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A NOVEL MYOSIN HEAVY CHAIN ISOFORM IN VASCULAR SMOOTH MUSCLE1 Yoko Okai-Matsuo, Hiromi Takano-Ohmuro*,Teruhiko Toyo-oka2, andTsuneaki Sugimoto The SecondDepartmentof Internal Medicine, Health Service Center,University of Tokyo and *Tokyo Metropolitan Institute of Medical Science,Tokyo, Japan Received
October
8,
1990
SuMMARY:Previasstuligd twomyosinheavychaini5&rmsinv~smo&h mmdeswith Sv gel-; MHCl (204kDa) and MHCz (2OOkDa). Wereport the existenceof a novel myosinheavy chain isoform,MI-K, (l%kDa), which was exdusivelyantainedinh&riorvenaca~ EqualamoontofMHClandMHC~wasobsewedin aortaandpnkmnaq artery, respectively.However,inferior venacavacontainedonly MHC3 Pro&Ayticart&twas&utedbyimmunobUiqoftissue~ witboutpllledqor SDS-polyacrylamide gel electrophoresisof myosin bandsisolatedby pyrophosphate gel elecbophoresir FmWmmre, c+chymotryptic deavagedMHCl, MHC2, and MHC3 displayed curk!mltpeplidenrmps,~tbeprinmrysbrududdifferenceaImngalltbree~ B 1991 Academic
Press,
Inc.
Multiple forms of myosin exist in skeletaland cardiac muscle,and their characteristics have beenclosely documented(1, 2). Recently, isoformsof myosin heavy chain (MHC) have beenidentified in smoothmuscleon sodiumdodecyl sulfate (SDS)-polyacrylamide gels. Several studies have determined two MHC isoforms in various smooth muscle tissues, and showed variation in the relative amount of these two isoforms among different species,organsor developmentalstages(3-6). Vascular smoothmuscle(VSM) showsa functional diversity. which is important for the regulation of the circulatory system(7). Moreover. different vesselsreceive varied pressure/volumeload and innervation. Thesefacts raisea questionasto the uniformity of lA part of this study was financially supportedby the Ministries of Education, Science and Culture, and of Health and Welfare, Japan; Ca signal Workshop in Cardiovascular Systems; Sankyo Life Science Foundation and Research Foundation for Clinical Pharmacology:UeharaMemorial Foundation. An abstractof this paperwas presentedin the 62nd Scientific Sessionof American Heart Association Meeting in New Orleans, on Nov. 15, 1989. 2Address correspondenceto T. Toyo-oka, M.D., The Second Department of Internal Medicine, Tokyo University Hospital. University of Tokyo, Hongo 7-3-l. Bunkyo-ku, Tokyo 113. Japan. Abbreviations: anti-SMM, anti-smooth muscle myosin antibodies: CBB. Coomassie Brilliant Blue R250; ELISA, enzyme-linked immunosorbentassay; IVC, inferior vena cava; MHC, myosin heavy chain; PAGE, polyacrylamide gel electrophoresis: PPi. pyrophosphate; RLC. regulatory light chain; SDS, sodium dodecyl sulfate: VSM, vascular smoothmuscle.
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MHC isoform composition in various vessels. Previous investigations have resulted in determination of two MHC isoforms in VSM (3. 5, 8, 9). which was often solely represented by arteries. The purpose of the present study is to compare MHC isoforms among various vessels, especially between artery and vein, which have distinct difference in their physiological characteristics.
