we performed immunofluorescence microscopy using our antibody to the seven-amino acid insert. As shown in Fig. 7, the smooth muscle of the CCSM reacts with antibody from Sigma (total myosin) that recognizes all known smooth muscle MHC isoforms (Fig. 7E). In addition, the CCSM also reacts with the antibody to the seven-amino acid insert (Fig. 7F). The aorta smooth muscle reacts strongly with the antibody that recognizes total myosin (Fig. 7B); however, it does not react with the antibody specific for the seven-amino acid insert (Fig. 7C). The faint fluorescence observed in Fig. 7C is the autofluorescence coming from the connective tissue fibers between the smooth muscle cells. As a positive control, we show that the antibody against the seven-amino acid insert reacts strongly with bladder smooth muscle (Fig. 7G), which contains smooth muscle MHC that is completely inserted as predicted by RT-PCR and Western blotting (Figs. 2A and 6). Omission of the second antibody or replacing the primary antibody (7-amino acid antibody) with preimmune serum resulted in no significant immunofluorescence from the tissue sections (data not shown). These immunofluorescence data demonstrate the specificity of our antibody in tissue sections and demonstrate for the first time that the CCSM cells do express the inserted SMB MHC isoform.


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Fig. 7.   Localization of the SMB isoform in the corpus cavernosum smooth muscle (CCSM). Sections of aorta (A-C), corpus cavernosum tissue (D-F), and bladder (G) were either stained with hematoxylin and eosin (A and D) or treated with antibody against the total myosin (B and E) or antibody against the 7-amino acid peptide (C, F, and G). Immunofluorescence microscopy reveals that both aortic and CCSM tissue reacted with antibody against the total myosin molecule (B and E). Aorta did not react with the antibody against the 7-amino acid insert (C), whereas CCSM and bladder smooth muscle tissue did (F and G). Faint fluorescence observed in C is the autofluorescence coming from the connective tissue fibers between the smooth muscle cells. Bar, ~40 µm.

Expression of mRNA transcripts for 17-kDa MLC isoforms. To determine the LC17a/LC17b mRNA composition of the rabbit corpora smooth muscle myosin, we designed primers (P5 and P6; see Fig. 1) based on the partial LC17 mouse sequence deposited in GenBank (Halstones and Gunning, GenBank accession no. UO4443), since there is no published sequence for rabbit smooth muscle LC17. Figure 2C shows that these primers amplified two bands in the rabbit corpora (lane 3) of the approximate sizes (232 and 276 bp) predicted by the partial mouse cDNA sequence. To ensure that our products were indeed representing LC17 mRNA of rabbit, the amplified cDNAs contained in these two bands were subcloned and sequenced as outlined in METHODS. Sequence analysis of the LC17 cDNA bands revealed that both bands possessed identical nucleic acid sequences, with the exception of an insertion of 44 nucleotides in the LC17b isoform, which was not found in the LC17a isoform (data not shown). Therefore, the two bands in Fig. 2C appear to represent rabbit LC17a and LC17b mRNA. The top band represents LC17b, since the cDNA runs slower in the agarose gel, reflecting the 44-bp insertion in the 3' end of the mRNA.

Analysis of the PCR products obtained in Fig. 2C using total RNA from corpora smooth muscle tissue (lane 3) showed ~18% LC17b (n = 4). This value is similar to that for bladder smooth muscle myosin (lane 2) but lower than the value obtained for aortic smooth muscle that possesses almost equal levels of the two 17-kDa light chain isoforms (lane 4). Therefore, the composition of the essential light chain isoform at the level of mRNA expression in corpora smooth muscle is similar to that in bladder smooth muscle, which exhibits phasic characteristics.

Expression of LC17 isoforms at the protein level. Two-dimensional electrophoresis was performed as described in METHODS. The CCSM LC17 isoforms can be seen in the lower right portion of the gels shown in Fig. 8, A-C. As indicated in Fig. 8, the LC17a isoform migrates more to the acidic end of the gel, since it has been shown to possess a slightly more acidic isoelectric point than the LC17b isoform. Densitometric analysis of the gel revealed that the corporal tissue consists of ~13% LC17b (n = 6). Thus the level of protein expression of the essential light chains correlates well with expression at the mRNA level. Bladder smooth muscle, despite consisting of roughly the same mRNA isoform composition as the corpora, does not appear to express any detectable LC17b protein (data not shown). Tropomyosin and actin isoforms are labeled in all two-dimensional gels for orientation.


