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Identification of Myoglobin in Human Smooth Muscle

1998; Elsevier BV; Volume: 273; Issue: 36 Linguagem: Inglês

10.1074/jbc.273.36.23426

1083-351X

Yang Qiu, Lee Sutton, Austen Riggs,

Cardiovascular and exercise physiology

Myoglobin (Mb) has been believed to be absent generally from mammalian smooth muscle tissue. Examination of human rectal, uterine, bladder, colon, small intestine, arterial, and venous smooth muscle by immunohistochemical techniques shows that each of these tissues is immunopositive for both smooth muscle myosin and human Mb. Mb-specific primers were used for the polymerase chain reaction to generate cDNA from smooth muscle tissues. Southern hybridization with a Mb-specific probe gave a very strong signal with the cDNA from rectum, weaker signals from small intestine and uterus, a faint signal from colon, and no signal from bladder tissue. High performance liquid chromatography analysis coupled with sequence determination has shown that contaminating heme-binding serum albumin as well as hemoglobin in extracts of smooth muscle seriously compromise any heme-based or spectrophotometric assay of Mb. Combined affinity and size exclusion chromatography, however, provide the necessary resolution. The cDNA-derived amino acid sequence of human smooth muscle Mb was found to be identical to that of Mb from striated muscle. Myoglobin (Mb) has been believed to be absent generally from mammalian smooth muscle tissue. Examination of human rectal, uterine, bladder, colon, small intestine, arterial, and venous smooth muscle by immunohistochemical techniques shows that each of these tissues is immunopositive for both smooth muscle myosin and human Mb. Mb-specific primers were used for the polymerase chain reaction to generate cDNA from smooth muscle tissues. Southern hybridization with a Mb-specific probe gave a very strong signal with the cDNA from rectum, weaker signals from small intestine and uterus, a faint signal from colon, and no signal from bladder tissue. High performance liquid chromatography analysis coupled with sequence determination has shown that contaminating heme-binding serum albumin as well as hemoglobin in extracts of smooth muscle seriously compromise any heme-based or spectrophotometric assay of Mb. Combined affinity and size exclusion chromatography, however, provide the necessary resolution. The cDNA-derived amino acid sequence of human smooth muscle Mb was found to be identical to that of Mb from striated muscle. myoglobin bovine serum albumin diethylpyrocarbonate fluorescein isothiocyanate isomer I high performance liquid chromatography phosphate-buffered saline polymerase chain reaction sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis 3-[cyclohexylamino]-1-propanesulfonic acid. Ray Lankester (2Lankester E.R. Pflügers Arch. Gesamte Physiol. 1871; 4: 315-320Crossref Scopus (10) Google Scholar, 3Lankester E.R. Proc. Roy. Soc. Lond. B Biol. Sci. 1872; 21: 70-80Google Scholar) first observed intracellular hemoglobin (myoglobin, Mb)1 in mammalian striated muscle. He also reported Hb (Mb) in the rectal smooth muscle (sphincter) of man, but it appeared to be absent from other mammalian smooth muscle. Later, Biörck (4Biörck, G. (1949) Acta Med. Scand. 226, (suppl.) 1–216Google Scholar) found Mb in human uterine smooth muscle, and Jaisle and Huber (5Jaisle F. Huber K. Klin. Wochenschr. 1966; 44: 1182-1184Crossref PubMed Scopus (5) Google Scholar) reported a 3-fold increase in Mb in this tissue during pregnancy. However, neither Kagen and Gurevich (6Kagen L.J. Gurevich R. Immunology. 1967; 12: 667-673PubMed Google Scholar) nor Fasold et al. (7Fasold H. Riedl G. Jaisle F. Eur. J. Biochem. 1970; 15: 122-126Crossref PubMed Scopus (7) Google Scholar) detected any Mb in uterine tissue by immunological procedures. The techniques of Fasold et al.were reported to be capable of detecting skeletal muscle Mb at levels as low as 1 ng/g of muscle wet weight. Intrigued by Lankester's early observation, we have reexamined smooth muscle tissue from the human rectum and from other human tissues. We reasoned that if Mb did occur generally in smooth muscle, even at a low concentration, it might function not only in facilitated diffusion (8Wittenberg J.B. J. Gen. Physiol. 