Summary
The validity of the pyroantimonate method, which has been used for detecting intracellular Ca localization and translocation in smooth muscles, was examined by making cryosections of the relaxed anterior byssal retractor muscle (ABRM) of Mytilus edulis at various stages of procedures for preparing ordinary Epon-embedded sections and determining the elemental concentration ratios of the pyroantimonate precipitate, localized along the inner surface of the plasma membrane, with an energy dispersive X-ray microanalyzer. The concentration of Ca (relative to that of Sb) in the precipitate stayed constant after the procedures of fixation, dehydration and Epon-embedding, while the concentrations of K, Mg, Na and Os showed their respective characteristic changes after the above procedures, being lower than that of Ca in the Epon-embedded sections. The presence of Ca in the precipitate was also demonstrated with an electron energy-loss spectrometer. The localization of Ca underneath the plasma membrane was also observed in the cryosections of the ABRM fibers prepared after mild fixation with acrolein vapor without using pyroantimonate. These results indicate that the pyroantimonate precipitate serves as a valid measure of intracellular Ca localization.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Atsumi S, Sugi H (1976) Localization of calcium-accumulating structures in the anterior byssal retractor muscle of Mytilus edulis and their role in the regulation of active and catch contraction. J Physiol (London) 257:549–560
Ebashi S, Endo M (1968) Calcium ion and muscle contraction. Prog Biophys Mol Biol 18:123–183
Egerton RF (1986) Electron energy-loss spectroscopy in the electron microscope. Plenum Press, New York
Fukuoka H, Takagi T, Nagai H, Hotta K, Suzuki S, Sugi H (1980) Localization and translocation of intracellular calcium in smooth muscle cells of bovine cerebral artery. J Electron Microsc 29:266–269
Garfield RE, Henderson RM, Daniel EE (1972) Evaluation of the pyroantimonate technique for localization of tissue sodium. Tissue Cell 4:575–589
Hayat MA (1975) Positive staining for electron microscopy. Van Nostrand Reinhold, New York
Iwamoto H, Suzuki S, Mizobe H (1988) Regulatory mechanism of contraction in the proboscis retractor muscle of a sipunculid, Phasocolosoma scolops. Cell Tissue Res 253:15–21
Klein RL, Yen SS, Thureson-Klein Å (1972) Critique on the K-pyroantimonate method for semiquantitative estimation of cations in conjunction with electron microscopy J Histochem Cytochem 20:65–78
Komnick H (1962) Electronenmikroscopische Lokalisation von Na+ und Cl− in Zellen und Geweben. Protoplasma 55:414–418
Komnick H, Komnick U (1963) Electronenmikroscopische Untersuchungen zur funktionellen Morphologie des Ionentransportes in der Sarzdrüse von Larus argentatus. Z Zellforsch Mikrosk Anat 60:163–208
Kowarski D, Shuman H, Somlyo AP, Somlyo AV (1985) Calcium release by noradrenaline from central sarcoplasmic reticulum in rabbit main pulmonary artery smooth muscle. J Physiol (London) 366:153–175
Legato MJ, Langer GA (1969) The subcellular localization of calcium ion in mammalian myocardium. J Cell Biol 41:401–423
Mizuhira V (1976) Elemental analysis of biological specimens by electron probe X-ray microanalysis. Acta Histochem Cytochem 9:69–87
Nakayama K, Suzuki S, Sugi H (1986) Physiological and ultrastructural studies on the mechanism of stretch-induced contractile activation in rabbit cerebral artery smooth muscle. Jpn J Physiol 36:745–760
Russ JC (1974) The direct element ratio model for quantitative analysis of thin sections. In: Hall T, Echlin P, Kaufman R (eds) Microprobe analysis as applied to cells and tissues. Academic Press, London New York, pp 269–276
Russ JC (1977) Analyzing calcium in the presence of antimony. EDAX EDITor 7(4):18–20
Shiina S-I, Mizuhira V, Amakawa T, Futaesaku Y (1970) An analysis of the histochemical procedure for sodium ion detection. J Histochem Cytochem 18:644–649
Simson JAV, Spicer SS (1975) Selective subcellular localization of cations with variants of the potassium (pyro)antimonate technique. J Histochem Cytochem 23:575–598
Somlyo AP (1985a) Cell calcium measurement with electron probe and energy loss analysis. Cell Calcium 6:197–212
Somlyo AP (1985b) Excitation-contraction coupling and the ultrastructure of smooth muscle. Circ Res 57:497–507
Somlyo AV, Shuman H, Somlyo AP (1977) Elemental distribution in striated muscle and the effects of hypertonicity. Electron probe analysis of cryosections. J Cell Biol 74:828–857
Sugi H, Daimon T (1977) Translocation of intracellularly stored calcium during the contraction-relaxation cycle in guinea-pig taenia coli. Nature 269:436–438
Sugi H, Suzuki S (1982) Physiological and ultrastructural studies on the intracellular calcium translocation during contraction in invertebrate smooth muscles. In: Twarog BM, Devine RJC, Dewey MM (eds) Basic biology of muscles: A comparative approach. Raven Press, New York, pp 359–370
Sugi H, Suzuki S, Daimon T (1982) Intracellular calcium translocation during contraction in vertebrate and invertebrate smooth muscles as studied by the pyroantimonate method. Can J Physiol Pharmacol 60:576–587
Suzuki S (1982) Physiological and cytochemical studies on activator calcium in contraction by smooth muscle of a sea cucumber, Isostichopus badionotus. Cell Tissue Res 222:11–24
Suzuki S, Sugi H (1978) Ultrastructural and Physiological studies on the longitudinal body wall muscle of Dolabella auricularia. II. Localization of intracellular calcium and its translocation during mechanical activity. J Cell Biol 79:467–478
Suzuki S, Sugi H (1982a) Physiological and ultrastructural studies on the longitudinal retractor muscle of a sea cucumber Stichopus japonicus. II. Intracellular localization and translocation of activator calcium during mechanical activity. J Exp Biol 97:113–119
Suzuki S, Sugi H (1982b) Mechanisms of intracellular calcium translocation in muscle. In: Anghileri LJ, Tuffet-Anghileri AM (eds) The role of calcium in biological systems, vol 1. CRC Press, Boca Raton, Florida, pp 201–217
Tisher CC, Weavers BA, Cirksena WJ (1972) X-ray microanalysis of pyroantimonate complexes in rat kidney. Am J Pathol 69:255–270
Wick SM, Hepler PK (1982) Selective localization of intracellular Ca2+ with potassium antimonate. J Histochem Cytochem 30:1190–1204
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Suzuki, S., Sugi, H. Evaluation of the pyroantimonate method for detecting intracellular calcium localization in smooth muscle fibers by the X-ray microanalysis of cryosections. Histochemistry 92, 95–101 (1989). https://doi.org/10.1007/BF00490226
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00490226