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Fig. 8.   Two-dimensional gel electrophoresis of smooth muscle total protein extracts. Two-dimensional gel electrophoresis was performed on rabbit corpus cavernosum protein extracts as described in METHODS. A: CCSM at the basal tone. B: CCSM treated with 4 mM EGTA. C: CCSM maximally stimulated with 200 µM phenylephrine. The pH range of the isoelectric focusing gel is from 4 to 6.5. Positions of the unphosphorylated (U) and phosphorylated (P) 20-kDa regulatory light chain and the essential 17-kDa a (a) and b (b) light chains on the gels are indicated. For orientation purposes, the stained regions of the gel corresponding to tropomyosin and actin are also labeled. Positions of molecular mass markers that were run in second dimension are indicated at left in A. All 3 protein extracts were prepared in a manner to prevent phosphorylation changes induced by endogenous kinase or phosphatase activity as described in METHODS.

Phosphorylation level of LC20. Tissue samples were processed for IEF followed by SDS-PAGE in a slab gel in the second dimension under conditions that prevented the activities of MLC kinase and phosphatase (see METHODS). A typical migration of the LC20 after two-dimensional electrophoresis is shown in Fig. 8. The phosphorylated and unphosphorylated LC20 were identified in the two-dimensional gels based on their isoelectric point and molecular size. In another experiment, the CCSM extract in IEF sample buffer was mixed with purified bladder myosin phosphorylated with [gamma -32P]ATP using the endogenous kinase, and the mixture was processed for two-dimensional gel electrophoresis. The phosphorylated LC20 comigrated with exogenous LC20 of purified bladder myosin phosphorylated with [gamma -32P]ATP as the Coomassie blue spots and the signal obtained after autoradiography overlapped (data not shown). The intensity of the monophosphorylated LC20, as well as the small spot that is more acidic than the monophosphorylated LC20, is decreased on incubation of the CCSM in EGTA (Fig. 8B). On the basis of the Ca2+-dependent appearance of these spots, their molecular size, and their cross-reactivity with the monoclonal antibody (Sigma) against LC20 (data not shown), these spots are identified as monophosphorylated and diphosphorylated LC20.

The myosin in the rabbit corporal tissue at the basal level of tone is ~10% (n = 6), thus existing in a predominantly unphosphorylated state (Fig. 8A). This percentage is calculated as the percentage of monophosphorylated LC20 divided by the total unphosphorylated plus monophosphorylated LC20 (30). Satellite spots were not included in the calculation of MLC phosphorylation levels. This can be compared with ~25% (n = 4), the basal level of phosphorylation of the rabbit urinary bladder body. On maximal stimulation (Fig. 8C), induced by 200 µM phenylephrine, the LC20 of the CCSM only reaches a phosphorylation level of ~23% (lower than the bladder at rest).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In this study, we examined the expression of the myosin isoforms in the myocytes in the corpus cavernosum penis both at the mRNA and protein levels using RT-PCR, quantitative competitive RT-PCR, one- and two-dimensional gel electrophoresis, and Western blot analysis. Furthermore, we localized the MHC isoforms in situ by immunofluorescence microscopy. To our knowledge, these are the first detailed analyses of the myosin isoforms in the myocytes in the smooth muscle tissue of corpus cavernosum penis. The results clearly demonstrate that rabbit CCSM possesses a myosin isoform composition somewhat intermediate between the bladder and aortic smooth muscles, considered to exhibit tonic and phasic characteristics, respectively, although experimental conditions affect the tonic and phasic behavior of a smooth muscle. Our findings suggest that the myosin isoform composition contributes, at least partially, to the unique ability of the CCSM to exist most of the time in a state of high resting tone and to relax rapidly on stimulation for penile erection to occur.

The data from RT-PCR show that ~31% of the mRNAs for MHC isoform in the CCSM contain a 21-nt insert (SMB) in the 5' end of the mRNA that encodes the region near the ATP binding site. The majority of the mRNA, however, is the SMA type (Fig. 2A). Results obtained from the quantitative competitive RT-PCR experiments allowed us to determine that the absolute concentration of SMB transcripts in the CCSM is ~4.5-fold lower than in the bladder (Fig. 4B). Data from Western blot analysis using antibody specific to the seven-amino acid insert near the ATP binding region show that the SMB mRNA is translated into protein in the CCSM (Fig. 6). The presence of this seven-amino acid insert is correlated with higher actin-activated Mg2+-ATPase activity of the avian gizzard smooth muscle myosin, and myosin from this tissue moves actin filaments in vitro at a higher velocity than that for aortic smooth muscle myosin (17). Although it was originally thought that all arterial smooth muscle myosin consists of completely noninserted (SMA) myosin, a recent study shows that the smaller muscular arteries of rabbits (femoral, saphenous) and rats (tail) consist of predominantly the inserted SMB type heavy chain isoform (8). Furthermore, actin-activated ATPase activity of myosin isolated from small muscular arteries and the maximum velocity of shortening of this smooth muscle are twofold higher than that of the aortic muscle (8). Bladder myocytes contain only the myosin isoforms with the seven-amino acid insert, since they reveal predominantly (~99.5%) this isoform both at the mRNA and protein levels.

