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Nitric oxide synthase I (NOS I) is a costameric enzyme in rat skeletal muscle
Acta histochem. 100,451-462 (1998) © Gustav Fischer Verlag
Atla hisl•••ita
Nitric oxide synthase I (NOS I) is a costameric enzyme in rat skeletal muscle Reinhart Gossrau* Institute of Anatomy, Department of Molecular Anatomy and Cell Biology, University Clinic Benjamin Franklin, Free University of Berlin, Konigin-Luise-Str. 15, 0-14195 Berlin, Germany Accepted 20 August 1998
Summary Previously, we and others have reported association of nitric oxide (NO)generating nitric oxide synthase I (NOS I) with dystrophin in the subsarcolemmal cytoskeleton of striated muscle fibers. Since this structure shows a costameric organization the detailed distribution of NOS I and other molecules inside and outside the subsarcolemmal cytoskeleton was investigated. Using catalytic histochemistry and immunohistochemistry on rat skeletal muscle NOS I was colocalized in the costameres together with dystrophin, p-dystroglycan, a-, p- and y-sarcoglycan, p1-integrin, vinculin, paxillin and caveolin-3. Additionally, only NOS appeared in uncharacterized subsarcolemmal wave-like structures. These data show 1) a growing family of proteins assembled in the costameres including NOS I as described here for the first time, 2) expanded distribution patterns for NOS I, and 3) therefore, presumably uneven NO concentrations within the skeletal muscle fibers which may have implications for NO function. Key words: Nitric oxide synthase I - NOS I - costameres - skeletal muscle
Introduction Since our observation in 1992 of NADPH diaphorase localization in the sarcolemma region of muscle fibers (Gossrau et al., 1993) many groups including ours described NO-generating NOS I, to which this diaphorase activity is related, in myofibers of numerous mammalian and non-mammalian speCorrespondence to: R. Gossrau *Dedicated to Robert E. Smith, MD, the visionary of proteases and protease inhibitors
452
R. Gossrau
cies (for references, see Gossrau 1998). The enzyme is a component of the subsarcolemmal cytoskeleton and associated with the sarcolemma via the a1-synthrophin-dystrophin complex (for a recent review, see Reid 1998). Other investigations have shown that the subsarcolemmal cytoskeleton has a differential distribution and forms a lattice with the costameres as central ring- or rib-like structures encircling the myofibers perpendicularly to their longitudimil axis (for reviews, see Engel et aI., 1994; Watkins et aI., 1997). Therefore, in the present study the spatial relationship of NOS I to this lattice in combination with known or unknown lattice members was analyzed showing for the first time NOS I as a costameric protein and, in addition, this molecule in so far unknown subsarcolemmal structures in rat myofibers.
Materials and Methods Animals tissue, pretreatment. Adult Wistar rats (n =7) were used. The animals were of our own breeding stock, kept under standardized laboratory conditions, sacrificed in deep ether anaesthesia and tongue and neck muscles were rapidly excised. The muscles were frozen in liquid nitrogen, mounted on cryostat tables, cut at a thickness of 10 11m in a cryostat, mounted onto poly-L-Iysine-coated glass slides and used either unfixed after air-drying or fixed by immersion in 4% formaldehyde or 100% acetone for 10 min at 4°C.
