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. 1974 Jun 1;61(3):723–742. doi: 10.1083/jcb.61.3.723

ELECTRON MICROSCOPY AND ELECTRON PROBE ANALYSIS OF MITOCHONDRIAL CATION ACCUMULATION IN SMOOTH MUSCLE

A P Somlyo 1, A V Somlyo 1, C E Devine 1, P D Peters 1, T A Hall 1
PMCID: PMC2109306  PMID: 4836390

Abstract

The contractile responses to barium and the ultrastructure and ionic composition of mitochondria were studied in vascular smooth muscle. In normal rabbit portal anterior mesenteric vein (PAMV) and main pulmonary artery (MPA) smooth muscle mitochondria were frequently associated with the surface vesicles. The average distance between the outer mitochondrial and inner surface vesicle membrane was 4–5 nm. Ba contractures of MPA were tonic and of PAMV were phasic. Incubation of MPA and PAMV with Ba resulted in the accumulation of mitochondrial granules, followed in the MPA by massive mitochondrial swelling. Oligomycin and anoxia inhibited the appearance of mitochondrial electron-opaque granules and prevented the Ba-induced mitochondrial swelling in the MPA. Electron probe analysis of mitochondria in PAMV incubated with Ba and containing granules showed characteristic Ba signals over the mitochondria. Electron probe X-ray microanalysis also showed a highly significant (P < 0.001) correlation of P with mitochondrial Ba, in an estimated elemental ratio of approximately 3 Ba/4 P. Mitochondrial granules were still prominent after block staining of the osmium-fixed, Ba-loaded PAMV, but electron probe microanalysis showed no Ba, but only U, emissions. Tissues incubated with strontium had electron-opaque mitochondrial granules and deposits in the sarcoplasmic reticulum. X-ray microanalysis of mitochondria containing granules showed the presence of characteristic Sr and Ca emissions. The presence of Sr was similarly verified in the sarcoplasmic reticulum. These findings indicate the energy dependent uptake of divalent cations, in association with phosphate, by mitochondria in vascular smooth muscle in situ and the possibility that mitochondria may contribute to the regulation of intracellular divalent cation levels in smooth muscle.