MATERIALS
AND METHODS
[II Materials Swine intrathoracic organs were freshly obtained from local slaughter house (n=6) and kept below 4 “C during all isolation procedures. Aorta, pulmonary artery. and inferior vena cava (IVC) were excised, cleaned in phosphate buffered saline and extensively removed of their adventitial and intimal layer. Purified myosins and whole tissue extracts were obtained from each tissue. (i) Purification of Myosins Myosins from each vessel were prepared by a slightly modified method ( 10) of Ebashi (11). in the presence of 10 pM leupeptin (12). 1 pM pepstatin A, 1 pM phenylmethylsulfonylfluoride, 1 mM ethylene glycol-bis (B-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA), and were subjected to SDS-polyacrylamide gel electrophoresis (PAGE; Ref. 13) as described below. (ii) Whole Tissue Extraction Whole tissue extracts of each vessel were obtained by crushing the media, freshly frozen in liquid nitrogen, and homogenizing them in three volumes of a buffer containing 7 % SDS. 3 % 2-mercaptoethanol, 0.1 M Tris-Cl (pH 6.8). 1 mM EGTA, and SO % glycerol. The homogenates were immediately boiled for 3 minutes, centrifuged at 10,O g for 15 minutes, and the supematants were subjected to SDS-PAGE. [II] Analysis of Myosin Heavy Chain Isoforms (i) SDS-polyacrylamide Gel Electrophoresis Purified myosins and whole tissue extracts were subjected to SDS-PAGE. 3.8 % acrylamide gels were employed in a buffer system according to Laemmli ( 13) to separate MHCs. (ii) Densitometry The SDS gels were stained with Coomassie Brilliant Blue R250 (CBB) and the bands of purified myosins were scanned with a densitometer (Cliniscan 2, Helena Laboratories). MHC band ratios were quantitated by averaging the area under the peaks in triplicates. (iii) Immunoblotting The SDS gels were subjected to immunoblotting. The gels were electroblotted onto nitrocellulose sheets in a buffer containing 25 mM Tris,192 mM glycine, 0.1 % SDS, and 20 % methanol for 4.5 hours at 8 V/cm. Each lane of the transblotted sheets were cut into two strips. one for protein staining with Amido Black and the other for immunoblotting (14) with anti-smooth muscle myosin antibodies (anti-SMM), which were produced in rabbits against bovine uterus myosin (Biomedical Technologies). The cross-reactivity of the rabbit antibodies to myosins from swine tissues were determined by enzyme-linked immunosorbent assay (ELISA; Ref. 15). The rabbit antibodies showed strong cross-reactivity with VSM myosins of swine aorta, pulmonary artery, and WC. (iv) Pyrophosphate-polyawylamide Gel Electrophoresis Myosins were subjected to pyrophosphate (PPi)-PAGE according to the method of Hoh et al. ( 16) with minor modifications (10). Each native myosin band obtained from different vessels were subsequently electrophoresed on 3.8 % SDS-polyacrylamide gels for analyzing MHC isoforms, as described previously (18) and stained with CBB. (v) Peptide Mapping Peptide mapping of fragments produced by a-chymotryptic digestion of each MHC isoform in aorta, pulmonary artery. and IVC was performed on a 12.5 % SDS slab gel with 4 % stacking gel. according to a minor modification ( 10) of Cleveland’s method (17). 1366
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RESULTS SDS-PAGE of purified myosin from aorta resolved two closely spaced protein bands which were assumed to be MHCs on 3.8 % gels (Fig. 1). The molecular mass of the slower
migrating band designated MHCt and the faster band designated MHC2 was
estimated at 204 kDa and 200 kDa, respectively. These isoforms have been described previously (3. 5). A similar result was obtained from pulmonary artery; the two MHC bands comigrated with aorta MHCs (Fig. 1). A significant difference was observed in IVC myosin on the SDS gels (Fig. 1). A single band of 196 kDa. designated MHCs, was recognized by CBB staining at similar protein loadings. MHCt and MHC:! could not be detected. These results were consistent among all six swine.
MHC,.
MHC*,
and MHC,
were identified
as MHCs
by
immunoblotting as described below. A protein band observed at 250 kDa was assumed to be filamin. Densitometric quantitation of MHC bands on CBB stained gels revealed that the ratio of MHCt and MHCl pulmonary
artery
(49f7
was near 1 in both aorta (57f2 VS. 43+2 %) and
vs. Slf7
%). In contrast,
MHC in IVC was occupied
exclusively by MHC3.
SDS-PAGE of whole tissue extracts resolved multiple protein bands (Fig. 2). which were electrophoretically blotted onto nitrocellulose sheets. An efficient transfer was verified by Amido Black staining of the sheets (Fig. 2) and CBB staining of the original gels. Immunoblotting of these sheets with anti-SMM identified two protein bands equivalent to MHC1 and MHC2 as MHCs in aorta and pulmonary
artery.
In IVC, a
single protein band equivalent to MHC3 could be detected as MHC (Fig. 2). These observations were consistent with the results obtained in purified myosins, and is a supportive evidence against the argument that the former observations were produced by proteolytic artifact during the purification procedures. P
A
F-
-250K
01
A
MHCI- *a, MCI'
-204K 7200K 196K
e
P
v
$‘ !