Table 1. Dilution and source of the primary antibodies Antibodies
Dilution
Dystrophin-2 p- Dystroglycan a-Sarcoglycan
1:20 1:100 1:100
p-Sarcoglycan y-Sarcoglycan Spectrin-2 pI-Integrin Vinculin Paxillin
1:100 1 :100 1:100 1:200 1:400 1:1000
a-Actinin Caveolin-3 monoclonal Caveolin-3 polyclonal
1:200 1 :200 1:200
NOS I polyclonal NOS I (aa 1-181) NOS I (aa 251-270) NOS I (aa 1409-1429)
1:1000 1:300 1: 1000 1:100
Source
Novocastra Laboratories Newcastle upon Tyne, UK
C. Loster, Berlin, Germany Sigma, Munich, Germany Transduction Laboratories Lexington, KY, USA Sigma, Munich, Germany Transduction Laboratories Lexington, KY, USA B. Mayer, Graz, Austria Sigma, Munich, Germany
NOS I in costameres
453
Histochemistry. Single incubations. Immunohistochemistry. The details of the dilution and source of the antibodies are given in Table 1. For immunofluorescence tissue sections were rinsed in phosphate-buffered saline (PBS; pH 7,4) containing 0.3% Triton X-lOO (PBS-Triton), immersed in 2% normal horse serum to block non-specific staining (30 min), washed in PBS-Triton, and incubated with the following antibodies: NOS I polyclonal, NOS I (N-terminus, aa 1-181) monoclonal, NOS I (N-domain, aa 251-270) polyclonal, NOS I (C-terminus, aa 1409-1429) polyclonal, dystrophin-2 (C-terminus) monoclonal, p-dystroglycan monoclonal, a-sarcoglycan monoclonal, p-sarcoglycan monoclonal, y-sarcoglycan monoclonal, spectrin-2 monoclonal, caveolin-3 monoclonal and polyclonal, Pl-integrin polyclonal, vinculin monoclonal, paxillin monoclonal, and a-actinin monoclonal. The primary antibodies were diluted in PBS-Triton and applied to sections overnight at room temperature. After extensive washing, the sites of antigen-antibody reaction were visualized by incubation with the corresponding secondary Cy-3- or Cy-2-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA), diluted 1: 100, for 1 h at room temperature. All sections were then washed and covered in a mixture of PBS and glycerol (1: 1). In control incubations, carried out by replacing primary antibodies with either PBS or a non-relevant immune serum, no specific staining was visible. As all antibodies are well characterized for rat skeletal muscles, we did not perform Western blots or absorption tests as further controls. Enzyme histochemistry. The NADPH diaphorase activity of NOS I was demonstrated according to Scherer et al. (1993) with 3 or 4 M urea as inhibitor for NOS 1unrelated diaphorases (Gossrau, unpublished observations) in the incubation medium containing 1 mg/ml P-NADPH, 0.25 mg/ml nitro blue tetrazolium (NBT), and 0.3% Triton X-100 (v/v) in 0.1 M phosphate buffer, pH 7.4. Incubations were carried out at 37°C for 15-60 min. In control experiments, incubation media without substrate were used and yielded negative results. Double incubations. In order to investigate the relationship between NOS I and the other molecules with regard to their costameric distribution tissue sections were first labeled for NOS I diaphorase activity and then incubated with the antibodies against the other molecules followed by fluorescence visualization with the corresponding Cy-3-labeled secondary antibodies. Alternatively, tissue sections were first immunoreacted for NOS I with either polyclonal or monoclonal antibodies followed by visualization of the NOS I antigen-antibody complexes with Cy-3-conjugated secondary antibodies and were then mixed with the antibodies for the remaining molecules, which were detected with the corresponding Cy-2-labeled secondary antibodies. All sections were viewed using conventional microscopes.
Results In longitudinal sections outside the region of the subsarcolemmal cytoskeleton NOS I diaphorase activity as well as NOS I (Fig. 1), dystrophin-2 (Fig. 2), p-dystroglycan a-, p-, and y-sarcoglycan (Fig. 3), spectrin-2 (Fig. 4), p1-integrin (Fig. 5), vinculin, paxillin (Fig. 6), and caveolin-3 immunoreactivities were visualized as focal concentrations at those sites where the myofibers appeared to be relaxed. In more contracted regions this uneven distribution was less obvious or absent but was found to be more regularly distributed. Double incubation for NOS I diaphorase activity and the other molecules mentioned above revealed colocalization of NOS I-generated for-
454
R. Gossrau
Figs. 1-4. Immunofluorescence is seen as focal (dot-like) accummulation (arrows) in the region of the sarcolemma of longitudinal sections. Arrowhead = subsarcolemmal lattice. Fig. 1. NOS I. Fig. 2. Dystrophin. Fig. 3. y-Sarcoglycan. Fig. 4. Spectrin. x 360
NOS I in costameres
455
mazan and immunofluorescence at sites with low or medium activity. However, in focal concentrations, where the amount of formazan was high, immunofluorescence for all molecules was significantl'y weaker or absent. For reasons of spatial relationship double incubation for NOS I diaphorase and the Z band protein a-actinin was performed indicating colocalization of the enzyme and a-actinin.