1965; 49: 57-74Crossref PubMed Scopus (3) Google Scholar,9Wittenberg J.B. Physiol. Rev. 1970; 50: 559-636Crossref PubMed Scopus (412) Google Scholar) and possibly in aiding oxidative phosphorylation (10Wittenberg B.A. Wittenberg J.B. Annu. Rev. Biophys. Biophys. Chem. 1990; 19: 217-241Crossref PubMed Scopus (145) Google Scholar) but also might play a role in the regulatory interaction of NO with these tissues. In particular, it might affect the kinetics of the dynamic cycle in which S-nitrosohemoglobin delivers NO to vascular tissues and causes vasodilation (11Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1464) Google Scholar). Samples of human smooth muscle from rectum, uterus, small intestine, colon, and bladder were frozen in liquid nitrogen and stored at −80 °C. Each of these samples was obtained during surgery with the permission of the patient. All of the surgical procedures involved removal of cancerous tissue, but the muscle cell samples studied here were not themselves cancerous. About 0.4 g of smooth muscle from each of these tissues was used for the mRNA preparation with the Fast Track mRNA Isolation Kit version 3.5 from Invitrogen (San Diego, CA). mRNA (10 μg) from each tissue was combined with an oligo dT 28-mer that has an XbaI site at its 5′ end (500 ng) in diethylpyrocarbonate sterile deionized water (DEPC-dH2O) prepared by adding 100 μl of DEPC (Sigma, catalog number D-5758) to 100 ml of deionized water, shaken, and incubated at 37 °C overnight and autoclaved. The annealing reaction was carried out by incubating the mixture at 65 °C for 5 min followed by cooling on ice. Next, 1 μl of RNasin (40 units/μl, Promega), 8 μl of 2.5 mm dNTP mix, 10 μl of 5× reverse transcription buffer from U. S. Biochemical Corp., 5 μl of 0.1m dithiothreitol (Life Technologies, Inc.), 14 μl of DEPC-dH2O, and 2 μl of 200 units/μl Moloney murine leukemia virus reverse transcriptase (U.S. Biochemical Corp.) were added to the annealing reaction solution. After incubating at 37 °C for an hour, 5 μl of a 3 m sodium acetate solution and 125 μl of 100% ethanol were added, and the resulting solution was kept at −20 °C overnight. After ethanol precipitation, the pellet was dissolved in 20 μl of DEPC-dH2O. A polymerization chain reaction (PCR) was carried out to amplify Mb cDNA. The coding sequence of Mb was obtained by using 2 μl of the mRNA·cDNA from the reverse transcription as template and two synthesized primers, 5′-GACTCTAGAATGGGGCTCAGCGACGGG-3′ and 5′-AGTTCTAGACTAGCCCTGGAAGCCCAG-3′, which are complementary to the 5′ and 3′ ends of the known human Mb coding sequence (12Weller P. Jeffreys A.J. Wilson V. Blanchetot A. EMBO J. 1984; 3: 439-446Crossref PubMed Scopus (86) Google Scholar) and contain an XbaI site. The 100-μl PCR reaction solution contained 1 × PCR buffer (10 × PCR buffer: 0.5m KCl, 0.1 m Tris-HCl, pH 8.0, and 15 mm MgCl2), 0.1 mg/ml bovine serum albumin (BSA), 200 μm dNTP, 100 pmol each of NH2-terminal and COOH-terminal primers, and 2.5 units of Promega Taq polymerase. Denaturation at 94 °C for 5 min was followed by 30 cycles of 1 min each at 94 °C for denaturing, 48 °C for annealing, and 72 °C for elongation followed by a final 15 min at 72 °C for complete elongation. The resulting PCR product was analyzed after running an aliquot on a 1.0% agarose gel. The PCR product (480 base pairs) obtained by amplification of Mb cDNA (see above) was digested withXbaI and inserted into pUC19 vector (New England Biolabs) cut with XbaI. The ligation of the target DNA with the vector was achieved with a DNA ligation kit from Takara Shuzo Co. (catalog number 6021). The resulting ligation solution was used to transform MAX efficiency DH5α competent cells (Life Technologies, Inc.). Plasmid DNA was extracted from positive clones, and the cDNA insert was sequenced by the dideoxy chain termination method (U.S. Biochemical Corp. SequenaseTM version 2.0 DNA Sequencing kit). A blot of mRNA from multiple human tissues (catalog number 7760-1), supplied by CLONTECH Laboratories, Inc., was used to test for Mb expression in other tissues. The blot included mRNA