Comprehensive Physiology Wiley Online Library

Isolated Membranes and Organelles from Vascular Smooth Muscle

Full Article on Wiley Online Library



Abstract

The sections in this article are:

1 Isolation Techniques
1.1 Homogenization
1.2 Fractionation
2 Identification of Isolated Fractions of Vascular Smooth Muscle
2.1 Mitochondria
2.2 Microsomal and Plasma Membrane Fractions
3 Functional Studies of Isolated Fractions of Vascular Smooth Muscle
3.1 Mitochondria
3.2 Microsomal and Plasma Membrane Fractions
4 Summary
Figure 1. Figure 1.

Fractionation scheme for isolation of mitochondria from bovine vascular smooth muscle. MPA, main pulmonary artery; MV, mesenteric vein; MSE, 225 mM mannitol, 75 mM sucrose, 5 mM EDTA, pH 7.2.

Adapted from Vallières et al.
Figure 2. Figure 2.

A: mitochondria isolated from main pulmonary artery. B: mitochondria isolated from mesenteric vein as described in Figure . × 15,800.

From Vallières et al.
Figure 3. Figure 3.

Distribution of markers and proteins in different fractions of rat myometrium. WGA, wheat germ agglutinin; PNS, postnuclear supernatant; MITO, mitochondria; SOL, soluble; MIC, microsomes; F1–F5, fractions isolated on sucrose density gradient of from 28% to 45% sucrose.

From Daniel et al.
Figure 4. Figure 4.

Rates of Ca2+ uptake by bovine main pulmonary artery and mesenteric vein mitochondria at various Ca2+ concentrations. Mitochondria (0.2–2.7 mg/ml) were incubated at 26°C in 100 mM sucrose, 50 mM KCl, 2 mM MgCl2, 5 mM sodium succinate, 3 μM rotenone, 5 mM sodium acetate, 20 mM 4‐morpholinopropanesulfonate (pH 7.2), and 100 μM murexide or 40 μM arsenazo. Rates were measured with arsenazo for Ca2+ concentrations ranging from 1 to 25 μM and with murexide for higher concentrations. A: mitochondria were isolated from main pulmonary artery as described in Figure ; •, in the presence of 0.2% bovine serum albumin; □, in the absence of albumin; ▵, after addition of Nagarse proteinase. Each point represents the average of two or three experiments on five preparations (•) or on two preparations (□, ▵). B: all points were obtained from two experiments with four preparations of mesenteric vein mitochondria.

From Vallières et al.
Figure 5. Figure 5.

Initial rate of Mg2+ release by mitochondria of bovine main pulmonary artery at various external [Mg2+]. Reaction mixtures contained 1.0–1.5 mg/ml of mitochondria. Transport of Mg2+ was measured at 25°C in 200 mM sucrose, 20 mM KCl, 30 mM 4‐morpholinopropanesulfonate, 3 μM rotenone (pH 7.1), and 30 μM eriochrome blue. Each point represents the average of three experiments on three mitochondrial preparations.

From Sloane et al.
Figure 6. Figure 6.

Calcium uptake of microsomal vesicles of rabbit aorta in a representative experiment. Incubation was at 37°C in 30 μmol tris‐HCl, 300 μmol KCl, 9 μmol Mg‐ATP, 15 μmol ammonium oxalate, 15 μmol sodium azide, 0.4 μCi 45CaCl2, and 0.3 or 0.06 μmol CaCl2. Curve a, 100 μM calcium, complete medium; curve b, 20 μM calcium, complete medium; curve c, 100 μM calcium, oxalate omitted; curve d, 100 μM calcium, Mg‐ATP omitted.

From Fitzpatrick et al. Science 176(4032): 305–306, 1972. Copyright 1972 by the American Association for the Advancement of Science
Figure 7. Figure 7.

A: effect of varying initial free calcium level on calcium uptake by vesicular fraction of vascular smooth muscle. Calcium uptake was determined at 37°C in an incubation solution consisting of 5 mM ATP, 5 mM MgCl2, 104 mM KCl, 18 mM imidazole (pH 7.0), 10 mM sodium azide, 10 mM potassium oxalate, 6 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase, and 0.05 μCi/ml 45CaCl2; 0.040 mM ethylene glycol‐bis(β‐aminoethyl ether)‐N,N′‐tetraacetic acid (EGTA) plus various amounts of CaCl2 were added to give the initial free calcium levels shown. B: Woolf plot of the vesicular calcium uptake shown in A. The average Km and Vmax for seven such determinations is given.