Figs. 5,6. For localization details see Figs. 1-4. Fig. 5. p1-Integrin. Fig. 6. Paxillin. x 450
456
R. Gossrau
Figs. 7-10. NOS I diaphorase is present in costameres (arrowheads) and finer interconnections (intercostameres) forming a lattice in longitudinal sections through the region of the subsarcolemmal cytoskeleton. Figs. 7-9. Infrahyoid muscle. Fig. 10. Tongue. x 360
NOS I in costameres
457
Figs. 11-14. Immunofluorescence is found in the subsarcolemmal lattice (arrows). Fig. 11. Dystrophin. Fig. 12. p-Dystroglycan. Figs. 13, 14. p-Sarcoglycan. x 360
458
R. Gossrau
Figs. 15-18. For localization details see Figs. 7-10. Immunofluorescence is shown in the subsarcolemmal lattice (arrows, arrowhead) consisting of costameres and intercostameres. Fig. 15. y-Sarcoglycan. Fig. 16. Vinculin. Figs. 17, 18. Caveolin-3. x 360
NOS I in costameres
459
Figs. 19-22. NOS I. Figs. 19, 21, 22. NOS I diaphorase. Fig. 20. NOS I immunofluorescence. Figs. 19, 20. NOS I is found in wave-like structures (arrowheads, short arrows) of infrahyoid muscles. Figs. 21, 22. In hypercontracted myofibers of the tongue the transversal costameres appear as thick accumulations of formazan. Arrows = costameres. x 360
460
R. Gossrau
In longitudinal sections tangentially to the sarcolemma and through the region of the subsarcolemmal cytoskeleton, all molecules investigated were visualized in dense transversal bands and in finer interconnections (Figs. 718). Again, colocalization was observed for NOS I diaphorase and the immunohistochemical demonstration of the other molecules if the determinable diaphorase activity was low or medium. In contrast to the other molecules, NOS I (immunoreactivity and activity) was observed in longitudinal sections in single or double wave-like bands in the region of the subsarcolemmal cytoskeleton which occasionally crossed the whole myofiber diameter (Figs. 19, 20). These structures appeared to be connected with transversal bands positive for NOS I protein and enzyme (diaphorase) activity. Furthermore, at obviously hypercontracted sites the transversal bands were significantly thicker (Figs. 21, 22).
Discussion The data presented here reveal a differential distribution of NOS I protein and activity in the surface of myofibers of rat tongue and neck muscles. Because NOS I was colocalized with several known marker molecules for the costameric compartments such as dystrophin, Pl-integrin and vinculin (for reviews, see Engel et aI., 1994; Watkins et aI., 1997) this enzyme is a further member of the costameres. Colocalization of all molecules was also found in the finer interconnections (which may be termed intercostameres) forming a subsarcolemmal lattice together with the costameres. Furthermore, this work adds caveolin-3, paxillin as well as a-, fi- and y-sarcoglycan to the family of proteins of the subsarcolemmal lattice whose most prominent structures are the costameres. Because these costameric molecules are also colocalized with a-actinin they are mainly expressed in the region of those Z discs which contact the sarcolemma region (peripheral or subsarcolemmal Z discs) but not in the remaining (central) Z discs. The presence of the sarcoglycans in the lattice suggests the possibility that they are not exclusively associated as transmembrane proteins with the dystroglycan-dystrophin axis (Tinsley et aI., 1997) in a yet unknown manner, but in addition contribute