from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. The human Mb cDNA from the PCR amplification was used as a probe, and the manufacturer's protocol was followed. The procedure of Schuder et al. (13Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google Scholar) was used in an attempt to quantify Mb in muscle tissue after removal of Hb by affinity chromatography. The affinity column was made by coupling the αβ dimer of human Hb to CNBr-activated Sepharose 4B. The Hb in the extract binds to the αβ dimer on the column, and Mb passes through unretarded (13Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google Scholar). A column (0.9 × 7 cm) of CNBr-activated Sepharose 4B (2 g, Pharmacia Fine Chemicals) was used as described (13Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google Scholar). Human Hb (8 μmol) 2All molar concentrations of Hb are on a heme basis. was used for the coupling reaction. The capacity of the column was determined by applying pure human Hb and then measuring the absorbance of the eluate at 420 nm. The column retained all of 0.3 μmol of Hb applied but 0.7 μmol of Hb exceeded the capacity and was detected in the eluate. Therefore, quantities of extract were applied that contained less than 0.3 μmol of Hb. The column was regenerated with 2.0 mNaCl, which removes bound Hb. The extraction of rectal skeletal muscle, rectal smooth muscle, and uterine tissue generally followed Schuderet al. (13Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google Scholar). Frozen tissue (0.4–1.0 g) was cut into small pieces and ground under liquid nitrogen. The resulting tissue powder was weighed and extracted with 10 ml of 100 mm potassium phosphate buffer, pH 7.0, 5 mm Na2EGTA, incubated on ice for 5 min, and then centrifuged at 10,000–20,000 × g for 10 min. The clear supernatant Extract I was used for affinity chromatography. The remaining pellet was extracted with 100 mm NaCl, 10 mm Tris-Cl, pH 7.6, 1 mm EDTA, 2% SDS, and 100 μg/ml phenylmethylsulfonyl fluoride. This extract was centrifuged as above to give a second supernatant, Extract II. Extract I (4 ml) of rectal smooth muscle was applied to the column at 4 °C and developed with 10 mmpotassium phosphate buffer, pH 7.0. The flow was downward under gravity, and the flow rate was adjusted to be under 15 ml/h. The fractions containing apparent Mb were collected and pooled. Extract I of rectal skeletal muscle was similarly processed. The spectra of both deoxy-Mb and CO-Mb were measured as described (13Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google Scholar). The millimolar extinction coefficients were taken to be εmM = 207 mm−1 cm−1 at 424 nm (MbCO) and εmM = 121 mm−1 cm−1at 435 nm (deoxyMb), the values given for horse Mb (14Antonini E. Physiol. Rev. 1965; 45: 123-170Crossref PubMed Scopus (137) Google Scholar). We measured the absorbance ratio,A 424/A 435 = 0.820 for horse deoxy-Mb and then calculated the εmM value at 424 nm: 0.820 × 121 = 99.2 mm−1cm−1. The apparent concentration of Mb was calculated from the following equation.CMb=ACOMb−AdeoxyMbɛmMCOMb−ɛmMdeoxyMb(Eq. 1) where the εmM values are for 424nm. All HPLC was performed on a SynChropak RP-P C18 reverse phase column (250 × 4.6 mm, SynChrom, Inc., Lafayette, IN) driven by a Beckman model 332 gradient chromatography system. Extract I of rectal tissue and the eluate from the affinity column (see above) were applied separately to the HPLC column with the following gradient program: Buffer A 0.1% trifluoroacetic acid in water, Buffer B, 0.1% trifluoroacetic acid in acetonitrile, 0–5 min, 0% B; 5–15 min, 0–30% B; 15–115 min, 30–55% B; 115–125 min, 55–0% B. Absorbance of the eluate was monitored at 220 nm with an Hitachi model 100–10 spectrophotometer. Absorbing fractions were collected and lyophilized. The protein was redissolved in water and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (10% resolving gel and 3% stacking gel) (15Bollag D.M. Edelstein S.J. Protein Methods. Wiley-Liss, Inc., New York1991: 96-116Google Scholar). The separation of Mb from serum albumin in Extract I (after affinity chromatography) was examined with a Bio-Gel SEC 40XL size exclusion column (300 × 7.8 mm; Bio-Rad catalog number 125-0604). The