02
s
a b c
d e f
Figure SDS-PAGE patternsof swinevascularsmoothmuscle myosin on 3.8 8 gels. A. aorta: P. pulmonary artery; V. inferior vena cava: S, rabbit skeletal muscle: F, filamin: MHCt. MHC2. and MHCs, myosin heavy chain isoforms. Fieure 2. Combinations of SDS-PAGE pattern. electroblot, and immunoblot of whole tissue homogenates of aorta (A). pulmonary artery (P), and inferior vena cava (V). (a, d. g). 3.8 % SDS gels stained with CBB: (b, e, h). electroblots of 3.8 % gels stained with Amido Black: (c. f, i). immunoblots reacted with anti-SMM: MHC,. MHC,. and MHC,. myosin heavy chain isoforms. 1367
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A single myosin band of equal mobility was observed in aorta and IVC on PPi gels (Fig. 3a). In contrast. pulmonary artery myosin migrated into three bands, the slowest of which comigrated with the myosin bands in aorta and IVC (Fig. 3a). The myosin bands obtained from PPi-PAGE of aorta, pulmonary artery, and IVC were subjected to further analysis of their subunits with 3.8 % SDS-PAGE. The single myosin band of aorta, excised from PPi gel and run on a SDS gel, yielded two closely spaced MHC bands equivalent to MHCt and MHC;! (Fig. 3b). In pulmonary artery, each of the three myosin bands in PPi gel migrated into two heavy chain bands equivalent to MHC, and MHC2 (Fig. 3b). In contrast, the native myosin band obtained from PPiPAGE of IVC migrated into a single MHC band equivalent to MHC, on SDS gel (Fig. 3b). Peptide Mapping
of Heavy Chain Isoforms
The two MHC isoforms in aorta, pulmonary artery, and the single MHC isoform in WC separated with SDS-PAGE were subjected to peptide mapping according to Cleveland’s method. Fragments produced by a-chymotryptic
digestion were run on a
12.5 % SDS gel. The peptide maps of the two MHC isoforms in aorta showed large similarity but several differences were observed (Fig. 4). This observation confirms a former investigation (8). The two MHC isoforms in pulmonary artery produced same results as in aorta. Identical peptide maps were observed in MHCt of aorta and pulmonary artery. This observation also applied in case of MHC,. The peptide map
Figure
3,
a: PPi-PAGE of myosins in swine vascular smooth muscles. A, aorta; P,
pulmonaryartery: V, inferiorvenacava. A singlebandis observed in aortaandinferior vena cava. Myosin in pulmonary artery was resolved into three bands (Pt, 2, 3). b: SDS-PAGE of native myosin bands. Each myosin band was excised from the PPi gel
andsubjectedto 3.8 % SDS-PAGE. Note that eachlanecontainstwo MHC bandsin aortaandpulmonaryartery,but only onebandis observedin inferiorvenacava. MHCt, MHCz.andMHCs, myosin heavy chain isoforms. FiPure +, Peptide maps of each myosin heavy chain isoforms in swine aorta, pulmonary artery, and inferior vena cava. cx-chymotryptic cleavage products of aorta MHCI (a), aorta MHC2 (b), pulmonary artery MHC, (c), pulmonary artery MHC, (d), and inferior vena cava MHCs (e) were separated on a 12.5 % SDS gel. (a) and (c) show identical peptide maps. (b) and (d) are also identical. Single arrowheads point to major difference peptides between a and b. c and d. Double arrowheads indicate peptide fragments specific to (e). MHCt, MHC2. and MHCs differ in their peptide map patterns. The electrophoretic band corresponding to a-chymotrypsin is indicated by an asterisk.
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pattern of the single MHC isoform
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in IVC, MHCs, was quite different
COMMUNICATIONS
from either
MHCt or MHC2 (Fig. 4). Thus, MHC, differed from either of the previously identified isoforms, MHC, or MHC,, in its peptide composition.
DISCUSSION Previous studies (3-6) have determined two MHC isoforms in various smooth muscle tissues. Present observations revealed the presence of a novel MHC isoform in VSM. Moreover, different vessels were composed of entirely different MHC isoforms. Swine aorta and pulmonary artery contained equal amount of two MHC isoforms equivalent to 204 kDa and 200 kDa (MHCi
and MHCz), which were consistent with former reports
(3, 5, 8, 9). A distinctive feature was demonstrated in IVC. which exclusively contained MHC, isoform equivalent to 196 kDa. The possibility
that MHC2 and MHCs were produced by proteolytic degradation of
MHC, could be refuted by the following four reasons. (i) Myosin purification was performed in the presence of protease inhibitors and at below 4 “C. Samples prepared without the inhibitors denied its influence on isoform stoichiometry (data not shown). Direct homogenization of fresh tissues in SDS sample buffer without purification
(ii) step,
and the subsequent immunoblotting did not alter the results obtained in purified myosins. (iii) Each native myosin band observed in PPi-PAGE of purified aorta and pulmonary artery myosins yielded two heavy chain bands, MHCi and MHCZ on SDS gels. The native myosin band of IVC had the same mobility
as aorta on PPi gels. and yielded
MHC, on SDS gel. (iv) The three isoforms proved to be different polypeptides by the peptide mapping. These observations confirmed the presence of at least three MHC isoforms in VSMs differing in their primary structure. PPi-PAGE of pulmonary artery resolved three myosin bands, as opposed to aorta and IVC, which showed a single band. However, the heavy chain and light chain (data not shown) composition This demonstrates