together with the modified transmembrane protein caveolin-3 (for review, see Parton, 1996) to the organization of the costameres and intercostameres. In opposite to all the other lattice molecules, NOS I appears in additional wave-like structures associated with the costameres which suggests further localization possibilities of the enzyme in and/or outside the subsarcolemmaI cytoskeleton. Since NOS I generates NO, the data presented here point to uneven concentrations of this signal gas in the myofiber surface. Because of its physicochemical properties, NO should be involved in local effects, i.e. at or close to its generation site by NOS I rather than in more general fiber effects as has been described for vascular control, muscle fiber metabolism (downregulation of mitochondrial respiration, glyceraldehyde dehydrogenase and
NOS I in costameres
461
creatine kinase, upregulation of glucose uptake) and for contractile function (modulation of Ca2+ release channels, sarcoplasmic reticulum Ca2+- and myosin ATPase as well as myosin heavy chains and thus excitation-contraction coupling or relaxation-contraction processes; for a review, see Reid, 1998). Indeed, most of these observations were obtained by experiments which did not consider the highest NO concentration in the sarcolemma region and local accumulation of this gas. Therefore, it appears to be useful to include these uneven sarcolemmal concentrations of NO in interpretations about the functional role of NO in myofibers. Several of the molecules analyzed in the present investigation form complexes such as the dystrophin complex (built up by the dystroglycan and sarcoglycan axis and dystrophin-associated NOS I; for references, see Gossrau, 1998) and the integrin complex (consisting of integrin subunits, vinculin and paxillin and further molecules; for reviews, see Knudsen and Horwitz, 1994; Clark and Brugge, 1995) while for others, e. g. caveolin-3 complexes are not yet firmly established. Our study shows that all of them are appearently components of the costameric compartment of skeletal muscle. Because these molecules are involved in different complex cellular processes as signal and force transduction, adhesion as well as sarcolemma stabilization (for reviews, see Engel et al., 1994; Watkins et al., 1997) a coordinated supramolecular arrangement in the costameres seems reasonable where the precise function of the NO clouds remains to be elucidated. In conclusion, in the surface of myofibers of rats the NOS lINO signalling system shows an uneven distribution. One part of this system is concentrated in the costameres colocalized with other molecules relevant for skeletal muscle function. Therefore, the molecular costameric organization of the subsarcolemmal cytoskeleton should be highly important for undisturbed striated myofiber activity. The other part of the NOS lINO system appears to be concentrated in not yet specified structures which will hopefully be identified in the near future using high resolution confocal laser microscopy.
Acknowledgement I am thankful to Dr. B. Mayer, Graz, Austria and Dr. C. Laster, Berlin, Germany for the generous support with NOS I antibody and p1-integrin antibody, respectively, Ms H. Richter for technical assistance, Ms U. Sauerbier for photographic work, Mr G. Planitzer for typewriting the manuscript and Dr. 0. Baum for fruitful discussions and linguistic corrections.