buffer used was 100 mm KPO4, 1 mm Na2EDTA at pH 7.0 running at 25 °C. The column eluate was monitored at either 280 or 415 nm with an Hitachi spectrophotometer (model 100–10) with an HPLC system previously described (16Zhu H. Ownby D.W. Riggs C.K. Nolasco N.J. Stoops J.K. Riggs A.F. J. Biol. Chem. 1996; 271: 30007-30021Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). A reference mixture of bovine serum albumin (Sigma catalog number A-8531) and horse Mb (Sigma catalog number M-0630) was also examined in this way. Proteins in both Extract I and Extract II of rectal smooth muscle tissue were run on SDS-PAGE. The gel was electro-blotted to ProtranTM Nitrocellulose membrane (BA79, 0.1-μm pore size, Schleicher & Schuell). The transferring buffer contained 10 mm CAPS at pH 11 and 10% methanol. The chemiluminescent ECLTM Western blotting System (Amersham Pharmacia Biotech) with the manufacturer's protocol was used for the detection of immobilized specific antigens conjugated with horseradish peroxidase-labeled antibodies. The primary antibody was rabbit anti-human Mb (Sigma catalog number M-8648, diluted 1:500). The secondary antibody was horseradish peroxidase-labeled donkey anti-rabbit whole antibody (Amersham Pharmacia Biotech catalog number NA934, diluted 1:1500). Biopsy samples of human tissues were trimmed of connective tissue and pinned to balsa wood sticks between two muscles from the hind limb of an adult rat (soleus and extensor digitorum longus). These tissues were then frozen in isopentane cooled to −60 °C with liquid nitrogen. Cryostat sections (10 μm) were collected on gelatin-subbed slides and stored dessicated at −20 °C. For immunolabeling, slides were incubated for 30 min at room temperature in phosphate-buffered saline (PBS, 137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mmKH2PO4, pH 7.4) containing 0.2% BSA, incubated in primary antibody diluted in PBS + BSA overnight at 4 °C, washed three times, 5 min each with PBS + BSA, incubated in secondary antibody diluted in PBS + BSA for 1 h at room temperature, washed three times, 5 min each with PBS, and coverslipped using FITC Guard (Testog, Chicago, IL). Primary antibodies were rabbit anti-human Mb (Sigma catalog number M-8648, diluted 1:500); monoclonal mouse anti-smooth muscle myosin (Sigma catalog number M-7786, diluted 1:250); monoclonal anti-human sarcomeric myosin heavy chain (clone A4.1025, Developmental Studies Hybridoma Bank, Johns Hopkins University, used as undiluted supernatant). Secondary antibodies were FITC-conjugated sheep anti-mouse Ig-G, F(ab′)2 fragment (Sigma catalog number F-2266, diluted 1:200) and rhodamine-conjugated goat anti-rabbit IgG, F(ab′)2 fragment (Cappel, Durham, NC, catalog number 55671, diluted 1:200). In some cases double immunolabeling was performed by applying a mixture of mouse anti-smooth muscle myosin and rabbit anti-Mb antibodies followed by a mixture of FITC-conjugated anti-mouse and rhodamine-conjugated anti-rabbit secondary antibodies. Slides were examined with a Nikon Optiphot epifluorescence microscope equipped with rhodamine and fluorescein filters. Images were acquired with an integrating CCD camera connected to a Macintosh computer containing a frame grabber and NIH image software. Images were cropped and labeled by use of Adobe Photoshop and Canvas software and printed with a Sony color printer. cDNA encoding human Mb was amplified by PCR from the total cDNA prepared from different tissues. Specificity was achieved by using primers constructed on the basis of the gene-derived NH2- and COOH-terminal sequences (12Weller P. Jeffreys A.J. Wilson V. Blanchetot A. EMBO J. 1984; 3: 439-446Crossref PubMed Scopus (86) Google Scholar). The same mass of tissue (∼0.4 g) was used from each source for Southern hybridization (Fig. 1) so that the signal should reflect approximately the relative quantity of Mb mRNA in each tissue. The strongest signal was clearly from rectal smooth muscle tissue with much less from tissue of the small intestine and uterus. Only a very weak signal was obtained from colon tissue, and none was detected from bladder tissue. Northern hybridization of mRNA from diverse tissues probed with cDNA for human Mb gave