of each native myosin band in pulmonary artery were identical. that the difference
in mobility
on PPi gel was not produced by
heterogeneity of the heavy chains or light chains but by post-translational modifications. We have previously reported that the myosins alter their electrophoretic mobility on PPi gels according to difference in phosphorylated degree of their regulatory light chain (RLC; Ref. 18, 19). The slowest, the intermediate, and the fastest migrating band corresponded to myosins with 2 unphosphorylated RLC, 1 unphosphorylated and 1 monophosphorylated RLC, and 2 mono-phosphorylated RLC, respectively. Our present observations could be caused by various phosphorylated states among different vessels. The peptide maps revealed that MHC, was a different polypeptide from either of the previously identified MHC isoforms, MHC i or MHC,. This extends the observations by Eddinger et al., who have shown MHCi and MHCz to differ in their peptide compositions in swine stomach muscle (8). Their studies have been supported by a nucleotide sequence study. which has suggested the difference between MHCi and MHC2 to lie in the carboxy terminus of MHC in rabbit uterus (20). The peptide map of 1369
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MHC, was also distinguishable from nonmuscle MHC, which has been shown to exist in several smooth muscle tissues (Ref. 21, data not shown). Different regions of VSM has its specific physiological characteristics. IVC has distinctive functional roles, regulating the preload of the heart. and receives markedly low pressure load compared to aorta or pulmonary artery. Experimental studies which have displayed force-velocity
constants in various VSMs revealed a significant
difference
between arteries (22) and veins (7). including the maximum shortening velocity. Our study has demonstrated that IVC contained an entirely different myosin isoform from aorta or pulmonary artery. This may produce difference in contractile functions, such as mentioned above. Meanwhile. pressure load has been suggested to alter myosin isoform composition in cardiac muscle (23). but its effect on VSM is unknown. The functional characteristics of MHC isoforms based on structural variations, and factors which regulate their expression awaits to be elucidated.
REFERENCES 1. Hoh, J.F.Y. and Yeoh. G.P.S. (1979) Nature 280. 321-323 2. Hoh, J.F.Y., McGrath. P.A. and Hale, P.T. ( 1978) J. Mol. Cell. Cardiof. 10, 1053-1076 3. Rovner, A.S., Thompson, M.M. and Murphy. R.A. ( 1986) Am. J. Physiol. 250 (Cell Physiol. 19). C861-C870 4. Cavaille, F., Janmot, C., Ropert, S. and d’Albis, A. ( 1986) Eur. J. Biochem. 160, 507-5 13 5. Kawamoto, S. and Adelstein, R.S. (1987) J. Biol, Chem. 262, 7282-7288 6. Mohammad, M.A. and Sparrow, M.P. (1988) FEBS Let?. 228, 109-l 12 7. Hellstrand. P. and Paul, R.J. (1982) Vascular smooth muscle: Metabolic, ionic, and contractile mechanisms. ~~1-35, Academic Press. New York 8. Eddinger, T.J. and Murphy, R.A. (1988) Biochemistry 27, 3807-3811 9. Sparrow. M.P., Mohammad, M.A., Amer. A.. Hellstrand, P. and Ruegg, J.C. (1988) Pjluegers Arch. 412, 624-633 10. Takano-Ohmuro. H., Obinata, T., Mikawa. T. and Masaki, T. (1983) J. Biochem. 93, 903-908 11. Ebashi. S. (1976) J. Biochem. 79, 229-231 12. Toyo-oka. T., Shimizu. T. and Masaki. T. (1978) Biochem. Biophys. Res. Commun. 82, 484-491 13. Laemmli, U.K. (1970) Nature 227, 680-685 14. Towbin. H.. Staehelin, T. and Gordon, J. (1979) Proc. Nat/. Acad. Sci. USA 76, 4350-4354 15. Voller, A.. Bidwell, D.E.. and Bartlett, A. (1979) The enzyme linked immunosorbent assay (ELISA). A guide with abstracts of microplate application. Dynatech Laboratories Inc., VA 16. Hoh, J.F.Y., McGrath, P.A. and White, RI. (1976) Biochem. J. 157, 87-95 17. Cleveland. D.W., Fischer. S.G., Kirschner, M.W. and Laemmli, U.K. (1977) J. Biol. Chem. 252, 1102-1106 18. Takano-Ohmuro, H. and Kohama, K. (1986) J. Biochem. 100, 259-268 19. Takano-Ohmuro, H. and Kohama, K. (1986) J. Biochem. 100, 1681-1684 20. Nagai, R., Kuro-0, M., Babij, P. andPeriasamy.M. (1989) J. Biol. Chem.264, 9734-9737 21. Gaylinn, B.D.. Eddinger, T.J., Martino, P.A., Monical, P.L.. Hunt, D.F. and Murphy, R.A. (1989) Am. J. Physiol. 257 (Cell Physiol. 26). C997-Cl004 22. Herlihy. J.T. and Murphy. R.A. (1974) Circ. Res. 34. 461-466 23. Litten, R.Z.. Martin, B.J., Low, R.B. andAlpert, N.R. (1982) Circ. Res.50, 856-864
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A NOVEL MYOSIN HEAVY CHAIN ISOFORM IN VASCULAR SMOOTH MUSCLE1 Yoko Okai-Matsuo, Hiromi Takano-Ohmuro*,Teruhiko Toyo-oka2, andTsuneaki Sugimoto The SecondDepartmentof Internal Medicine, Health Service Center,University of Tokyo and *Tokyo Metropolitan Institute of Medical Science,Tokyo, Japan Received
October
8,
1990
SuMMARY:Previasstuligd twomyosinheavychaini5&rmsinv~smo&h mmdeswith Sv gel-; MHCl (204kDa) and MHCz (2OOkDa). Wereport the existenceof a novel myosinheavy chain isoform,MI-K, (l%kDa), which was exdusivelyantainedinh&riorvenaca~ EqualamoontofMHClandMHC~wasobsewedin aortaandpnkmnaq artery, respectively.However,inferior venacavacontainedonly MHC3 Pro&Ayticart&twas&utedbyimmunobUiqoftissue~ witboutpllledqor SDS-polyacrylamide gel electrophoresisof myosin bandsisolatedby pyrophosphate gel elecbophoresir FmWmmre, c+chymotryptic deavagedMHCl, MHC2, and MHC3 displayed curk!mltpeplidenrmps,~tbeprinmrysbrududdifferenceaImngalltbree~ B 1991 Academic
Press,
Inc.