References Clark EA, and Brugge IS (1995) Integrins and signal transduction pathways: the road taken. Science 268: 233-239 Engel AG, Yamamoto M, and Fischbeck KH (1994) Dystrophinopathies In: Engel AG, Franzini-Armstrong C (Eds) Myology. Basic and clinical, 2nd ed, Vol 2. McGraw-Hill, New York, St. Louis, pp 1133-1187
462
R. Gossrau
Gossrau R (1998) Caveolin-3 and nitric oxid synthase I in healthy and diseased muscle. Acta histochem 100: 99-112 Gossrau R, Grozdanovic Z, and Miehe B (1993) Histochemical visualization of nitric oxide synthase using NADPH and nitro BT. Symposium of the Czechoslovak Society of Histochemistry and Cytochemistry, Hradec Knilove, Czechoslovakia, December 6-9,1992. Histochem J 25: 879 Knudsen KA, and Horwitz AF (1994) The plasma membrane of muscle fiber: adhesion molecules In: Engel AG, Franzini-Armstrong C (Eds) Myology. Basic and clinical, 2nd ed, Voll. McGraw-Hill, New York, St. Louis, pp 223-241 Parton RG (1996) Caveolae and caveolins. Cur Opin Cell BioI 8: 542-548 Reid MB (1998) Role of nitric oxide in skeletal muscle: synthesis, distribution and functional importance. Acta Physiol Scand 162: 401-409 Scherer-Singler U, Vincent SR, Kimura H, and McGeer EG (1983) Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry. 1. Neurosci Meth 9: 229-234 Tinsley JM, Blake DJ, and Davis KE (1997) Dystrophin, utrophin and their associated proteins. In: Brown SC, Lucy JA (Eds) Dystrophin. Gene, protein and cell biology. Cambridge University Press, Cambridge, pp 56-78 Watkins SC, Swartz DR, and Byers TJ (1997) Localization of dystrophin in skeleton, cardiac and smooth muscle. In: Brown SC, Lucy JA (Eds) Dystrophin. Gene, protein and cell biology. Cambridge University Press, Cambridge, pp 79-104
Atla hisl•••ita
Nitric oxide synthase I (NOS I) is a costameric enzyme in rat skeletal muscle Reinhart Gossrau* Institute of Anatomy, Department of Molecular Anatomy and Cell Biology, University Clinic Benjamin Franklin, Free University of Berlin, Konigin-Luise-Str. 15, 0-14195 Berlin, Germany Accepted 20 August 1998
Summary Previously, we and others have reported association of nitric oxide (NO)generating nitric oxide synthase I (NOS I) with dystrophin in the subsarcolemmal cytoskeleton of striated muscle fibers. Since this structure shows a costameric organization the detailed distribution of NOS I and other molecules inside and outside the subsarcolemmal cytoskeleton was investigated. Using catalytic histochemistry and immunohistochemistry on rat skeletal muscle NOS I was colocalized in the costameres together with dystrophin, p-dystroglycan, a-, p- and y-sarcoglycan, p1-integrin, vinculin, paxillin and caveolin-3. Additionally, only NOS appeared in uncharacterized subsarcolemmal wave-like structures. These data show 1) a growing family of proteins assembled in the costameres including NOS I as described here for the first time, 2) expanded distribution patterns for NOS I, and 3) therefore, presumably uneven NO concentrations within the skeletal muscle fibers which may have implications for NO function. Key words: Nitric oxide synthase I - NOS I - costameres - skeletal muscle
Introduction Since our observation in 1992 of NADPH diaphorase localization in the sarcolemma region of muscle fibers (Gossrau et al., 1993) many groups including ours described NO-generating NOS I, to which this diaphorase activity is related, in myofibers of numerous mammalian and non-mammalian speCorrespondence to: R. Gossrau *Dedicated to Robert E. Smith, MD, the visionary of proteases and protease inhibitors
452
R. Gossrau
cies (for references, see Gossrau 1998). The enzyme is a component of the subsarcolemmal cytoskeleton and associated with the sarcolemma via the a1-synthrophin-dystrophin complex (for a recent review, see Reid 1998). Other investigations have shown that the subsarcolemmal cytoskeleton has a differential distribution and forms a lattice with the costameres as central ring- or rib-like structures encircling the myofibers perpendicularly to their longitudimil axis (for reviews, see Engel et aI., 1994; Watkins et aI., 1997). Therefore, in the present study the spatial relationship of NOS I to this lattice in combination with known or unknown lattice members was analyzed showing for the first time NOS I as a costameric protein and, in addition, this molecule in so far unknown subsarcolemmal structures in rat myofibers.
Materials and Methods Animals tissue, pretreatment. Adult Wistar rats (n =7) were used. The animals were of our own breeding stock, kept under standardized laboratory conditions, sacrificed in deep ether anaesthesia and tongue and neck muscles were rapidly excised. The muscles were frozen in liquid nitrogen, mounted on cryostat tables, cut at a thickness of 10 11m in a cryostat, mounted onto poly-L-Iysine-coated glass slides and used either unfixed after air-drying or fixed by immersion in 4% formaldehyde or 100% acetone for 10 min at 4°C.