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Selected References

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  1. Altura B. M., Altura B. T. Differential effects of substrate depletion on drug-induced contractions of rabbit aorta. Am J Physiol. 1970 Dec;219(6):1698–1705. doi: 10.1152/ajplegacy.1970.219.6.1698. [DOI] [PubMed] [Google Scholar]
  2. Barasa A., Godina G., Buffa P., Pasquali-Ronchetti I. Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo. I. The effect of fluoroacetate: a study by phase-contrast microscopy and time-lapse cinemicrography. Z Zellforsch Mikrosk Anat. 1973 Mar 29;138(2):187–210. doi: 10.1007/BF00306607. [DOI] [PubMed] [Google Scholar]
  3. Batra S. C., Daniel E. E. Effect of multivalent cations and drugs on Ca uptake by the rat myometrial microsomes. Comp Biochem Physiol A Comp Physiol. 1971 Feb 1;38(2):285–300. doi: 10.1016/0300-9629(71)90055-7. [DOI] [PubMed] [Google Scholar]
  4. Batra S. The role of mitochondrial calcium uptake in contraction and relaxation of the human myometrium. Biochim Biophys Acta. 1973 May 30;305(2):428–432. doi: 10.1016/0005-2728(73)90188-6. [DOI] [PubMed] [Google Scholar]
  5. Baudouin-Legros M., Meyer P. Effects of angiotensin, catecholamines and cyclic AMP on calcium storage in aortic microsomes. Br J Pharmacol. 1973 Feb;47(2):377–385. doi: 10.1111/j.1476-5381.1973.tb08335.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bielawski J., Lehninger A. L. Stoichiometric relationships in mitochondrial accumulation of calcium and phosphate supported by hydrolysis of adenosine triphosphate. J Biol Chem. 1966 Oct 10;241(19):4316–4322. [PubMed] [Google Scholar]
  7. Bülbring E., Tomita T. Effect of calcium, barium and manganese on the action of adrenaline in the smooth muscle of the guinea-pig taenia coli. Proc R Soc Lond B Biol Sci. 1969 Mar 11;172(1027):121–136. doi: 10.1098/rspb.1969.0015. [DOI] [PubMed] [Google Scholar]
  8. CARAFOLI E. ACTIVE ACCUMULATION OF SR2+ BY RAT-LIVER MITOCHONDRIA. 3. STIMULATION OF RESPIRATION BY SR2+ AND ITS STOICHIOMETRY. Biochim Biophys Acta. 1965 Jan 4;97:107–117. doi: 10.1016/0304-4165(65)90274-6. [DOI] [PubMed] [Google Scholar]
  9. CARAFOLI E. ACTIVE ACCUMULATION OF SR2+ BY RAT-LIVER MITOCHONDRIA. II. COMPETITION BETWEEN CA2+ AND SR2+. Biochim Biophys Acta. 1965 Jan 4;97:99–106. doi: 10.1016/0304-4165(65)90273-4. [DOI] [PubMed] [Google Scholar]
  10. CARAFOLI E., WEILAND S., LEHNINGER A. L. ACTIVE ACCUMULATION OF SR2+ BY RAT-LIVER MITOCHONDRIA. I. GENERAL FEATURES. Biochim Biophys Acta. 1965 Jan 4;97:88–98. doi: 10.1016/0304-4165(65)90272-2. [DOI] [PubMed] [Google Scholar]
  11. CHANCE B. THE ENERGY-LINKED REACTION OF CALCIUM WITH MITOCHONDRIA. J Biol Chem. 1965 Jun;240:2729–2748. [PubMed] [Google Scholar]
  12. Caplan A. I., Carafoli E. The effect of Sr2+ on swelling and ATP-linked contraction of mitochondria. Biochim Biophys Acta. 1965 Jul 8;104(2):317–329. doi: 10.1016/0304-4165(65)90338-7. [DOI] [PubMed] [Google Scholar]
  13. Carsten M. E. Role of calcium binding by sarcoplasmic reticulum in the contraction and relaxation of uterine smooth muscle. J Gen Physiol. 1969 Apr;53(4):414–426. doi: 10.1085/jgp.53.4.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Coleman J. R., Nilsson J. R., Warner R. R., Batt P. Electron probe analysis of refractive bodies in Amoeba proteus. Exp Cell Res. 1973 Jan;76(1):31–40. doi: 10.1016/0014-4827(73)90415-1. [DOI] [PubMed] [Google Scholar]
  15. Coleman J. R., Nilsson J. R., Warner R. R., Batt P. Qualitative and quantitative electron probe analysis of cytoplasmic granules in Tetrahymena pyriformis. Exp Cell Res. 1972 Sep;74(1):207–219. doi: 10.1016/0014-4827(72)90499-5. [DOI] [PubMed] [Google Scholar]
  16. DANIEL E. E. ON ROLES OF CALCIUM STRONTIUM AND BARIUM IN CONTRACTION AND EXCITABILITY OF RAT UTERINE MUSCLE. Arch Int Pharmacodyn Ther. 1963 Dec 1;146:298–349. [PubMed] [Google Scholar]