From Ford and Hess , by permission of the American Heart Association, Inc
Figure 8. Figure 8.

Distribution of calcium uptake activity and enzyme markers in six fractions obtained by centrifuging microsomal vesicles in a sucrose density gradient. A: representative data from guinea pig intestinal smooth muscle. B: representative data from rabbit aorta. Specific activity of calcium uptake was measured from 5 to 15 min after start of incubation. Specific activity of sodium potassium adenosine triphosphatase (ATPase) is in μmol inorganic phosphate (Pi)/mg protein per 15 min. Specific activity of 5′‐nucleotidase is in μmol Pi/mg protein per 30 min. Specific activity of NADH oxidase is in μmol NADH/mg protein per min.

From Hurwitz et al. Science 179 (71): 384–386, 1973. Copyright 1973 by the American Association for the Advancement of Science


Figure 1.

Fractionation scheme for isolation of mitochondria from bovine vascular smooth muscle. MPA, main pulmonary artery; MV, mesenteric vein; MSE, 225 mM mannitol, 75 mM sucrose, 5 mM EDTA, pH 7.2.

Adapted from Vallières et al.


Figure 2.

A: mitochondria isolated from main pulmonary artery. B: mitochondria isolated from mesenteric vein as described in Figure . × 15,800.

From Vallières et al.


Figure 3.

Distribution of markers and proteins in different fractions of rat myometrium. WGA, wheat germ agglutinin; PNS, postnuclear supernatant; MITO, mitochondria; SOL, soluble; MIC, microsomes; F1–F5, fractions isolated on sucrose density gradient of from 28% to 45% sucrose.

From Daniel et al.


Figure 4.

Rates of Ca2+ uptake by bovine main pulmonary artery and mesenteric vein mitochondria at various Ca2+ concentrations. Mitochondria (0.2–2.7 mg/ml) were incubated at 26°C in 100 mM sucrose, 50 mM KCl, 2 mM MgCl2, 5 mM sodium succinate, 3 μM rotenone, 5 mM sodium acetate, 20 mM 4‐morpholinopropanesulfonate (pH 7.2), and 100 μM murexide or 40 μM arsenazo. Rates were measured with arsenazo for Ca2+ concentrations ranging from 1 to 25 μM and with murexide for higher concentrations. A: mitochondria were isolated from main pulmonary artery as described in Figure ; •, in the presence of 0.2% bovine serum albumin; □, in the absence of albumin; ▵, after addition of Nagarse proteinase. Each point represents the average of two or three experiments on five preparations (•) or on two preparations (□, ▵). B: all points were obtained from two experiments with four preparations of mesenteric vein mitochondria.

From Vallières et al.


Figure 5.

Initial rate of Mg2+ release by mitochondria of bovine main pulmonary artery at various external [Mg2+]. Reaction mixtures contained 1.0–1.5 mg/ml of mitochondria. Transport of Mg2+ was measured at 25°C in 200 mM sucrose, 20 mM KCl, 30 mM 4‐morpholinopropanesulfonate, 3 μM rotenone (pH 7.1), and 30 μM eriochrome blue. Each point represents the average of three experiments on three mitochondrial preparations.

From Sloane et al.


Figure 6.

Calcium uptake of microsomal vesicles of rabbit aorta in a representative experiment. Incubation was at 37°C in 30 μmol tris‐HCl, 300 μmol KCl, 9 μmol Mg‐ATP, 15 μmol ammonium oxalate, 15 μmol sodium azide, 0.4 μCi 45CaCl2, and 0.3 or 0.06 μmol CaCl2. Curve a, 100 μM calcium, complete medium; curve b, 20 μM calcium, complete medium; curve c, 100 μM calcium, oxalate omitted; curve d, 100 μM calcium, Mg‐ATP omitted.

From Fitzpatrick et al. Science 176(4032): 305–306, 1972. Copyright 1972 by the American Association for the Advancement of Science


Figure 7.