Table 1. Dilution and source of the primary antibodies Antibodies
Dilution
Dystrophin-2 p- Dystroglycan a-Sarcoglycan
1:20 1:100 1:100
p-Sarcoglycan y-Sarcoglycan Spectrin-2 pI-Integrin Vinculin Paxillin
1:100 1 :100 1:100 1:200 1:400 1:1000
a-Actinin Caveolin-3 monoclonal Caveolin-3 polyclonal
1:200 1 :200 1:200
NOS I polyclonal NOS I (aa 1-181) NOS I (aa 251-270) NOS I (aa 1409-1429)
1:1000 1:300 1: 1000 1:100
Source
Novocastra Laboratories Newcastle upon Tyne, UK
C. Loster, Berlin, Germany Sigma, Munich, Germany Transduction Laboratories Lexington, KY, USA Sigma, Munich, Germany Transduction Laboratories Lexington, KY, USA B. Mayer, Graz, Austria Sigma, Munich, Germany
NOS I in costameres
453
Histochemistry. Single incubations. Immunohistochemistry. The details of the dilution and source of the antibodies are given in Table 1. For immunofluorescence tissue sections were rinsed in phosphate-buffered saline (PBS; pH 7,4) containing 0.3% Triton X-lOO (PBS-Triton), immersed in 2% normal horse serum to block non-specific staining (30 min), washed in PBS-Triton, and incubated with the following antibodies: NOS I polyclonal, NOS I (N-terminus, aa 1-181) monoclonal, NOS I (N-domain, aa 251-270) polyclonal, NOS I (C-terminus, aa 1409-1429) polyclonal, dystrophin-2 (C-terminus) monoclonal, p-dystroglycan monoclonal, a-sarcoglycan monoclonal, p-sarcoglycan monoclonal, y-sarcoglycan monoclonal, spectrin-2 monoclonal, caveolin-3 monoclonal and polyclonal, Pl-integrin polyclonal, vinculin monoclonal, paxillin monoclonal, and a-actinin monoclonal. The primary antibodies were diluted in PBS-Triton and applied to sections overnight at room temperature. After extensive washing, the sites of antigen-antibody reaction were visualized by incubation with the corresponding secondary Cy-3- or Cy-2-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA), diluted 1: 100, for 1 h at room temperature. All sections were then washed and covered in a mixture of PBS and glycerol (1: 1). In control incubations, carried out by replacing primary antibodies with either PBS or a non-relevant immune serum, no specific staining was visible. As all antibodies are well characterized for rat skeletal muscles, we did not perform Western blots or absorption tests as further controls. Enzyme histochemistry. The NADPH diaphorase activity of NOS I was demonstrated according to Scherer et al. (1993) with 3 or 4 M urea as inhibitor for NOS 1unrelated diaphorases (Gossrau, unpublished observations) in the incubation medium containing 1 mg/ml P-NADPH, 0.25 mg/ml nitro blue tetrazolium (NBT), and 0.3% Triton X-100 (v/v) in 0.1 M phosphate buffer, pH 7.4. Incubations were carried out at 37°C for 15-60 min. In control experiments, incubation media without substrate were used and yielded negative results. Double incubations. In order to investigate the relationship between NOS I and the other molecules with regard to their costameric distribution tissue sections were first labeled for NOS I diaphorase activity and then incubated with the antibodies against the other molecules followed by fluorescence visualization with the corresponding Cy-3-labeled secondary antibodies. Alternatively, tissue sections were first immunoreacted for NOS I with either polyclonal or monoclonal antibodies followed by visualization of the NOS I antigen-antibody complexes with Cy-3-conjugated secondary antibodies and were then mixed with the antibodies for the remaining molecules, which were detected with the corresponding Cy-2-labeled secondary antibodies. All sections were viewed using conventional microscopes.