  17. Devine C. E., Somlyo A. V., Somlyo A. P. Sarcoplasmic reticulum and excitation-contraction coupling in mammalian smooth muscles. J Cell Biol. 1972 Mar;52(3):690–718. doi: 10.1083/jcb.52.3.690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Devine C. E., Somlyo A. V., Somlyo A. P. Sarcoplasmic reticulum and mitochondria as cation accumulation sites in smooth muscle. Philos Trans R Soc Lond B Biol Sci. 1973 Mar 15;265(867):17–23. doi: 10.1098/rstb.1973.0005. [DOI] [PubMed] [Google Scholar]
  19. Drahota Z., Gazzotti P., Carafoli E., Rossi C. S. A comparison of the effects of different divalent cations on a number of mitochondrial reactions linked to ion translocation. Arch Biochem Biophys. 1969 Mar;130(1):267–273. doi: 10.1016/0003-9861(69)90033-2. [DOI] [PubMed] [Google Scholar]
  20. Edwards C., Lorković H., Weber A. The effect of the replacement of calcium by strontium on excitation-contraction coupling in frog skeletal muscle. J Physiol. 1966 Oct;186(2):295–306. doi: 10.1113/jphysiol.1966.sp008035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goodford P. J., Wolowyk M. W. Localization of cation interactions in the smooth muscle of the guinea-pig taenia coli. J Physiol. 1972 Aug;224(3):521–535. doi: 10.1113/jphysiol.1972.sp009911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Greenawalt J. W., Carafoli E. Electron microscope studies on the active accumulation of Sr++ by rat-liver mitochondria. J Cell Biol. 1966 Apr;29(1):37–61. doi: 10.1083/jcb.29.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hackenbrock C. R., Caplan A. I. Ion-induced ultrastructural transformations in isolated mitochondria. The energized uptake of calcium. J Cell Biol. 1969 Jul;42(1):221–234. doi: 10.1083/jcb.42.1.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Harigaya S., Schwartz A. Rate of calcium binding and uptake in normal animal and failing human cardiac muscle. Membrane vesicles (relaxing system) and mitochondria. Circ Res. 1969 Dec;25(6):781–794. doi: 10.1161/01.res.25.6.781. [DOI] [PubMed] [Google Scholar]
  25. Hermsmeyer K., Sperelakis N. Decrease in K+ conductance and depolarization of frog cardiac muscle produced by Ba++. Am J Physiol. 1970 Oct;219(4):1108–1114. doi: 10.1152/ajplegacy.1970.219.4.1108. [DOI] [PubMed] [Google Scholar]
  26. Hoff H. F. A comparison of the fine-structural localization of nucleoside phosphatase activity in large intracranial blood vessels and the thoracic aorta of rabbits. Histochemie. 1968;13(2):183–191. doi: 10.1007/BF00266579. [DOI] [PubMed] [Google Scholar]
  27. Horn R. S., Fyhn A., Haugaard N. Mitochondrial calcium uptake in the perfused contracting rat heart and the influence of epinephrine on calcium exchange. Biochim Biophys Acta. 1971 Mar 2;226(2):459–466. doi: 10.1016/0005-2728(71)90112-5. [DOI] [PubMed] [Google Scholar]
  28. Hurwitz L., Fitzpatrick D. F., Debbas G., Landon E. J. Localization of calcium pump activity in smooth muscle. Science. 1973 Jan 26;179(4071):384–386. doi: 10.1126/science.179.4071.384. [DOI] [PubMed] [Google Scholar]
  29. Jones A. W., Somlyo A. P., Somlyo A. V. Potassium accumulation in smooth muscle and associated ultrastructural changes. J Physiol. 1973 Jul;232(2):247–273. doi: 10.1113/jphysiol.1973.sp010268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Karaki H., Ikeda M., Urakawa N. Effects of external calcium and some metabolic inhibitors on barium-induced tension changes in guinea pig taenia coli. Jpn J Pharmacol. 1967 Dec;17(4):603–612. doi: 10.1254/jjp.17.603. [DOI] [PubMed] [Google Scholar]