A question that arises when analyzing complex tissue samples containing multiple cell types (such as the corpus cavernosum) is the source of the cell type for the mRNA or protein. The primers used for RT-PCR are specific for smooth muscle myosin, since we have shown that they do not amplify cDNA reverse transcribed from other cell types such as liver cells (8). This fact would rule out mRNA contamination from other cell types besides smooth muscle. Still, there could be contamination from the smooth muscle cells of the vasculature within the corporal tissue. In a prior publication we have shown that, although large vascular structures such as the aorta do not contain the SMB (inserted) form of MHC, the smaller more muscular arteries such as the rabbit saphenous artery and the tail artery from the rat contain almost completely SMB (9). Thus it is possible that SMB from the small arteries in the corpora could contribute a minor portion of the 31% SMB level determined for the corpora. Interestingly, the amount of the SMB mRNA from liver (which also contains small arteries) was undetectable under the PCR conditions used; hence it is unlikely that all of the 31% of the SMB mRNA are from the small arteries in the corpus cavernosum tissue.

Immunofluorescence microscopy using highly specific antibody against the seven-amino acid peptide reveals that this isoform is present in the myocytes in the CCSM (Fig. 7). Interestingly, the CCSM possesses the SMB to a lesser extent than the small muscular arteries, despite its vascular nature and embryonic origin. Thus the corpus cavernosum penis is an example of a smooth muscle that contains myosin isoforms present in aorta smooth muscle, which exhibits tonic characteristics, and in bladder smooth muscle, which is considered to be a phasic muscle, although the tonic or phasic behavior of a smooth muscle depends on other factors.

The data from this study also show that the SM2/SM1 isoform ratio in the CCSM is ~1.1:1. Thus the SM2/SM1 myosin isoform ratio of the CCSM is more similar to that of the rabbit aorta (1:1) than the value of 1.7:1 found for adult bladder in this paper and by others (8, 38). Interestingly, the small muscular arteries, although predominantly inserted, still possess relatively equal amounts of SM2 and SM1 isoforms (8) as the aortic smooth muscle, showing a similarity in the distribution of the tail isoforms in the myocytes in the arterial system. The expression of these COOH-terminal isoforms at the protein level correlates well with the mRNA values. Although CCSM contain a significant amount of the NH2-terminal inserted SMB myosin isoform, the predominant isoform in the bladder, the ratio of the SM2/SM1 is similar to that present in the arterial myosin. Therefore, it appears that the factors that modulate the expression of SMB in CCSM cells do so without altering the expression pattern of the COOH-terminal SM2/SM1 isoforms.

Our sequence analysis of the LC17 cDNA bands revealed that both bands possessed identical nucleic acid sequences, with the exception of an insertion of 44 nucleotides in the LC17b isoform, which was not found in the LC17a isoform. The rabbit LC17b sequence was determined to be ~89% similar to the mouse LC17b sequence at the nucleic acid level and thus predictably similar at the protein level. Human (89%), bovine (90%), and chicken (75%) LC17b were also found to be quite homologous to the rabbit sequence.

Numerous studies have shown that the levels of LC17a seem to correlate with the ability of smooth muscle cells to generate more force (25). Corporal smooth muscle was found to possess ~18% mRNA coding for LC17b; thus 82% of the mRNA codes for LC17a (Fig. 2C). This correlated well with the expression of LC17b in the corpora at the protein level (13%; Fig. 8, A-C). Like the level of NH2-terminal inserted SMB, this value was intermediate between aorta (45%) and bladder smooth muscle (0%) but closer to that of the bladder. Thus the relative amount of NH2-terminal inserted myosin isoform SMB appears to correlate with the amount of LC17a. However, at the physiological level, the NH2-terminal insertion is more important than the essential light chain. Kelley et al. (17) showed that exchanging the light chains between the avian aorta and gizzard (containing SMA and SMB, respectively) was ineffective in altering ATPase activity or in vitro motility of actin filament over the heads of myosin isoform, two physiologically relevant myosin functions (17). Recently, expressed recombinant myosin containing this seven-amino acid insertion has been shown to have an actin-activated myosin ATPase activity that is higher than that of the native rabbit aorta myosin or the recombinant myosin without this insert (34).

The phosphorylated and unphosphorylated LC20 were identified in the two-dimensional gels based on their isoelectric point and by Western blotting (data not shown) using monoclonal antibody against LC20. Furthermore, the phosphorylated LC20 comigrated with LC20 of purified bladder myosin phosphorylated with [gamma -32P]ATP. The intensity of the small spots identified as the monophosphorylated and diphosphorylated LC20 showed diminished intensity on incubation in EGTA. If they were isoforms of LC20, depleting the Ca2+ concentration should have no effect on their isoelectric point. Based on the Ca2+-dependent appearance of these spots and their molecular size and cross-reactivity with the LC20 antibody, these spots are identified as monophosphorylated and diphosphorylated LC20.