Results In longitudinal sections outside the region of the subsarcolemmal cytoskeleton NOS I diaphorase activity as well as NOS I (Fig. 1), dystrophin-2 (Fig. 2), p-dystroglycan a-, p-, and y-sarcoglycan (Fig. 3), spectrin-2 (Fig. 4), p1-integrin (Fig. 5), vinculin, paxillin (Fig. 6), and caveolin-3 immunoreactivities were visualized as focal concentrations at those sites where the myofibers appeared to be relaxed. In more contracted regions this uneven distribution was less obvious or absent but was found to be more regularly distributed. Double incubation for NOS I diaphorase activity and the other molecules mentioned above revealed colocalization of NOS I-generated for-
454
R. Gossrau
Figs. 1-4. Immunofluorescence is seen as focal (dot-like) accummulation (arrows) in the region of the sarcolemma of longitudinal sections. Arrowhead = subsarcolemmal lattice. Fig. 1. NOS I. Fig. 2. Dystrophin. Fig. 3. y-Sarcoglycan. Fig. 4. Spectrin. x 360
NOS I in costameres
455
mazan and immunofluorescence at sites with low or medium activity. However, in focal concentrations, where the amount of formazan was high, immunofluorescence for all molecules was significantl'y weaker or absent. For reasons of spatial relationship double incubation for NOS I diaphorase and the Z band protein a-actinin was performed indicating colocalization of the enzyme and a-actinin.
Figs. 5,6. For localization details see Figs. 1-4. Fig. 5. p1-Integrin. Fig. 6. Paxillin. x 450
456
R. Gossrau
Figs. 7-10. NOS I diaphorase is present in costameres (arrowheads) and finer interconnections (intercostameres) forming a lattice in longitudinal sections through the region of the subsarcolemmal cytoskeleton. Figs. 7-9. Infrahyoid muscle. Fig. 10. Tongue. x 360
NOS I in costameres
457
Figs. 11-14. Immunofluorescence is found in the subsarcolemmal lattice (arrows). Fig. 11. Dystrophin. Fig. 12. p-Dystroglycan. Figs. 13, 14. p-Sarcoglycan. x 360
458
R. Gossrau
Figs. 15-18. For localization details see Figs. 7-10. Immunofluorescence is shown in the subsarcolemmal lattice (arrows, arrowhead) consisting of costameres and intercostameres. Fig. 15. y-Sarcoglycan. Fig. 16. Vinculin. Figs. 17, 18. Caveolin-3. x 360
NOS I in costameres
459
Figs. 19-22. NOS I. Figs. 19, 21, 22. NOS I diaphorase. Fig. 20. NOS I immunofluorescence. Figs. 19, 20. NOS I is found in wave-like structures (arrowheads, short arrows) of infrahyoid muscles. Figs. 21, 22. In hypercontracted myofibers of the tongue the transversal costameres appear as thick accumulations of formazan. Arrows = costameres. x 360
460
R. Gossrau
In longitudinal sections tangentially to the sarcolemma and through the region of the subsarcolemmal cytoskeleton, all molecules investigated were visualized in dense transversal bands and in finer interconnections (Figs. 718). Again, colocalization was observed for NOS I diaphorase and the immunohistochemical demonstration of the other molecules if the determinable diaphorase activity was low or medium. In contrast to the other molecules, NOS I (immunoreactivity and activity) was observed in longitudinal sections in single or double wave-like bands in the region of the subsarcolemmal cytoskeleton which occasionally crossed the whole myofiber diameter (Figs. 19, 20). These structures appeared to be connected with transversal bands positive for NOS I protein and enzyme (diaphorase) activity. Furthermore, at obviously hypercontracted sites the transversal bands were significantly thicker (Figs. 21, 22).