signals only for skeletal muscle and heart mRNA (Fig. 2). Although no signal was detected in the other tissues, the Northern hybridization is much less sensitive than the Southern hybridization prepared with PCR-amplified cDNA, so the negative finding could mean only that an insufficient quantity of mRNA was present in the experiment. We examined tissues with smooth muscle from rectum, uterus, colon, small intestine, bladder, arteries, and veins with immunohistochemical techniques. Differently labeled fluorescent antibodies to both human smooth muscle myosin and Mb were used. The results reveal that human rectal muscle is immunopositive for both smooth muscle myosin and for Mb (Fig. 3 a). A cryostat section passing longitudinally (panels A and B) and two sections passing orthogonally (panels C and D and panels E and F) through a bundle of smooth muscle fibers were double-immunolabeled; immunoreactivity to smooth muscle myosin was detected by using an FITC-labeled second antibody and to Mb by using a rhodamine-conjugated second antibody. Views through the fluorescein filter set are shown in panels A, C, and E, and views through the rhodamine filter set are shown in panels B, D, and F (Fig. 3 a). The tissue sections in panels A, B, C, and D (Fig. 3 a) received a mixture of anti-smooth muscle myosin and anti-Mb antibodies, followed by a mixture of the two secondary antibodies. The sections in panels E and F received no primary antibody but were incubated with the mixture of the two secondary antibodies. Thus, the sections in panels E and F serve as negative controls. Although some labeling of connective tissue is obvious in the absence of primary antibody, the muscle fibers themselves are unlabeled. Additional controls (also not shown) showed that these smooth muscle fibers were unreactive with an antibody to sarcomeric myosin and that rat skeletal muscles were unreactive with the anti-smooth muscle myosin. Very similar results were obtained for artery and vein tissue (Fig. 3 b), for uterine tissue (Fig. 3 c), and for colon, small intestine, and bladder (data not shown). The PCR-amplified cDNA for Mb from human rectal smooth muscle was cloned into pUC19 and sequenced. The deduced amino acid sequence was found to be identical to that of Mb from striated muscle (12Weller P. Jeffreys A.J. Wilson V. Blanchetot A. EMBO J. 1984; 3: 439-446Crossref PubMed Scopus (86) Google Scholar). The same tissue preparation also yielded PCR-amplified cDNA encoding part of the heavy chain of smooth muscle myosin, thus confirming the identification of the tissue. The principle of the procedure (see "Materials and Methods") is to use an affinity column that selectively removes Hb but not Mb. Extract I (4 ml) of rectal smooth muscle containing less than 0.05 μmol of Hb was applied to the column. The amount of Hb in the aliquot of Extract I was much less than the capacity of the affinity column, which can bind at least 0.3 μmol of Hb. Fig. 4 shows the HPLC pattern obtained for Extract I before (Fig. 4 a) and after (Fig. 4 b) passage through the column. The α and β chains of Hb (about half of the total protein) have clearly been removed by the column. Quantitative correspondence of the HPLC absorbance with the spectrophotometric determination can be determined as follows with the use of hemin and protein extinction coefficients previously determined (17Ownby D.W. Zhu H. Schneider K. Beavis R.C. Chait B.T. Riggs A.F. J. Biol. Chem. 1993; 268: 13539-13547Abstract Full Text PDF PubMed Google Scholar). The quantity of human Hb in the original solution, determined from the α and β chain absorbances in Fig. 4 a, was found to be 41.8 nmol. The total heme (measured as hemin) in Fig. 4 a corresponds to 73.4 nmol in the original solution. The difference (73.4 − 41.8) should give the heme attributed to Mb, 31.6 nmol, which corresponds to 174 μmol of Mb/kg tissue. Although this value is within 8% of the quantity of Mb, 188 μmol, determined by difference spectrophotometry of Extract I after affinity chromatography, SDS electrophoresis of fraction 4 in Fig. 4 bshowed that it is largely composed of a 66-kDa polypeptide, and only traces of 17-kDa protein are present (data not shown). Fraction 