Multiple forms of myosin exist in skeletaland cardiac muscle,and their characteristics have beenclosely documented(1, 2). Recently, isoformsof myosin heavy chain (MHC) have beenidentified in smoothmuscleon sodiumdodecyl sulfate (SDS)-polyacrylamide gels. Several studies have determined two MHC isoforms in various smooth muscle tissues, and showed variation in the relative amount of these two isoforms among different species,organsor developmentalstages(3-6). Vascular smoothmuscle(VSM) showsa functional diversity. which is important for the regulation of the circulatory system(7). Moreover. different vesselsreceive varied pressure/volumeload and innervation. Thesefacts raisea questionasto the uniformity of lA part of this study was financially supportedby the Ministries of Education, Science and Culture, and of Health and Welfare, Japan; Ca signal Workshop in Cardiovascular Systems; Sankyo Life Science Foundation and Research Foundation for Clinical Pharmacology:UeharaMemorial Foundation. An abstractof this paperwas presentedin the 62nd Scientific Sessionof American Heart Association Meeting in New Orleans, on Nov. 15, 1989. 2Address correspondenceto T. Toyo-oka, M.D., The Second Department of Internal Medicine, Tokyo University Hospital. University of Tokyo, Hongo 7-3-l. Bunkyo-ku, Tokyo 113. Japan. Abbreviations: anti-SMM, anti-smooth muscle myosin antibodies: CBB. Coomassie Brilliant Blue R250; ELISA, enzyme-linked immunosorbentassay; IVC, inferior vena cava; MHC, myosin heavy chain; PAGE, polyacrylamide gel electrophoresis: PPi. pyrophosphate; RLC. regulatory light chain; SDS, sodium dodecyl sulfate: VSM, vascular smoothmuscle.
1365
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MHC isoform composition in various vessels. Previous investigations have resulted in determination of two MHC isoforms in VSM (3. 5, 8, 9). which was often solely represented by arteries. The purpose of the present study is to compare MHC isoforms among various vessels, especially between artery and vein, which have distinct difference in their physiological characteristics.