A: effect of varying initial free calcium level on calcium uptake by vesicular fraction of vascular smooth muscle. Calcium uptake was determined at 37°C in an incubation solution consisting of 5 mM ATP, 5 mM MgCl2, 104 mM KCl, 18 mM imidazole (pH 7.0), 10 mM sodium azide, 10 mM potassium oxalate, 6 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase, and 0.05 μCi/ml 45CaCl2; 0.040 mM ethylene glycol‐bis(β‐aminoethyl ether)‐N,N′‐tetraacetic acid (EGTA) plus various amounts of CaCl2 were added to give the initial free calcium levels shown. B: Woolf plot of the vesicular calcium uptake shown in A. The average Km and Vmax for seven such determinations is given.

From Ford and Hess , by permission of the American Heart Association, Inc


Figure 8.

Distribution of calcium uptake activity and enzyme markers in six fractions obtained by centrifuging microsomal vesicles in a sucrose density gradient. A: representative data from guinea pig intestinal smooth muscle. B: representative data from rabbit aorta. Specific activity of calcium uptake was measured from 5 to 15 min after start of incubation. Specific activity of sodium potassium adenosine triphosphatase (ATPase) is in μmol inorganic phosphate (Pi)/mg protein per 15 min. Specific activity of 5′‐nucleotidase is in μmol Pi/mg protein per 30 min. Specific activity of NADH oxidase is in μmol NADH/mg protein per min.