4 was electroblotted onto a polyvinylidene difluoride membrane for sequencing by the Microanalysis Facility of the University of Texas with a model 477A Applied Biosystems Sequencer. The 20-residue sequence obtained showed that the 66-kDa protein is serum albumin. Therefore, most of the apparent Mb in the spectrophotometric assay must be attributed to heme-binding serum albumin and not Mb. Heme-binding by serum albumin was tested with the Schuder et al. (13Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google Scholar) spectrophotometric assay for Mb content as follows. Hemin (Fluka, 1 μl of 12.4 mm stock in 0.1 N NaOH) was added to 4 ml of 10 mm phosphate buffer, pH 7, containing 8 nmol of BSA (Sigma) to give a 1.5 molar ratio of hemin to albumin. An apparent Mb content, 0.66 μm, was determined spectrophotometrically as described by Schuder et al. (13Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google Scholar) (see "Materials and Methods"). In contrast to the smooth muscle analysis, Extract I of rectal skeletal muscle did not show a large quantity of serum albumin. Analysis of fraction 7 in Fig. 4 c showed that it consists of Mb and that serum albumin was absent (data not shown). Analysis of Extracts I and II of rectal smooth muscle tissue after SDS-PAGE showed that the two extracts have different protein band patterns (Fig. 5 a). Additional cellular proteins were obtained in Extract II. When Extract I and Extract II were probed with anti-Mb antibody on a Western blot, a single band of about 17 kDa was detected in both Extracts I and II (Fig. 5 b). We conclude that a low concentration of Mb is present in both Extracts I and II and that Extract I did not extract all the protein. Application of a mixture of BSA (66 kDa), horse Mb (17 kDa), and human Hb (65 kDa) to a Bio-Rad size exclusion column failed to isolate the Mb because of the partial dissociation of the human Hb tetramers to dimers, which caused the Mb and Hb peaks to overlap (data not shown). However, separation of horse Mb from BSA alone did occur (Fig. 6 a). When Extract I, after removal of Hb with the affinity column (see above), was applied to the SEC column (Fig. 6 b), a small shoulder on the human serum albumin peak corresponded in position to Mb. The amount of protein in this shoulder is roughly estimated to be about 10% of the serum albumin. Comparison of the quantity of serum albumin shown in Fig. 6allows us to estimate the quantity of Mb to be 0.16 mg/g wet tissue, a rough estimate at best because we are at the limit of detectability for the Mb. This difficulty does not compromise the estimation of Mb in skeletal muscle tissue because the latter contains a much larger quantity of Mb. Our results confirm the early report by Lankester (1870) that Mb occurs in human rectal smooth muscle (2Lankester E.R. Pflügers Arch. Gesamte Physiol. 1871; 4: 315-320Crossref Scopus (10) Google Scholar, 3Lankester E.R. Proc. Roy. Soc. Lond. B Biol. Sci. 1872; 21: 70-80Google Scholar), but only at very low concentrations. We show by immunohistological techniques that Mb is also present in all other smooth muscle tissues examined: colon, small intestine, uterus, bladder, arteries, and veins. Table I summarizes the presence and content of Mb in various tissues. However, attempts to quantify the Mb in the rectal tissue with existing protocols failed because they depended on the assumption that the only interfering heme-containing protein is Hb (13Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google Scholar). The procedure of de Duve (20de Duve C. Acta Chem. Scand. 1948; 2: 264-289Crossref PubMed Google Scholar), which relies entirely on determination of hemin as the pyridine hemochromogen derivative, would also fail to measure Mb accurately. The reason for these failures is that substantial quantities of hemin-binding serum albumin can be present. The protocols do not distinguish between heme or hemin from Mb and from serum albumin. The procedure of Schuder et al. (13Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google Scholar) adequately removes contaminating Hb by affinity chromatography but does not remove serum albumin. Therefore affinity separation followed by size exclusion chromatography (Fig. 6) should be the method of choice to separate both of these contaminants from the much smaller Mb. Size exclusion chromatography alone is less satisfactory because of the tetramer to dimer dissociation of Hb.Table IMyoglobin reported for various tissuesMuscle TissueMb contentReferencemg/g wet weightHuman smooth Rectal+ immunohistochemical, mRNAPresent results Uterine smooth+ immunohistochemical, mRNAPresent results Uterine smooth0.2–0.65Jaisle F. Huber K. Klin. Wochenschr. 