Discussion The data presented here reveal a differential distribution of NOS I protein and activity in the surface of myofibers of rat tongue and neck muscles. Because NOS I was colocalized with several known marker molecules for the costameric compartments such as dystrophin, Pl-integrin and vinculin (for reviews, see Engel et aI., 1994; Watkins et aI., 1997) this enzyme is a further member of the costameres. Colocalization of all molecules was also found in the finer interconnections (which may be termed intercostameres) forming a subsarcolemmal lattice together with the costameres. Furthermore, this work adds caveolin-3, paxillin as well as a-, fi- and y-sarcoglycan to the family of proteins of the subsarcolemmal lattice whose most prominent structures are the costameres. Because these costameric molecules are also colocalized with a-actinin they are mainly expressed in the region of those Z discs which contact the sarcolemma region (peripheral or subsarcolemmal Z discs) but not in the remaining (central) Z discs. The presence of the sarcoglycans in the lattice suggests the possibility that they are not exclusively associated as transmembrane proteins with the dystroglycan-dystrophin axis (Tinsley et aI., 1997) in a yet unknown manner, but in addition contribute together with the modified transmembrane protein caveolin-3 (for review, see Parton, 1996) to the organization of the costameres and intercostameres. In opposite to all the other lattice molecules, NOS I appears in additional wave-like structures associated with the costameres which suggests further localization possibilities of the enzyme in and/or outside the subsarcolemmaI cytoskeleton. Since NOS I generates NO, the data presented here point to uneven concentrations of this signal gas in the myofiber surface. Because of its physicochemical properties, NO should be involved in local effects, i.e. at or close to its generation site by NOS I rather than in more general fiber effects as has been described for vascular control, muscle fiber metabolism (downregulation of mitochondrial respiration, glyceraldehyde dehydrogenase and
NOS I in costameres
461
creatine kinase, upregulation of glucose uptake) and for contractile function (modulation of Ca2+ release channels, sarcoplasmic reticulum Ca2+- and myosin ATPase as well as myosin heavy chains and thus excitation-contraction coupling or relaxation-contraction processes; for a review, see Reid, 1998). Indeed, most of these observations were obtained by experiments which did not consider the highest NO concentration in the sarcolemma region and local accumulation of this gas. Therefore, it appears to be useful to include these uneven sarcolemmal concentrations of NO in interpretations about the functional role of NO in myofibers. Several of the molecules analyzed in the present investigation form complexes such as the dystrophin complex (built up by the dystroglycan and sarcoglycan axis and dystrophin-associated NOS I; for references, see Gossrau, 1998) and the integrin complex (consisting of integrin subunits, vinculin and paxillin and further molecules; for reviews, see Knudsen and Horwitz, 1994; Clark and Brugge, 1995) while for others, e. g. caveolin-3 complexes are not yet firmly established. Our study shows that all of them are appearently components of the costameric compartment of skeletal muscle. Because these molecules are involved in different complex cellular processes as signal and force transduction, adhesion as well as sarcolemma stabilization (for reviews, see Engel et al., 1994; Watkins et al., 1997) a coordinated supramolecular arrangement in the costameres seems reasonable where the precise function of the NO clouds remains to be elucidated. In conclusion, in the surface of myofibers of rats the NOS lINO signalling system shows an uneven distribution. One part of this system is concentrated in the costameres colocalized with other molecules relevant for skeletal muscle function. Therefore, the molecular costameric organization of the subsarcolemmal cytoskeleton should be highly important for undisturbed striated myofiber activity. The other part of the NOS lINO system appears to be concentrated in not yet specified structures which will hopefully be identified in the near future using high resolution confocal laser microscopy.
Acknowledgement I am thankful to Dr. B. Mayer, Graz, Austria and Dr. C. Laster, Berlin, Germany for the generous support with NOS I antibody and p1-integrin antibody, respectively, Ms H. Richter for technical assistance, Ms U. Sauerbier for photographic work, Mr G. Planitzer for typewriting the manuscript and Dr. 0. Baum for fruitful discussions and linguistic corrections.
References Clark EA, and Brugge IS (1995) Integrins and signal transduction pathways: the road taken. Science 268: 233-239 Engel AG, Yamamoto M, and Fischbeck KH (1994) Dystrophinopathies In: Engel AG, Franzini-Armstrong C (Eds) Myology. Basic and clinical, 2nd ed, Vol 2. McGraw-Hill, New York, St. Louis, pp 1133-1187
462
R. Gossrau
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