From Hurwitz et al. Science 179 (71): 384–386, 1973. Copyright 1973 by the American Association for the Advancement of Science
References
 1. Allfrey, V. The isolation of subcellular components. In: The Cell, edited by J. Bracket and A. E. Mirsky. New York: Academic, 1959, vol. 1, p. 193–290.
 2. Brierley, G., E. Murer, E. Bachmann, and D. E. Green. Studies on ion transport. II. The accumulation of inorganic phosphate and magnesium ions by heart mitochondria. J. Biol. Chem. 238: 3482–3489, 1963.
 3. Carafoli, E., and M. Crompton. The regulation of intracellular calcium by mitochondria. Ann. NY Acad. Sci. 307: 269–284, 1978.
 4. Carsten, M. E., and J. D. Miller. Purification and characterization of microsomal fractions from smooth muscle. In: Excitation‐Contraction Coupling in Smooth Muscle, edited by R. Casteels, T. Godfraind, and J. C. Rüegg. New York: Elsevier North‐Holland, 1977, p. 155–163.
 5. Chappell, J. B., and S. V. Perry. Biochemical and osmotic properties of skeletal muscle mitochondria. Nature 173: 1094, 1954.
 6. Clyman, R. I., V. C. Manganiello, C. J. Lovell‐Smith, and M. Vaughan. Calcium uptake by subcellular fractions of human umbilical artery. Am. J. Physiol. 231: 1074–1081, 1976.
 7. Crompton, M. V., M. Capano, and E. Carafoli. Respiration‐dependent efflux of magnesium ions from heart mitochondria. Biochem. J. 154: 735–742, 1976.
 8. Daniel, E. E., C. Y. Kwan, M. A. Matlib, D. Crankshaw, and A. Kidwai. Characterization and Ca2+‐accumulation by membrane fractions from myometrium and artery. In: Excitation‐Contraction Coupling in Smooth Muscle, edited by R. Casteels, T. Godfraind, and J. C. Rüegg. New York: Elsevier North‐Holland, 1977, p. 181–188.
 9. Devine, C. E., A. V. Somlyo, and A. P. Somlyo. Sarcoplasmic reticulum and excitation‐contraction coupling in mammalian smooth muscle. J. Cell Biol. 52: 690–718, 1972.
 10. Devynck, M.‐A., M.‐G. Pernollet, P. Meyer, S. Fermandjian, and P. Fromageot. Angiotensin receptors in smooth muscle cell membranes. Nature New Biol. 245: 55–58, 1973.
 11. Ebashi, S. Calcium binding activity of vesicular relaxing factor. J. Biochem. Tokyo 50: 236–244, 1961.
 12. Filo, R. S., D. F. Bohr, and J. C. Ruegg. Glycerinated skeletal and smooth muscle: calcium and magnesium dependence. Science 147: 1581–1583, 1965.
 13. Fitzpatrick, D. F., E. J. Landon, G. Debbas, and L. Hurwitz. A calcium pump in vascular smooth muscle. Science 176: 305–306, 1972.
 14. Ford, G. D., and M. L. Hess. Calcium‐accumulating properties of subcellular fractions of bovine vascular smooth muscle. Circulation Res. 37: 580–587, 1975.
 15. Greville, G. D. A scrutiny of Mitchell's chemiosmotic hypothesis of respiratory chain and photosynthetic phosphorylation. Current Topics Bioenergetics 3: 1–78, 1970.
 16. Haley, N. J., H. Shio, and S. Fowler. Characterization of lipid‐laden aortic cells from cholesterol‐fed rabbits. Lab. Invest. 37: 287–296, 1977.
 17. Hartshorne, D. J., and M. Aksoy. Introduction: biochemistry of the contractile proteins in smooth muscle. A survey of current knowledge. In: The Biochemistry of Smooth Muscle, edited by N. L. Stephens. Baltimore: Univ. Park, 1977, p. 363–378.
 18. Hasselbach, W., and M. Makinose. Über den Mechanismus des Calciumtransportes durch die Membranen des sarkoplasmatischen Reticulums. Biochem. Z. 339: 94–111, 1963.
 19. Hess, M. L., and G. D. Ford. Calcium accumulation by subcellular fractions from vascular smooth muscle. J. Mol. Cellular Cardiol. 6: 275–282, 1974.
 20. Hurwitz, L., D. F. Fitzpatrick, G. Debbas, and E. J. Landon. Localization of calcium pump activity in smooth muscle. Science 179: 384–386, 1973.
 21. Janis, R. A., D. J. Crankshaw, and E. E. Daniel. Control of intracellular Ca2+ activity in rat myometrium. Am. J. Physiol. 232: C50–C58, 1977 or
 22. Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 1: C50–C58, 1977.
 23. Kidwai, A. M. Isolation of plasma membrane from smooth, skeletal, and heart muscle. Methods Enzymol. 31: 134–144, 1974.
 24. Lehninger, A. L., B. Reynafarje, A. Vercesi, and W. P. Tew. Transport and accumulation of calcium in mitochondria. Ann. NY Acad. Sci. 307: 160–176, 1978.
 25. Martonosi, A., and R. Feretos. Sarcoplasmic reticulum. I. The uptake of Ca++ by sarcoplasmic reticulum fragments. J. Biol. Chem. 239: 648–658, 1964.
 26. Martonosi, A., and R. Feretos. Sarcoplasmic reticulum. II. Correlation between adenosine triphosphatase activity and Ca++ uptake. J. Biol. Chem. 239: 659–668, 1964.
 27. Page, E., L. P. McCallister, and B. Power. Stereological measurements of cardiac ultrastructures implicated in excitation‐contraction coupling. Proc. Natl. Acad. Sci. US 68: 1465–1466, 1971.