MATERIALS
AND METHODS
[II Materials Swine intrathoracic organs were freshly obtained from local slaughter house (n=6) and kept below 4 “C during all isolation procedures. Aorta, pulmonary artery. and inferior vena cava (IVC) were excised, cleaned in phosphate buffered saline and extensively removed of their adventitial and intimal layer. Purified myosins and whole tissue extracts were obtained from each tissue. (i) Purification of Myosins Myosins from each vessel were prepared by a slightly modified method ( 10) of Ebashi (11). in the presence of 10 pM leupeptin (12). 1 pM pepstatin A, 1 pM phenylmethylsulfonylfluoride, 1 mM ethylene glycol-bis (B-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA), and were subjected to SDS-polyacrylamide gel electrophoresis (PAGE; Ref. 13) as described below. (ii) Whole Tissue Extraction Whole tissue extracts of each vessel were obtained by crushing the media, freshly frozen in liquid nitrogen, and homogenizing them in three volumes of a buffer containing 7 % SDS. 3 % 2-mercaptoethanol, 0.1 M Tris-Cl (pH 6.8). 1 mM EGTA, and SO % glycerol. The homogenates were immediately boiled for 3 minutes, centrifuged at 10,O g for 15 minutes, and the supematants were subjected to SDS-PAGE. [II] Analysis of Myosin Heavy Chain Isoforms (i) SDS-polyacrylamide Gel Electrophoresis Purified myosins and whole tissue extracts were subjected to SDS-PAGE. 3.8 % acrylamide gels were employed in a buffer system according to Laemmli ( 13) to separate MHCs. (ii) Densitometry The SDS gels were stained with Coomassie Brilliant Blue R250 (CBB) and the bands of purified myosins were scanned with a densitometer (Cliniscan 2, Helena Laboratories). MHC band ratios were quantitated by averaging the area under the peaks in triplicates. (iii) Immunoblotting The SDS gels were subjected to immunoblotting. The gels were electroblotted onto nitrocellulose sheets in a buffer containing 25 mM Tris,192 mM glycine, 0.1 % SDS, and 20 % methanol for 4.5 hours at 8 V/cm. Each lane of the transblotted sheets were cut into two strips. one for protein staining with Amido Black and the other for immunoblotting (14) with anti-smooth muscle myosin antibodies (anti-SMM), which were produced in rabbits against bovine uterus myosin (Biomedical Technologies). The cross-reactivity of the rabbit antibodies to myosins from swine tissues were determined by enzyme-linked immunosorbent assay (ELISA; Ref. 15). The rabbit antibodies showed strong cross-reactivity with VSM myosins of swine aorta, pulmonary artery, and WC. (iv) Pyrophosphate-polyawylamide Gel Electrophoresis Myosins were subjected to pyrophosphate (PPi)-PAGE according to the method of Hoh et al. ( 16) with minor modifications (10). Each native myosin band obtained from different vessels were subsequently electrophoresed on 3.8 % SDS-polyacrylamide gels for analyzing MHC isoforms, as described previously (18) and stained with CBB. (v) Peptide Mapping Peptide mapping of fragments produced by a-chymotryptic digestion of each MHC isoform in aorta, pulmonary artery. and IVC was performed on a 12.5 % SDS slab gel with 4 % stacking gel. according to a minor modification ( 10) of Cleveland’s method (17). 1366
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RESULTS SDS-PAGE of purified myosin from aorta resolved two closely spaced protein bands which were assumed to be MHCs on 3.8 % gels (Fig. 1). The molecular mass of the slower
migrating band designated MHCt and the faster band designated MHC2 was
estimated at 204 kDa and 200 kDa, respectively. These isoforms have been described previously (3. 5). A similar result was obtained from pulmonary artery; the two MHC bands comigrated with aorta MHCs (Fig. 1). A significant difference was observed in IVC myosin on the SDS gels (Fig. 1). A single band of 196 kDa. designated MHCs, was recognized by CBB staining at similar protein loadings. MHCt and MHC:! could not be detected. These results were consistent among all six swine.
MHC,.
MHC*,
and MHC,
were identified
as MHCs
by
immunoblotting as described below. A protein band observed at 250 kDa was assumed to be filamin. Densitometric quantitation of MHC bands on CBB stained gels revealed that the ratio of MHCt and MHCl pulmonary
artery
(49f7
was near 1 in both aorta (57f2 VS. 43+2 %) and
vs. Slf7
%). In contrast,
MHC in IVC was occupied
exclusively by MHC3.
SDS-PAGE of whole tissue extracts resolved multiple protein bands (Fig. 2). which were electrophoretically blotted onto nitrocellulose sheets. An efficient transfer was verified by Amido Black staining of the sheets (Fig. 2) and CBB staining of the original gels. Immunoblotting of these sheets with anti-SMM identified two protein bands equivalent to MHC1 and MHC2 as MHCs in aorta and pulmonary
artery.
In IVC, a
single protein band equivalent to MHC3 could be detected as MHC (Fig. 2). These observations were consistent with the results obtained in purified myosins, and is a supportive evidence against the argument that the former observations were produced by proteolytic artifact during the purification procedures. P
A
F-
-250K
01
A
MHCI- *a, MCI'
-204K 7200K 196K
e
P
v
$‘ !