1966; 44: 1182-1184Crossref PubMed Scopus (5) Google Scholar Uterine smooth06Kagen L.J. Gurevich R. Immunology. 1967; 12: 667-673PubMed Google Scholar, 7Fasold H. Riedl G. Jaisle F. Eur. J. Biochem. 1970; 15: 122-126Crossref PubMed Scopus (7) Google Scholar Colon+ immunohistochemicalPresent results Bladder+ immunohistochemicalPresent results Colon07Fasold H. Riedl G. Jaisle F. Eur. J. Biochem. 1970; 15: 122-126Crossref PubMed Scopus (7) Google Scholar Small intestine+ immunohistochemical, mRNAPresent resultsPigeon smooth Gizzard9.013Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google ScholarHuman striated Rectal3.2Present results Pectoral1.75Jaisle F. Huber K. Klin. Wochenschr. 1966; 44: 1182-1184Crossref PubMed Scopus (5) Google Scholar Pectoral7.67Fasold H. Riedl G. Jaisle F. Eur. J. Biochem. 1970; 15: 122-126Crossref PubMed Scopus (7) Google Scholar Intercostal4.96Kagen L.J. Gurevich R. Immunology. 1967; 12: 667-673PubMed Google Scholar Cardiac4.36Kagen L.J. Gurevich R. Immunology. 1967; 12: 667-673PubMed Google Scholar Rectus abdominalis12.37Fasold H. Riedl G. Jaisle F. Eur. J. Biochem. 1970; 15: 122-126Crossref PubMed Scopus (7) Google ScholarRat, cat, and beef Cardiac2.6–5.413Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google ScholarBat Pectoral5.213Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google ScholarPigeon Breast4.913Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google Scholar Cardiac3.613Schuder S. Wittenberg J.B. Haseltine B. Wittenberg B.A. Anal. Biochem. 1979; 92: 473-481Crossref PubMed Scopus (50) Google ScholarSeal Pectoral33–421-aCalculated on the basis of O2 capacity with 1.32 cm3 O2/g Mb, assuming a molecular weight of ∼17,000.18Andersen H.T. Physiol. Rev. 1966; 46: 212-243Crossref PubMed Scopus (232) Google Scholar Diaphragm20–211-aCalculated on the basis of O2 capacity with 1.32 cm3 O2/g Mb, assuming a molecular weight of ∼17,000.18Andersen H.T. Physiol. Rev. 1966; 46: 212-243Crossref PubMed Scopus (232) Google ScholarPorpoise Psoas481-bSpectrophotometric assay of extracted MbCO. The values given are for dried muscle. Approximate wet weight values were estimated with the assumption that the muscle is 70% water.19Blessing M.H. Comp. Biochem. Physiol. 1971; 41A: 475-480Google Scholar Heart10 ± 11-bSpectrophotometric assay of extracted MbCO. The values given are for dried muscle. Approximate wet weight values were estimated with the assumption that the muscle is 70% water.19Blessing M.H. Comp. Biochem. Physiol. 1971; 41A: 475-480Google Scholar1-a Calculated on the basis of O2 capacity with 1.32 cm3 O2/g Mb, assuming a molecular weight of ∼17,000.1-b Spectrophotometric assay of extracted MbCO. The values given are for dried muscle. Approximate wet weight values were estimated with the assumption that the muscle is 70% water. Open table in a new tab Southern hybridization with cDNA for human Mb gave a small but clear signal with tissue of the small intestine and uterus, a very weak signal from the colon, and none for the bladder, whereas the rectal smooth muscle gave a very strong signal. Several possible explanations for the large differences in mRNA may be suggested. Mb might be expressed only in special, as yet unspecified, metabolic circumstances. Alternatively, a small subset of cells in the rectal smooth muscle tissue might have a substantial content of Mb, but the analysis of a relatively large mass of tissue would provide only a diluted estimate of the Mb. The latter possibility is enhanced by studies of another tissue, the esophageal sphincter of the opossum, which has been extensively used as a model for the esophageal sphincter in man (21Christensen J. Freeman B.W. Miller J.K. Gastroenterology. 