  31. Lehninger A. L. Mitochondria and calcium ion transport. Biochem J. 1970 Sep;119(2):129–138. doi: 10.1042/bj1190129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Northover B. J. The effect of drugs on the constriction of isolated depolarized blood vessels in response to calcium or barium. Br J Pharmacol. 1968 Oct;34(2):417–428. doi: 10.1111/j.1476-5381.1968.tb07062.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. PEACHEY L. D. ELECTRON MICROSCOPIC OBSERVATIONS ON THE ACCUMULATION OF DIVALENT CATIONS IN INTRAMITOCHONDRIAL GRANULES. J Cell Biol. 1964 Jan;20:95–111. doi: 10.1083/jcb.20.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Patriarca P., Carafoli E. A study of the intracellular transport of calcium in rat heart. J Cell Physiol. 1968 Aug;72(1):29–37. doi: 10.1002/jcp.1040720106. [DOI] [PubMed] [Google Scholar]
  35. Podolsky R. J., Hall T., Hatchett S. L. Identification of oxalate precipitates in striated muscle fibers. J Cell Biol. 1970 Mar;44(3):699–702. doi: 10.1083/jcb.44.3.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sakamoto Y., Kuriyama H. The relationship between the electrical and mechanical activity of the guinea-pig stomach. Jpn J Physiol. 1970 Dec 15;20(6):640–656. doi: 10.2170/jjphysiol.20.640. [DOI] [PubMed] [Google Scholar]
  37. Somlyo A. P., Devine C. E., Somlyo A. V., North S. R. Sarcoplasmic reticulum and the temperature-dependent contraction of smooth muscle in calcium-free solutions. J Cell Biol. 1971 Dec;51(3):722–741. doi: 10.1083/jcb.51.3.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Somlyo A. P., Devine C. E., Somlyo A. V., Rice R. V. Filament organization in vertebrate smooth muscle. Philos Trans R Soc Lond B Biol Sci. 1973 Mar 15;265(867):223–229. doi: 10.1098/rstb.1973.0027. [DOI] [PubMed] [Google Scholar]
  39. Somlyo A. P. Excitation-contraction coupling in vertebrate smooth muscle: correlation of ultrastructure with function. Physiologist. 1972 Nov;15(4):338–348. [PubMed] [Google Scholar]
  40. Somlyo A. P., Somlyo A. V. Vascular smooth muscle. II. Pharmacology of normal and hypotensive vessels. Pharmacol Rev. 1970 Jun;22(2):249–353. [PubMed] [Google Scholar]
  41. Somlyo A. V., Somlyo A. P. Strontium accumulation by sarcoplasmic reticulum and mitochondria in vascular smooth muscle. Science. 1971 Nov 26;174(4012):955–958. doi: 10.1126/science.174.4012.955. [DOI] [PubMed] [Google Scholar]
  42. Somlyo A. V., Vinall P., Somlyo A. P. Excitation-contraction coupling and electrical events in two types of vascular smooth muscle. Microvasc Res. 1969 Oct;1(4):354–373. doi: 10.1016/0026-2862(69)90014-4. [DOI] [PubMed] [Google Scholar]
  43. Spurr A. R. A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res. 1969 Jan;26(1):31–43. doi: 10.1016/s0022-5320(69)90033-1. [DOI] [PubMed] [Google Scholar]
  44. Sulakhe P. V., Dhalla N. S. Excitation-contraction coupling in heart. IV. Energy-dependent calcium transport in the myocardium of developing rat. Life Sci I. 1970 Dec 1;9(23):1363–1370. [PubMed] [Google Scholar]
  45. Sutfin L. V., Holtrop M. E., Ogilvie R. E. Microanalysis of individual mitochondrial granules with diameters less than 1000 angstroms. Science. 1971 Nov 26;174(4012):947–949. doi: 10.1126/science.174.4012.947. [DOI] [PubMed] [Google Scholar]
  46. VASINGTON F. D., MURPHY J. V. Ca ion uptake by rat kidney mitochondria and its dependence on respiration and phosphorylation. J Biol Chem. 1962 Aug;237:2670–2677. [PubMed] [Google Scholar]
  47. Vainio H., Mela L., Chance B. Energy dependent bivalent cation translocation in rat liver mitochondria. Eur J Biochem. 1970 Feb;12(2):387–391. doi: 10.1111/j.1432-1033.1970.tb00863.x. [DOI] [PubMed] [Google Scholar]
  48. Van der Kloot W. G., Glovsky J. The uptake of Ca2+ and Sr2+ by fractions from lobster muscle. Comp Biochem Physiol. 1965 Aug;15(4):547–565. doi: 10.1016/0010-406x(65)90154-4. [DOI] [PubMed] [Google Scholar]
  49. Weavers B. A. The potentiality of EMMA-4, the analytical electron microscope, in histochemistry: a review. Histochem J. 1973 Mar;5(2):173–183. doi: 10.1007/BF01012560. [DOI] [PubMed] [Google Scholar]

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