02
s
a b c
d e f
Figure SDS-PAGE patternsof swinevascularsmoothmuscle myosin on 3.8 8 gels. A. aorta: P. pulmonary artery; V. inferior vena cava: S, rabbit skeletal muscle: F, filamin: MHCt. MHC2. and MHCs, myosin heavy chain isoforms. Fieure 2. Combinations of SDS-PAGE pattern. electroblot, and immunoblot of whole tissue homogenates of aorta (A). pulmonary artery (P), and inferior vena cava (V). (a, d. g). 3.8 % SDS gels stained with CBB: (b, e, h). electroblots of 3.8 % gels stained with Amido Black: (c. f, i). immunoblots reacted with anti-SMM: MHC,. MHC,. and MHC,. myosin heavy chain isoforms. 1367
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A single myosin band of equal mobility was observed in aorta and IVC on PPi gels (Fig. 3a). In contrast. pulmonary artery myosin migrated into three bands, the slowest of which comigrated with the myosin bands in aorta and IVC (Fig. 3a). The myosin bands obtained from PPi-PAGE of aorta, pulmonary artery, and IVC were subjected to further analysis of their subunits with 3.8 % SDS-PAGE. The single myosin band of aorta, excised from PPi gel and run on a SDS gel, yielded two closely spaced MHC bands equivalent to MHCt and MHC;! (Fig. 3b). In pulmonary artery, each of the three myosin bands in PPi gel migrated into two heavy chain bands equivalent to MHC, and MHC2 (Fig. 3b). In contrast, the native myosin band obtained from PPiPAGE of IVC migrated into a single MHC band equivalent to MHC, on SDS gel (Fig. 3b). Peptide Mapping
of Heavy Chain Isoforms
The two MHC isoforms in aorta, pulmonary artery, and the single MHC isoform in WC separated with SDS-PAGE were subjected to peptide mapping according to Cleveland’s method. Fragments produced by a-chymotryptic
digestion were run on a
12.5 % SDS gel. The peptide maps of the two MHC isoforms in aorta showed large similarity but several differences were observed (Fig. 4). This observation confirms a former investigation (8). The two MHC isoforms in pulmonary artery produced same results as in aorta. Identical peptide maps were observed in MHCt of aorta and pulmonary artery. This observation also applied in case of MHC,. The peptide map
Figure
3,
a: PPi-PAGE of myosins in swine vascular smooth muscles. A, aorta; P,
pulmonaryartery: V, inferiorvenacava. A singlebandis observed in aortaandinferior vena cava. Myosin in pulmonary artery was resolved into three bands (Pt, 2, 3). b: SDS-PAGE of native myosin bands. Each myosin band was excised from the PPi gel
andsubjectedto 3.8 % SDS-PAGE. Note that eachlanecontainstwo MHC bandsin aortaandpulmonaryartery,but only onebandis observedin inferiorvenacava. MHCt, MHCz.andMHCs, myosin heavy chain isoforms. FiPure +, Peptide maps of each myosin heavy chain isoforms in swine aorta, pulmonary artery, and inferior vena cava. cx-chymotryptic cleavage products of aorta MHCI (a), aorta MHC2 (b), pulmonary artery MHC, (c), pulmonary artery MHC, (d), and inferior vena cava MHCs (e) were separated on a 12.5 % SDS gel. (a) and (c) show identical peptide maps. (b) and (d) are also identical. Single arrowheads point to major difference peptides between a and b. c and d. Double arrowheads indicate peptide fragments specific to (e). MHCt, MHC2. and MHCs differ in their peptide map patterns. The electrophoretic band corresponding to a-chymotrypsin is indicated by an asterisk.
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pattern of the single MHC isoform
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in IVC, MHCs, was quite different
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from either
MHCt or MHC2 (Fig. 4). Thus, MHC, differed from either of the previously identified isoforms, MHC, or MHC,, in its peptide composition.
DISCUSSION Previous studies (3-6) have determined two MHC isoforms in various smooth muscle tissues. Present observations revealed the presence of a novel MHC isoform in VSM. Moreover, different vessels were composed of entirely different MHC isoforms. Swine aorta and pulmonary artery contained equal amount of two MHC isoforms equivalent to 204 kDa and 200 kDa (MHCi
and MHCz), which were consistent with former reports
(3, 5, 8, 9). A distinctive feature was demonstrated in IVC. which exclusively contained MHC, isoform equivalent to 196 kDa. The possibility
that MHC2 and MHCs were produced by proteolytic degradation of
MHC, could be refuted by the following four reasons. (i) Myosin purification was performed in the presence of protease inhibitors and at below 4 “C. Samples prepared without the inhibitors denied its influence on isoform stoichiometry (data not shown). Direct homogenization of fresh tissues in SDS sample buffer without purification
(ii) step,
and the subsequent immunoblotting did not alter the results obtained in purified myosins. (iii) Each native myosin band observed in PPi-PAGE of purified aorta and pulmonary artery myosins yielded two heavy chain bands, MHCi and MHCZ on SDS gels. The native myosin band of IVC had the same mobility