 28. Peters, T. J., and C. deDuve. Lysosomes of the arterial wall. II. Subcellular fractionation of aortic cells from rabbits with experimental atheroma. Exptl. Mol. Pathol. 20: 228–256, 1974.
 29. Peters, T. J., M. Müller, and C. deDuve. Lysosomes of the arterial wall. I. Isolation and subcellular fractionation of cells from normal rabbit aorta. J. Exptl. Med. 136: 1117–1139, 1972.
 30. Raeymaekers, L., F. Wuytack, S. Batra, and R. Casteels. A comparative study of the calcium accumulation by mitochondria and microsomes isolated from the smooth muscle of the guinea‐pig taenia coli. Pfluegers Arch. European J. Physiol. 368: 217–223, 1977.
 31. Scarpa, A. Transport across the mitochondrial membrane. In: Handbook of Biological Transport, edited by H. H. Ussing and D. C. Tosteson. Berlin: Springer‐Verlag, 1978, vol. 2, p. 263–356.
 32. Scarpa, A., and P. Graziotti. Mechanisms for intracellular calcium regulation in heart. I. Stopped flow measurements of Ca++ uptake by cardiac mitochondria. J. Gen. Physiol. 62: 756–772, 1973.
 33. Scarpa, A., and J. R. Williamson. Calcium‐binding and calcium transport by subcellular fractions of heart. In: Calcium Binding Proteins, edited by W. Drabikowski, H. Strzelecka‐Golaszewska, and E. Carafoli. Amsterdam: Elsevier, 1975, p. 547–585.
 34. Shibata, N., and W. Hollander. Studies on the role of arterial microsomes in the contractile function of the arteries. Exptl. Mol. Pathol. 21: 1–15, 1974.
 35. Sloane, B. F., A. Scarpa, and A. P. Somlyo. Vascular smooth muscle mitochondria: magnesium content and transport. Arch. Biochem. Biophys. 189: 409–416, 1978.
 36. Somlyo, A. P., C. E. Devine, A. V. Somlyo, and S. R. North. Sarcoplasmic reticulum and the temperature‐dependent contraction of smooth muscle in calcium‐free solutions. J. Cell Biol. 51: 722–741, 1971.
 37. Somlyo, A. P., A. V. Somlyo, H. Shuman, B. F. Sloane, and A. Scarpa. Electron probe analysis of calcium compartments in cryo sections of smooth and striated muscles. Ann. NY Acad. Sci. 307: 523–544, 1978.
 38. Stauber, W. T., A. M. Hedge, and B. A. Schottelius. Alterations in lysosomes, catalase‐containing organelles, mitochondria and plasma membrane fragments from hypertensive rat aorta and caudal artery. Blood Vessels 16: 17–25, 1979.
 39. Tada, M., T. Yamamoto, and Y. Tonomura. Molecular mechanism of active calcium transport by sarcoplasmic reticulum. Physiol. Rev. 58: 1–79, 1978.
 40. Vallières, J., A. Scarpa, and A. P. Somlyo. Subcellular fractions of smooth muscle. Isolation, substrate utilization and Ca++ transport by main pulmonary artery and mesenteric vein mitochondria. Arch. Biochem. Biophys. 170: 659–669, 1975.
 41. Vallières, J., M. Fortier, A. V. Somlyo, and A. P. Somlyo. Isolation of plasma membranes from rabbit myometrium. Intern. J. Biochem. 9: 487–498, 1978.
 42. Verity, M. A., and J. A. Bevan. Membrane adenosine triphosphatase activity of vascular smooth muscle. Biochem. Pharmacol. 18: 327–338, 1969.
 43. Wainio, W. W. The Mammalian Mitochondrial Respiratory Chain. New York: Academic, 1970. (Molecular Biol. Ser.)
 44. Webb, R. C., and R. C. Bhalla. Calcium sequestration by subcellular fractions isolated from vascular smooth muscle: effect of cyclic nucleotides and prostaglandins J. Mol. Cellular Cardiol. 8: 145–157, 1976.
 45. Weber, A., R. Herz, and I. Reiss. On the mechanism of the relaxing effect of fragmented sarcoplasmic reticulum. J. Gen. Physiol. 46: 679–702, 1963.
 46. Wei, J.‐W., R. A. Janis, and E. E. Daniel. Calcium accumulation and enzymatic activities of subcellular fractions from aortas and ventricles of genetically hypertensive rats. Circulation Res. 39: 133–140, 1976.
 47. Yamashita, K., K. Aoki, K. Takikawa, and K. Hotta. Calcium uptake, release and Mg‐ATPase activity of sarcoplasmic reticulum from arterial smooth muscle. Japan. Circulation J. 40: 1175–1181, 1976.
 48. Zelck, U., and U. Karnstedt. ATP‐dependent Ca2+ binding by mitochondrial and microsomal fractions isolated from different smooth muscles: influence of cyclic nucleotides. In: Excitation‐Contraction Coupling in Smooth Muscles, edited by R. Casteels, T. Godfraind, and J. C. Rüegg. New York: Elsevier North‐Holland, 1977, p. 171–180.
 49. Zelck, U., U. Karnstedt, and E. Albrecht. Calcium uptake and calcium release by subcellular fractions of smooth muscle. II. Kinetics of calcium uptake by microsomes and mitochondria from pig coronary artery and guinea pig ileum. Acta Biol. Med. Ger. 34: 981–986, 1975.

Contact Editor

Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite

Bonnie F. Sloane. Isolated Membranes and Organelles from Vascular Smooth Muscle. Compr Physiol 2011, Supplement 7: Handbook of Physiology, The Cardiovascular System, Vascular Smooth Muscle: 121-132. First published in print 1980. doi: 10.1002/cphy.cp020205