The level of myosin phosphorylation in the CCSM is ~10% at the basal tone. In this state the muscle is contracted (the penis is flaccid). On further stimulation by phenylephrine [a concentration shown to produce maximal stimulation in the CCSM (21)], the level of phosphorylation rises to ~23%. A higher basal level of phosphorylation (~25%) is found in the bladder smooth muscle compared with the CCSM. Thus MLC kinase might be less active in the CCSM at the basal level of tone than in bladder smooth muscle. Alternatively, the level of phosphatase activity in the CCSM and urinary bladder smooth muscle may account for a difference in the resting level of MLC phosphorylation in these two smooth muscles.

The difference in the level of phosphorylation of these two muscles could also be due to a difference in the cytosolic Ca2+ in these muscles at the resting tone. The intracellular concentration of Ca2+ in the CCSM cell has been reported to be ~60-80 nM at rest (7, 39). The Ca2+ concentration is similar to what has been found for the rabbit pulmonary artery (~106 nM), which is a tonic type muscle, and for the resting ileum (~79 nM), which is a phasic type smooth muscle (12). These two tissues exhibited resting phosphorylation levels of 3 and 7%, respectively. Interestingly, the rabbit bladder has been found to possess a Ca2+ concentration of ~200 nM at rest (22), a slightly higher Ca2+ concentration than CCSM at rest.

In general, contraction of smooth muscle in response to agonist is caused by the production of inositol 1,4,5-trisphosphate (IP3), which releases Ca2+ from intracellular sarcoplasmic reticulum stores (16, 20). MLC kinase is activated by the rise in cytosolic Ca2+, which binds to calmodulin. This leads to phosphorylation of MLC, allowing actin-activated ATPase activity, cross-bridge cycling, and the development of force or shortening of the muscle. It has been shown that agonists also augment G protein-dependent downregulation of MLC phosphatase activity, resulting in an increase in the level of MLC phosphorylation (19). In view of the high resting tone exhibited by corporal smooth muscle, it is surprising that the level of phosphorylation of the LC20 at rest is <10%. Despite the low level of phosphorylation, it is difficult to rule out the possibility that myosin phosphorylation is not required for the maintenance of resting tone, since dephosphorylation by depletion of Ca2+ causes a relaxation of the CCSM (9). However, it is unlikely that myosin phosphorylation is the only mechanism that maintains the high resting tone in the CCSM. Other signal transduction mechanisms may also be involved in keeping the CCSM at a high resting tone.

Khalil and Morgan (18) showed that the phenylephrine-induced contraction of the ferret aorta is Ca2+ independent, involving a Ca2+-independent epsilon  isozyme of protein kinase C (13). The finding that the resting tone and the light chain phosphorylation decrease in the absence of Ca2+ (9) suggests that the mechanism for maintenance of tone at the resting level, to keep the penis in the flaccid state, is Ca2+ dependent. However, the possibility that the high resting tone in CCSM is also modulated by a mechanism that functions in the absence of Ca2+ or at a very low level of Ca2+ (resting level) and complements phosphorylation-mediated regulation cannot be ruled out. It has been suggested that the Ca2+-independent contraction in ferret aorta smooth muscle is mediated via the thin-filament-associated protein, calponin, which is a substrate for protein kinase C-epsilon (14). The high level of force at the resting tone, achieved with a very low (~10%) level of MLC phosphorylation, may be due to the cooperativity of the myosin molecules in the thick filaments (36).

In conclusion, while there is much to be learned regarding the mechanisms that regulate the high basal tone and rapid relaxation of the CCSM that are required for detumescence and tumescence of the penis, respectively, the unique myosin isoform composition of the corporal smooth muscle may be related to its unique function. This study is the first to examine myosin, the molecular motor that regulates the diameter of the cavernous space allowing the corpus cavernosum to increase in size and rigidity during penile erection. Future studies on corporal smooth muscle should include the proteins that regulate myosin phosphorylation and cross-bridge cycling (e.g., MLC kinase/phosphatase, PKC, caldesmon, etc.), as well as the composition of myosin isoforms in the corpora from normal humans and from patients with erectile dysfunction.

    ACKNOWLEDGEMENTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-39740 to S. Chacko. M. E. DiSanto was a postdoctoral fellow funded by the American Foundation for Urologic Disease and by NIDDK Training Grant DK-02196.

    FOOTNOTES

Address for reprint requests: S. Chacko, Dept. of Pathobiology, Univ. of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104.

Received 7 July 1997; accepted in final form 10 June 1998.

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Discussion
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