as aorta on PPi gels. and yielded
MHC, on SDS gel. (iv) The three isoforms proved to be different polypeptides by the peptide mapping. These observations confirmed the presence of at least three MHC isoforms in VSMs differing in their primary structure. PPi-PAGE of pulmonary artery resolved three myosin bands, as opposed to aorta and IVC, which showed a single band. However, the heavy chain and light chain (data not shown) composition This demonstrates
of each native myosin band in pulmonary artery were identical. that the difference
in mobility
on PPi gel was not produced by
heterogeneity of the heavy chains or light chains but by post-translational modifications. We have previously reported that the myosins alter their electrophoretic mobility on PPi gels according to difference in phosphorylated degree of their regulatory light chain (RLC; Ref. 18, 19). The slowest, the intermediate, and the fastest migrating band corresponded to myosins with 2 unphosphorylated RLC, 1 unphosphorylated and 1 monophosphorylated RLC, and 2 mono-phosphorylated RLC, respectively. Our present observations could be caused by various phosphorylated states among different vessels. The peptide maps revealed that MHC, was a different polypeptide from either of the previously identified MHC isoforms, MHC i or MHC,. This extends the observations by Eddinger et al., who have shown MHCi and MHCz to differ in their peptide compositions in swine stomach muscle (8). Their studies have been supported by a nucleotide sequence study. which has suggested the difference between MHCi and MHC2 to lie in the carboxy terminus of MHC in rabbit uterus (20). The peptide map of 1369
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MHC, was also distinguishable from nonmuscle MHC, which has been shown to exist in several smooth muscle tissues (Ref. 21, data not shown). Different regions of VSM has its specific physiological characteristics. IVC has distinctive functional roles, regulating the preload of the heart. and receives markedly low pressure load compared to aorta or pulmonary artery. Experimental studies which have displayed force-velocity
constants in various VSMs revealed a significant
difference
between arteries (22) and veins (7). including the maximum shortening velocity. Our study has demonstrated that IVC contained an entirely different myosin isoform from aorta or pulmonary artery. This may produce difference in contractile functions, such as mentioned above. Meanwhile. pressure load has been suggested to alter myosin isoform composition in cardiac muscle (23). but its effect on VSM is unknown. The functional characteristics of MHC isoforms based on structural variations, and factors which regulate their expression awaits to be elucidated.
REFERENCES 1. Hoh, J.F.Y. and Yeoh. G.P.S. (1979) Nature 280. 321-323 2. Hoh, J.F.Y., McGrath. P.A. and Hale, P.T. ( 1978) J. Mol. Cell. Cardiof. 10, 1053-1076 3. Rovner, A.S., Thompson, M.M. and Murphy. R.A. ( 1986) Am. J. Physiol. 250 (Cell Physiol. 19). C861-C870 4. Cavaille, F., Janmot, C., Ropert, S. and d’Albis, A. ( 1986) Eur. J. Biochem. 160, 507-5 13 5. Kawamoto, S. and Adelstein, R.S. (1987) J. Biol, Chem. 262, 7282-7288 6. Mohammad, M.A. and Sparrow, M.P. (1988) FEBS Let?. 228, 109-l 12 7. Hellstrand. P. and Paul, R.J. (1982) Vascular smooth muscle: Metabolic, ionic, and contractile mechanisms. ~~1-35, Academic Press. New York 8. Eddinger, T.J. and Murphy, R.A. (1988) Biochemistry 27, 3807-3811 9. Sparrow. M.P., Mohammad, M.A., Amer. A.. Hellstrand, P. and Ruegg, J.C. (1988) Pjluegers Arch. 412, 624-633 10. Takano-Ohmuro. H., Obinata, T., Mikawa. T. and Masaki, T. (1983) J. Biochem. 93, 903-908 11. Ebashi. S. (1976) J. Biochem. 79, 229-231 12. Toyo-oka. T., Shimizu. T. and Masaki. T. (1978) Biochem. Biophys. Res. Commun. 82, 484-491 13. Laemmli, U.K. (1970) Nature 227, 680-685 14. Towbin. H.. Staehelin, T. and Gordon, J. (1979) Proc. Nat/. Acad. Sci. USA 76, 4350-4354 15. Voller, A.. Bidwell, D.E.. and Bartlett, A. (1979) The enzyme linked immunosorbent assay (ELISA). A guide with abstracts of microplate application. Dynatech Laboratories Inc., VA 16. Hoh, J.F.Y., McGrath, P.A. and White, RI. (1976) Biochem. J. 157, 87-95 17. Cleveland. D.W., Fischer. S.G., Kirschner, M.W. and Laemmli, U.K. (1977) J. Biol. Chem. 252, 1102-1106 18. Takano-Ohmuro, H. and Kohama, K. (1986) J. Biochem. 100, 259-268 19. Takano-Ohmuro, H. and Kohama, K. (1986) J. Biochem. 100, 1681-1684 20. Nagai, R., Kuro-0, M., Babij, P. andPeriasamy.M. (1989) J. Biol. Chem.264, 9734-9737 21. Gaylinn, B.D.. Eddinger, T.J., Martino, P.A., Monical, P.L.. Hunt, D.F. and Murphy, R.A. (1989) Am. J. Physiol. 257 (Cell Physiol. 26). C997-Cl004 22. Herlihy. J.T. and Murphy. R.A. (1974) Circ. Res. 34. 461-466 23. Litten, R.Z.. Martin, B.J., Low, R.B. andAlpert, N.R. (1982) Circ. Res.50, 856-864
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