1973; 64: 1119-1125Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 22Christensen J. Proc. Soc. Exp. Biol. Med. 1982; 170: 194-202Crossref PubMed Scopus (15) Google Scholar, 23Christensen J. Conklin J.L. Freeman B.W. Am. J. Physiol. 1973; 225: 1265-1270Crossref PubMed Scopus (68) Google Scholar, 24Percy W.H. Sutherland J. Christensen J. Dig. Dis. Sci. 1991; 36: 1057-1065Crossref PubMed Scopus (2) Google Scholar). This sphincter may be similar to the rectal smooth muscle sphincter of man. The esophageal sphincter tissue of the opossum has several properties that distinguish it from the smooth muscle of the nearby esophageal body: 1) The rate of oxygen consumption of the sphincter tissue is higher than the esophageal body (25Schulze-Delrieu K. Crane S.A. Am. J. Physiol. 1982; 242: G258-G262PubMed Google Scholar). 2) The tonic contraction of the sphincter is entirely aerobic and cannot be maintained anaerobically. In contrast, the esophageal body contractions can be partially maintained anaerobically (22Christensen J. Proc. Soc. Exp. Biol. Med. 1982; 170: 194-202Crossref PubMed Scopus (15) Google Scholar). 3) The apparent mitochondrial profile area is larger in the sphincter cells than in the esophageal body (26Christensen J. Roberts R.L. Gastroenterology. 1983; 85: 650-656Abstract Full Text PDF PubMed Scopus (18) Google Scholar). 4) Lactic dehydrogenase type I isozyme is present in the sphincter but not in the esophageal body (27Prasad R. Mukhopadhyay A. Prasad N. Experientia. 1978; 34: 484-485Crossref PubMed Scopus (8) Google Scholar). These observations show that the aerobic metabolism of the sphincter tissue is unique. Under aerobic conditions the muscle is tonically contracted and is only briefly relaxed to allow swallowing. The aerobic demand would make the presence of Mb advantageous. No similar metabolic studies of rectal tissue appear to have been made. The presence of mRNA for Mb in small intestine but not colon may be correlated with the different physiological functions of the tissues. Perhaps even a small amount of Mb may be advantageous in small intestine tissue but not colon. Peristaltic waves in the small intestine move at 1–2 cm/s over long periods of time, and the villi, present at a density of 10–40/mm2, have smooth muscle and contract independently almost continuously every 10 s (28Bloom W. Fawcett D.W. Textbook of Histology. 9th Ed. W. B. Saunders, Philadelphia, PA1968: 574Google Scholar). In contrast, the colon is devoid of villi, and muscular activity is infrequent. Our finding of some Mb in smooth muscle of arteries and veins suggests that it may play a role in limiting the time of vasodilation induced by nitric oxide delivered by S-nitrosohemoglobin (11Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1464) Google Scholar). 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Acad. Sci. U. S. A. 1994; 91: 8137-8141Crossref PubMed Scopus (622) Google Scholar) has suggested that the action of NO may be restricted by Mb to localized areas of heart and skeletal muscle, a newly identified function for Mb. Similar localization may also occur in smooth muscle tissue. Mb should protect against the toxic effects of high NO production associated with tissue damage by accelerating its destruction. The proposed role of Mb in NO metabolism requires that the MetMb product be recycled. Ascorbate-mediated redox cycling of Mb has been shown to be capable of serving this function (34Galaris D. Cadenas E. Hochstein P. Arch. Biochem. Biophys. 1989; 273: 497-504Crossref PubMed Scopus (104) Google Scholar). Although the ascorbate content of human smooth muscle is not known, it is accumulated in many tissues and can reach concentrations of 300–800 μm in heart muscle and ∼200 μm in skeletal muscle (35Hornig D. Ann. N. Y. Acad. Sci. 1975; 258: 103-118Crossref PubMed Scopus (375) Google Scholar). We are grateful to surgeons John S. Mangione, Michael Breen, and Gladstone McDowell for providing the tissue samples and Wesley Thompson for valuable discussions. We thank James Christensen for valuable discussions regarding the opossum. We thank Thomas L. Vandergon for DNA from Cerebratulus lacteus. We thank Claire Riggs for critical reading.